Table of contents :

acute myeloid leukemias  FAB classification M0 / undifferentiated M1 M2 M3 / acute promyelocytic leukemia  M4 / myelomonocytic M5 / monocytic M6 / erythroleukemia M7 / megakaryocytic mixed lineage leukemias WHO classification : acute myeloid leukemia with recurrent genetic abnormalities acute myeloid leukemia with multilineage dysplasia acute myeloid leukemia and myelodysplastic syndromes, therapy related acute myeloid leukemia, not otherwise categorized (AML-NOC)

chronic myeloproliferative disorders (CMPDs) chronic myeloid leukemia  polycythemia vera  essential thrombocytemia  chronic idiopathic myelofibrosis  chronic eosinophilic leukemia (CEL) (and the hypereosinophilic syndrome (HES)) chronic neutrophilic leukemia

Pathogenesis :

• mutations in tyrosine kinases => proliferation defects => acute or chronic leukemia => acute leukemia
• down-regulation of HAT and upregulation of HDAC => differentiation defects => acute leukemia

normocytic and normochromic anemia, leukocytosis with relative neutropenia, thrombocytosis with thrombocytopathy

Clinical course :

• acute myeloproliferative disorders (AMPDs)
• acute myeloid (or myelogenous) leukemias (AML) / acute granulocytic leukemia / acute non-lymphoid leukemias (ANLL) represents a group of clonal hematopoietic stem cell disorders in which both failure to differentiate and overproliferation in the stem cell compartment result in accumulation of non-functional cells termed myeloblasts (in bone marrow > 30%). According to Rabbits master genes theory AML is an accumulation rather than a proliferation disease. Only 1 in 1 million leukemic blasts appears to be a true stem cell, according to the capacity to propagate and sustain human leukemia in immunologically susceptible mice^ref
• type I blast : no granules, fine chromatine, high nucleus/cytoplasm ratio and commonly evident nucleoli
• type II blast : rare azurophilic granules and smaller nucleus/cytoplasm ratio
• promyelocytes : eccentric nucleus, Golgi apparatus, addensed chromatin with conserved nucleolus, many granules and low nucleus/cytoplasm ratio
• myeloblastoma : a focal malignant tumor, observed in AML, composed of myeloblasts or early myeloid precursors occurring outside of the bone marrow
• chloroma / chloroleukemia / chloromatous or granulocytic sarcoma : a malignant green-colored tumor arising from myeloid tissue, associated with myelogenous leukemia and occurring anywhere in the body. Besides containing green pigment, which has no clear metabolic role and is principally myeloperoxidase, chloroma tissue demonstrates a bright red fluorescence under ultraviolet light
Epidemiology : most types affect primarily middle-aged to elderly people; incidence ranges from < 1 per 100,000/year in babies to 2.5-3.5 per 100,000/year in children aged 15 years to 10 per 100,000/year in people aged > 70 years; more common in males than females
Aetiology :
• de novo AML : usually unknown
• environmental factors
• radiation
• benzene
• inherited conditions
• identical or nonidentical sibling with AML
• Down syndrome
• Fanconi anemia
• Bloom syndrome
• Ataxia-pancytopenia
• Wiskott-Aldrich syndrome
• dyskeratosis congenita
• combined immunodeficiency syndrome
• congenital agranulocytosis
• D-trisomy
• familial AML
• Werner syndrome (progeria)
• neurofibromatosis type I
• Schwachman syndrome
• chromosome 21q disorder
• secondary AML
• therapy-related AML (t-AML) (chemotherapy (and radiotherapy to a lesser extent)^ref for lymphoproliferative syndromes) (13%)^ref, especially etoposide and mitoxantrone-related APL^ref
• t-APL (3%)
Until the early 1980s, most illnesses developed 3 to 10 years after prolonged use of alkylating agents, and presented as tMDS with complete or partial deletion of chromosome 7 or 5, often with other cytogenetic abnormalities (classic tMDS-tAML)^ref. More recently, tAML has been described after the use of topoisomerase II inhibitors (epipodophyllotoxins such as etoposide or teniposide, and anthracycline or anthracene dione). These illnesses often developed early (12 to 36 months) after onset of chemotherapy, usually had no preleukemic phase, and showed normal karyotype or cytogenetic rearrangements specific to de novo AML^ref. In the latter, 11q23 rearrangements predominated, whereas t(8;21), inv(16), and t(15;17) were less often seen, and other rearrangements were rarely observed^{ref1, ref2}. tMDS and tAML have also been reported after autologous stem-cell transplantation^ref. In addition, prolonged use of hydroxyurea in myeloproliferative disorders might increase the patients’ risk of progression to AML^ref
• chronic myeloproliferative syndromes
• myelodysplastic syndromes
• bi- or tricytopenia with hyperplastic marrow
• paroxysmal nocturnal hemoglobinuria
• aplastic anemia
• eosinophilic fasciitis
• multiple myeloma
Pathogenesis : an activating mutation in a signal transduction pathway and a mutation in a transcription factor are required for leukemogenesis.
• defined karyotype (45-50%)
• nucleophosmin exon-12 gene mutations^ref (27.5%^ref-35%; 45.7%^ref-62% of normal karyotype AMLs) : founding mutation, 19 different out-of-frame mutations. Normal nucleophosmin is a chaperon of preribosomal particles from nucleus to cytoplasm, has a role in centrosome duplication, enhances acetylation-dependent chromatin transcription^ref and inactivates p53 and p19ARF in the cytoplasm. NPM1 mutations are significantly underrepresented in patients younger than 35yr. NPM1 alterations were associated with FLT3-ITD mutations, even if restricted to patients with normal mutations : 66% also have mutated FLT3. NPM1 mutations positively correlate with AML with high WBC counts, normal karyotypes and FLT3 internal tandem duplication (ITD) mutations. NPM1 mutations associate inversely with the occurrence of CEBPA- and N-RAS mutations. With respect to gene expression profiling, AML cases with an NPM1 mutation cluster in specific subtypes of AML with previously established gene expression signatures, are highly associated with a homeobox gene-specific expression signature and can be predicted with high accuracy. Patients with intermediate cytogenetic risk AML without FLT3 ITD mutations but with NPM1 mutations have a significantly better OS and EFS than those without NPM1 mutations^ref. Finally, in multivariable analysis NPM1 mutations express independent favorable prognostic value with regard to overall-, event free- and disease free survival^ref
• activating FLT3 mutations (24-27% of de novo AMLs^ref, 42% of acute promyelocytic leukemia (APL), 17% of secondary AML^ref, and 4% of t-AML) :
• internal tandem duplication (ITD) in the juxtamembrane (JM) domain-coding sequence (FLT3-length mutations (FLT3-LM) / FLT3-ITD) can usually be detected by gel electrophoresis of the PCR products generated from amplifying exons 14 and 15 of the FLT3 gene, in which most ITDs occur. The PCR primers will generate a wild-type band and a larger FLT3 ITD band, which can be preparated by gel electrophoresis. ITD is  found in
• 20-25% of adult AML, more common in cases with MLL intragenic abnormalities (33%) than those with MLL translocation (8%)
• at a lower frequency in childhood AML
FLT3-ITD is associated with leukocytosis and a poor prognosis, especially in patients with normal karyotype.
• point mutations in the protein tyrosine kinase domain (TKD) (FLT3-TKD)
• D835 : mutation (D835Y >> D835V, D835H, D835E, and D835N)/deletion are found in 6% of adult AML; they are present in > 20% of cases with MLL intragenic abnormalities compared with 10% of AML with MLL translocation and 5% of adult AML with normal MLL status^ref.
• Y842H
The detection of FLT3 point mutations is more complicted, but can also be achieved by PCR. The double-strand PCR product is denaturated, then run on a single-strand conformational polymorphism (SSCP) gel, which allows the single strands to re-anneal, forming a unique secondary structure. A base-pair mutation results in a different secondary structure, which migrates differently on the gel than the structure of the wild-type sequence. The abnormal bands can then be excised from the gel and sequenced for confirmation.
Anyway FLT3 mutations are not primary mutations. There is some evidence that the ratio of mutant to wild-types alleles might determine the pathogenesis of FLT3 mutations. This can be assessed by fluorescent-labelled PCR, whereby the labelled PCR products are separatede according to size by migration through a capillary system, with the intensity of the products (relating to relative abundance) determined by laser excitation. Normally FLT3 has an activating phosphorylation at Y416 and a deactivating phosphorylation at Y527.
• translocations => chimeric fusion proteins => more immunogenic => good prognosis
• t(8;21)(q22;q22) (8-12%) fuses the AML1 / RUNX1 / core-binding factor 2 (CBF2) DNA-binding transcription factor to the ETO corepressor that associates with HDAC complexes. AML1 is homologous to the runt gene of Drosophila : it binds as a heterodimer, partnered with CBFß, and upregulates CD13, GM-CSF, MPO, IL-3, and the TcR.  Instead ETO encodes the human homologue of the Drosophila Nervy protein. Analyses have demonstrated that AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. The loss of the molecular events of AML1-ETO C-terminal NCoR/SMRT-interacting domain transforms AML1-ETO into a potent leukemogenic protein. Contrary to full-length AML1-ETO, the truncated form promotes in vitro growth and does not obstruct the cell-cycle machinery. These observations suggest a previously uncharacterized mechanism of tumorigenesis, in which secondary mutation(s) in molecular events disrupting the function of a domain of the oncogene promote the development of malignancy^ref. Mutations in AML1-ETO and FLT3 length mutation (FLT3-LM) are 2 of the most common genetic alterations seen in patients with AML, but neither mutation alone can cause leukemia in animal models : AML1-ETO cooperates with FLT3-LM, causing hematopoietic progenitor cells to become malignant and potently triggering rapid and aggressive acute leukemia in mice. Moreover, in a series of 135 patients with AML1-ETO-positive AML, the most frequently identified class of additional mutations affected genes involved in signal transduction pathways including FLT3-LM or mutations of KIT and NRAS^ref. Associated with loss of Y in males or of X in females over half the cases. Present in about 40% of myelomonocytic leukemia. High frequency of granulocytic sarcomas
• inv16 => MYH11-core-binding factor b (CBFB) (7-10%)
• t(15;17)(q22;q11-q21) fuses RARa-PML (in AML M3 / APL) (10%)
• 11q23 abnormalities (mixed lineage leukemia (MLL)-var) (10-12%) : frequently altered in ALL, primary AML, and especially in AML secondary to the use of topoisomerase II inhibitors. MLL is homologous to the trithorax gene of Drosophila and displays many features of a transcription factor and of a DNA methyl transferase. There are >50 known partner genes with which it is able to form in-frame fusions.
• fusion with chromosome 1 :
• MLL-MLLT11 / AF1Q (1q21)
• fusion with chromosome 4 :
• MLL-AFF1 / AF4 (4q21) (the most prevalent MLL fusion protein) binds to the CDKN1B promoter in vivo and depending on the background cell type, MLL-AF4 inhibits or activates CDKN1B expression^ref
• fusion with chromosome 6 : MLL-MLLT4 / AF6 (6q27)
• fusion with chromosome 9 :
• MLL-MLLT3 / AF9 (9q22) (7%). Associated with monocytic leukemia
• MLL-FNBP1 / FBP17 (9q34)
• fusion with chromosome 10 :
• MLL-ABI1 (10p11.2)
• MLL-MLLT10 / AF10 (10p12)
• fusion with chromosome 19 :
• MLL-ELL (19p13.1) (90%)
• MLL-MLLT1 / ENL (19p13.3) (10%)
• MLL-MLLT7 / AFX1 (Xq13.1)
• MLL-CBP
• MLL-GAS7 (17p31)
• MLL-SEPT5 / PNUTL1 (22q11.21)
• MLL-SEPT6 (Xq24)
• MLL-SEPT9 / MSF (17q25)
• MLL-RARa (15q22)
• [MLL-AFF1 / MLLT2 (4q21)
• MLL-EPS15 / MLLT5 (1p32)
• MLL-MLLT6 (17q21)]
Minor translocations :
• AML1 / RUNX1-EVI1
• ETV6-ABL
• ETV6-CDX2
• ETV6-CHIC2
• ETV6-MN1
• ETV6-EVI1
• FGFR1-FOP
• FUS-ERG
• MOZ-CBP
• NPM1-MLF1
• NUP98-HOXA9
• NUP214-DEK t(6:9) is usually seen in young patients and carries a poor prognosis. Its normal function is unknown, but DEK localizes to the nucleus.
• RARa-NUMA1
• RARa-ZNF145
• RBM15-MAL
• PDGFRB-TRIP11
• TCF3-PBX1
• TCL1A-TRA
• trisomy 8 (8% isolated, 12% with additional mutations)
• 5q, 5mono (3-4%)
• 7q-, 7mono (4-6%)
• H3 methylase hDOT1L activates Hox genes, whose increased activity is closely tied to AML^{ref1, ref2}.
• 20q del : DATF1 / death inducer-obliterator (Dido) gene gives rise to at least 3 polypeptides (Dido1, Dido2, and Dido3) through alternative splicing. Targeting of murine Dido caused a transplantable disease whose symptoms and signs suggested MDS/MPDs. Furthermore, 100% of human MDS/MPD patients analyzed showed Dido expression abnormalities, which was also found in other myeloid but not lymphoid neoplasms or in healthy donors. Dido might be one of the tumor suppressor genes at chromosome 20q and that the Dido-targeted mouse may be a suitable model for studying MDS/MPD diseases and testing new approaches to their diagnosis and treatment^ref.
• CEBPa mutated in 7-10% of AML (mainly M1-M2, not carrying the t(8;21) or the inv16 abnomalities, as they down-regulate it)^ref
• SALL4, a human homolog to Drosophila spalt, is a novel zinc finger transcriptional factor essential for development. SALL4 isoforms (SALL4A and SALL4B) are constitutively expressed in human primary AML (N=81). SALL4B transgenic mice developed MDS-like features and subsequently AML that was transplantable. Increased apoptosis associated with dysmyelopoiesis was evident in transgenic mouse marrow and CFU assays. Both isoforms could bind to b-catenin and synergistically enhanced the Wnt/b-catenin signaling pathway. The constitutive expression of SALL4 causes MDS/AML, most likely through the Wnt/b-catenin pathway^ref.
• activating mutations in RAS, predominantly NRAS, are common in myeloid malignancies. Previous studies in animal models have shown that oncogenic NRAS is unable to induce myeloid malignancies effectively and it was suggested that oncogenic NRAS might only act as a secondary mutation in leukemogenesis. The leukemogenecity of NRAS was examined using an improved mouse bone marrow transduction and transplantation model. Oncogenic NRAS rapidly and efficiently induced CMML- or AML-like disease in mice, indicating that mutated NRAS can function as an initiating oncogene in the induction of myeloid malignancies. In addition to CMML and AML, NRAS induced mastocytosis in mice^ref
• 15% of AML have unknown founding mutations
• complex karyotype
• unknown karyotype (40%)
In univariate and multivariate analysis high levels of PKC, ERK, pERK, and pAKT, but not AKT, were adverse factors for survival as was the combination variable PKC-ERK2&pERK2-pAKT. Survival progressively decreased as the number of activated pathways increased. Patients were more likely to have none or all 3 pathways activated than was predicted based on the frequency of individual pathway activation strongly suggesting that cross-activation occurred^ref. Interaction between the b₁ integrin VLA4 on AML cells and fibronectin on bone-marrow stromal cells increases resistance to ainokis after treatment with daunorubicin and araC and allows the development of minimal residual disease (MRD)
Classification : several types are distinguished, named according to the stage in which abnormal proliferation begins. The evolution of the classification system in AML from morphology to cytogenetic/genetic-based reflects the recognition of the importance of subtype-specific biology^ref. French-American-British (FAB) classification, 1976^ref (a classification of acute leukemia produced by a 3-nation joint collaboration) directly relates to severity of prognosis :
• M0 : morphologically undistinguishable (look for rearrengement of BcR or TcR genes)

Laboratory examinations :
• cytomorphology :
• blasts > 20% of nucleated bone marrow cells
• blasts > 20% of nonerythroid (excluding also lymphocytes, plasma cells, macrophages and mast cells) bone marrow cells
• < 3% of blasts are positive for Sudan Black B or MPO at optic microscopy
• blasts recognized as myeloblasts through immunological markers or ultrastructural cytochemistry
• cytogenetics : significant association with high-risk cytogenetics and 11q23 abnormalities^ref. Trisomy 13 in a patient with unusual durable remission^ref
• immunophenotype :  CD13⁺33⁺34⁺, MPO⁺, HLA-DR⁺
• M1 (15-20%) :

Laboratory examinations :
• cytomorphology :
• blasts > 20% of bone marrow cells; poorly granular blasts
• blasts >= 90% of nonerythroid (excluding also lymphocytes, plasma cells, macrophages and mast cells) bone marrow cells
• > 3% of blasts positive for peroxidase or Sudan black B
• maturating bone marrow monocytes (from promonocyte to monocyte) < 10% of nonerythroid bone marrow cells
• maturating bone marrow granulocytes (from promyelocyte to PMN granulocyte) < 10% of nonerythroid cells
• cytogenetics : t(1;3)(p36;q21), inv(3)(q21;q26), ins(3;3)(q26;q21q26), t(3;3)(q21;q26), t(9;22)(q34;q11) or +11
• immunophenotype : CD11⁺13⁺33⁺19^+/- HLA-DR⁺
• M2 (25%) :
• M2 Baso / basophilic leukemia : a rare type of leukemia in which basophils predominate; both acute and chronic varieties have been observed. t(6;9) (p23;q33)
• M2 Eos ?
Laboratory examinations :
• cytomorphology :
• bone marrow blasts >= 20% of bone marrow cells; very granular blasts
• blasts 20-89% of nonerythroid bone marrow cells
• maturating bone marrow granulocytes (from promyelocyte to PMN granulocyte) > 10% of nonerythroid cells
• maturating bone marrow monocytes (from promonocyte to monocyte) < 20% of nonerythroid cells and lack of other criteria diagnostic of AML M4
• cytogenetics : +4, t(6;9)(p23;q34), t(7;11)(p15;p15), +8, t(8;21)(q22;q22) [=> AML1 / RUNX1/ETO], +9, t(9;22)(q34;q11), +11 or t(11;17)(q23;q25)
• immunophenotype : CD11⁺13⁺33⁺19^+/- HLA-DR⁺
• M3 / acute promyelocytic leukemia (PML) (APL) (8-12%)

Epidemiology : usually occurs in young adults.
Aetiology :
• de novo APL
• therapy-related APL (t-APL) develops usually less than 3 years after a primary neoplasm (especially breast carcinoma) treated in particular with DNA topoisomerase II inhibitors^ref (anthracyclines or mitoxantrone and less often etoposide^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9, ref10, ref11, ref12, ref13, ref14, ref15, ref16, ref17, ref18, ref19, ref20, ref21, ref22, ref23, ref24, ref25}). Characteristics and outcome of tAPL seem similar to those of de novo APL^ref
Pathogenesis : activation of RARa gene on chromosome 17q21

Retinoids may be key for myeloid differentiation. Vitamin A-deficient mice and humans were noted to have defects in hematopoiesis18,19 and retinoids can preferentially stimulate granulopoiesis^{ref1, ref2}. In the early 1980s, it was noted that retinoic acid (ATRA) could induce differentiation of myeloid cell lines such as HL60^ref and of primary cells from patients with APL^ref. The cloning of the RARs and other members of the nuclear receptor superfamily allowed for further detailed studies into the mechanism of action of ATRA. Among the genes encoding RARs^{ref1, ref2, ref3}, RARa is identified with myeloid development^{ref1, ref2, ref3}

• t(15;17)(q22;q11-q21) (90%) => PML/RARa fusion protein (90%)^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}.


Pathogenesis : PML/RARa fusion protein is cleaved by neutrophil elastase (NE), which is encoded by the ELA2 gene, whose transcription is activated in early myeloid development and maximally expressed at the early promyelocyte stage. Cleavage creates a modified form of the protein that is important for initiation of this cancer^ref . PML-RARa recruits histone deacetylase (HDACs) (except for the AHT mutant) to ...
• ... chromatin to inhibit the expression of RAR target genes, predisposing cells to tumorigenesis
• ... p53 (whose mutations are surprisingly rare in APL) : deacetylated p53 is more susceptible to proteasomal degradation via MDM2^ref
APL shows both differentiation and proliferation defects.

Although both C/EBPa and C/EBPe can significantly prolong survival in a mouse model of APL, they are not functionally equivalent in this capacity. Forced expression of C/EBPa or C/EBPe in combination with ATRA treatment has a synergistic effect on survival of leukemic mice compared to either therapy alone^ref.
• t(11;17)(q23;q21) : fused to promyelocytic leukemia zinc finger (PLZF) gene (M3 V), which are retinoid-resistant. The PLZF/RARA cases were characterized by a predominance of blasts with regular nuclei, an increased number of Pelger-like cells, and by expression of CD56 in 4 of 6 cases tested. Use of this classification system, combined with an analysis for CD56 / NCAM1 expression, should allow early recognition of APL cases requiring tailored molecular investigations^ref.


• t(5;17)(q35;q21) : fused to nucleophosmin (NPM). Lacks Auer rods.


• t(11;17)(q13;q21) : fused to nuclear matrix associated (NuMA)


• t17q21 : STAT5B
PML oncogenic domains (PODs) / Kremer nuclear bodies / nuclear dots (ND10) (Ø 0.3÷0.5 mm; 10÷30 per nucleus ; made up of PML, Sp100, p53, pRb1, Daxx and BLM) disappear during APL. If cells are CD7⁺ => worse prognosis
Symptoms & signs : decreased levels of coagulation factor V => activation of the coagulation cascade and the fibrinolytic system => disseminated intravascular coagulation (DIC) => thrombocytopenia, hypofibrinogenemia => hemorrhages^ref; cutaneous promyelocytic sarcoma at sites of vascular access and marrow aspiration^ref
Laboratory examinations : there were no major differences observed in morphology or immunophenotype between cases with the classic t(15;17) and those with the cryptic PML/RARA gene rearrangements
• cytomorphology : morphology of blasts (hypergranular cells with many Auer rods)^{ref1, ref2}
• conventional karyotype (when normal it does not exclude the presence of rearrangements due to insertions or more complex mechanisms, including 3-way and simple variant translocations^ref), FISH, anti-PML PG-M3 mAb immunocytochemistry (directed against the amino terminal portion of the human PML gene product), change of the nuclear staining pattern from speckled ( due to localization of the wild-type PML protein into discrete dots (5 to 20 per nucleus), named PML nuclear bodies) to microgranular (PML-RARa fusion protein)^ref. Additional lesions have no prognostic value : FLT3 alterations are associated in APL with more aggressive clinical features but these lesions may not play a major role in leukemia progression^ref.
• immunophenotype : CD2^+/-10^-11c⁺13⁺14^-15⁺19⁺20^-33⁺34^+/-HLA-DR^-
Therapy^ref :

	all-trans retinoic acid (ATRA) inhibits PML/RARa hybrid protein, preventing HDACs from inhibiting transcription of differentiation genes and leading to recovery 70-80% of treated patients. Higher concentrations of ATRA are required to detach the HDAC complexref	chemotherapy
CR	96%	76%
48-mo OS	80%

• induction therapy : taken together, replacing standard chemotherapy with ATO in newly diagnosed APL will require larger studies. However, for selected patients who cannot tolerate anthracycline—for example those with cardiac dysfunction, older adults with poor performance status, and possibly Jehovah's Witnesses—ATO (probably still in combination with ATRA) would be a very reasonable alternative to the standard ATRA/chemotherapy. Of interest, in this report, patients were initially enrolled in the study because they could not afford standard therapy since the cost of single-agent ATO was much lower. In countries where cost would prohibit standard treatment of APL, a cheaper ATO, produced under good quality control, would be effective in curing many APL patients.
• single agent all-trans retinoic acid (ATRA) induces hematologic complete remission (CR) in almost all patients, but very often fails to produce molecular remissions. ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia blasts by restoring PU.1 expression^ref, presumably through degradation of the PML-RARA fusion protein^ref
• all-trans retinoic acid (ATRA) + chemotherapy^ref :
• induction therapy : ATRA 45 mg/m² PO (divided into 2 doses) daily for 90 days or until complete remission (CR) + anthracycline^{ref1, ref2} :
• ATRA + idarubicin (AIDA) 12 mg/m² IV days 2, 4, 6, 8^ref or for 4 days beginning on day 5 of ATRA^ref
• ATRA + daunorubicin
As ATRA has few side effects, therapy should be initiated before genetic diagnosis to prevent hemorrhagic complications. Although many still employ cytarabine in the induction and postremission management of APL patients, its use is probably not necessary^ref. Finding positive myeloblasts at the of induction therapy doesn't justify salvage therapy. Add aggressive platelet transfusions, tranexamic acid and low-dose heparin to prevent hemorrhages. Past trials involved :
• daunorubicin alone^ref
• idarubicin alone^{ref1, ref2}
Induction toxicities (not graded) : fever (93%), bacteremia (22%), hemorrhage (67%), thrombocytopenia (19 days to recovery > 50,000). 12/123 deaths in induction. Neutropenia median days to recovery : 24. Emetogenic potential : days 2, 4, 6, 8 level 3.
• postremission or consolidation therapy (not generally thought effective in other AML subtypes) : 2 cycles including anthracyclines (intensity related to risk : WBC > 10,000, PLT > 40,000)
• low and intermediate risk :
• consolidation #1 : idarubicin 5 mg/m²/day IV days 1, 4 (grade 3-4 neutropenia : 28%). Emetogenic potential days 1-4 level 3
• consolidation #2 : mitoxantrone 10 mg/m²/day IV days 1-5 (grade 3-4 neutropenia : 94%). Emetogenic potential days 1-5 level 3
• consolidation #3 : idarubicin 12 mg/m² IV day 1 (grade 3-4 neutropenia : 17%). Emetogenic potential days 1 level 3
• high risk : ? + cytarabine + etoposide
• maintenance therapy for 2 years (not generally thought effective in other AML subtypes) : tretinoin 45 mg/m2/day days 1-15 (repeat every 3 months), methotrexate 15 mg/m2 IM every week, mercaptopurine 90 mg/m2/day PO daily. Anyway 15% relapse^ref. ATRA resistance is not explained by LBD mutations, so pharmacodynamic reasons are likely to exist.
Grade 3-4 toxicites : pulmonary (16%), oral (14%), ATRA syndrome (6% : definite; 20% : indeterminate), cardiac (6%), hepatic (5%), diarrhea (3%), dermatologic (2%), neurologic (2%), renal (1%)
• single-agent arsenic trioxide (ATO)^ref : a study from study China showed, using quantitative PCR, that the number of PML/RARa transcript copies was significantly lower after induction treatment with ATO than with ATRA in a small group of newly diagnosed patients^ref. Similar results were reported by a group in Iran^ref. ATO induced in 72 newly diagnosed patients a CR rate of 86% (although a minority of 8 patients received anthracycline for high white cell count), without evidence of disease resistance. Prior to the next treatment cycle, most (76%) of the studied CR patients were PCR negative for PML/RARa. However, of greater interest is that ATO alone, given as consolidation plus maintenance of 10 doses per month for 6 cycles, was very effective in maintaining the molecular remission. The 3-year EFS was 75%. Further, using the white cell and platelet count at diagnosis, Mathews et al identified 2 risk groups that are different from those reported by Sanz et al^ref. All 20 patients in the good-risk group were alive and in remission^ref. Although this approach is less toxic and the results are promising, single-agent ATO cannot yet be considered an alternative to well-studied standard APL treatment: the number of patients is much smaller and the duration of follow-up is relatively shorter than those studied with ATRA plus chemotherapy. Also, the outcome of those not considered good-risk patients may not be as positive as in the poor- and intermediate-risk patients classified by the criteria of Sanz et al^ref despite the difficulty in a direct comparison. An advantage of single-agent ATO was its limited toxicity, especially infrequent and very short occurrence of grade  neutropenia. However, this is probably gained by eliminating the intensive chemotherapy, raising the question of whether ATRA also needs to be avoided. One might be concerned with using a single agent in a disease that is already highly curable
• arsenic trioxide (ATO) + all-trans retinoic acid (ATRA) : synergism was demonstrated in APL animal models. Further, in newly diagnosed APL patients, MRD measured by quantitative PCR was significantly lower with ATRA + ATO induction than with ATO alone^ref. A smaller study showed a very high percentage of continued molecular remission even in high-risk patients with ATRA plus ATO, with the addition of gemtuzumab only for high-risk patients^ref
• all-trans retinoic acid (ATRA) + chemotherapy + arsenic trioxide (ATO) : a small randomized trial from China suggested that the combination of all 3 modalities—chemotherapy, ATRA, and ATO in different treatment phases—resulted in significantly better outcome than chemotherapy + ATRA^ref. A much larger U.S. intergroup randomized trial at MD Anderson studied whether adding 2 cycles of ATO consolidations to standard ATRA/chemotherapy would improve the overall outcome, but the results are pending.
• relapsed APL :
• single-agent arsenic trioxide (ATO) (Fowler's solution) is more efficient than ATRA in reducing the degree of minimal residual APL cells and induces molecular remissions in almost all patients^ref : leads to a 4-log reduction in the APL disease burden in heavily pre-treated patients and is now being evaluated to consolidate first remissions
• induction therapy : ATO 0.15 mg/kg/day IV over 2 hours daily; cumulative maximum of 60 doses.
• consolidation therapy : ATO 0.15 mg/kg/day IV over 2 hours 5 days per week; cumulative maximum of 25 doses
Grade 3-4 neutropenia (8%), coagulopathy (clinical : 48%; subclinical : 35%), APL syndrome (25%), grade 4 hyperglycemia (13%), grade 4 hyperkalemia (13%), grade 4 dyspnea (10%), grade 4 abdominal pain (8%), grade 4 headache (3%), grade 4 insomnia (3%), grade 3 neuropathy (3%) => 40% had at least 1 EKG with QT prolongation, acute renal failure occurred in 3 patients. Monitor for signs of the APL differentiation syndrome (ATRA syndrome). Baseline EKG should be performed. Monitor the QTc interval. Monitor the patients for signs and symptoms of arsenic toxicity; recommended antidotes include dimercaprol and penicillamine, leukocytosis (50%). Emetogenic potential level 3^ref
• anti-CD33 immunotoxin conjugate (gemtuzumab ozogamicin) (Mylotarg^®) : 40% of patients remain positive but have no increase relapse risk. Mylotarg + ATO in second remission patients would be an interesting pilot study.
• allogeneic HSCT is not recommended after the first remission, but only in patients not undergoing molecular remission using a poorly sensitive PCR assay after consolidation therapy
• therapeutic DNA cancer vaccine using PML/RARa as a TSA fused with the highly immunogenic tetanus toxoid fragment C gene (FrC) is as effective as ATRA and when the 2 therapeutics are combined mice can live 40% longer than with either treatment alone^ref
Follow-up :
• bone marrow aspiration biopsy every 3 months
• molecular monitoring : APL is the one subtype of AML in which it has proven useful after achievement of complete response (CR). Few patients who achieve PCR^- status after postremission chemotherapy relapse; whereas those with persistently detectable PML-RAR fusion transcripts have a 25% risk of relapse^ref, yet whether such relapses can be prevented with additional therapy remains unclear
Prognosis : while the cure rate in APL with "standard" therapy is favorable (60-70%), the problems of t-MDS^ref and late central nervous system (CNS) relapses^ref have recently been described. Sanz criteria^ref :
• low-risk (WBC count <= 10,000/ml, platelet count > 40 × 10⁹/L)
• intermediate-risk (WBC count <= 10,000/ml, platelets  40 × 10⁹/L)
• high-risk (WBC count > 10,000/ml)
• M4 / acute myelomonocytic leukemia / Naegeli's leukemia (25%) : both malignant monocytes (> 20%) and myeloblasts. Its chronic counterpart is considered a MDS/MPD (chronic myelomonocytic leukemia). Methicillin-resistant coagulase-negative Staphylococcus have been identified within neutrophils^ref

Epidemiology : usually affects middle aged to older adults.
• M4 Eo : inv(16)(p13;q22), t(16;16)(p13;p22) or del(16)(q22) => fusion protein CBFB-smooth muscle myosin heavy chain (SMMHC) / MYH11 in which the CBFß transcription factor is fused to the tail domain of a smooth muscle myosin heavy chain.
Laboratory examinations :
• cytomorphology :
• blasts >= 30% of bone marrow cells
• blasts >= 30% of nonerythroid bone marrow cells
• maturating bone marrow granulocytes (from myeloblasts to PMN granulocytes) >= 20% of nonerythoid cells
• significant monocyte component as seen by one of the following criteria :
• bone marrow monocyte component (from monoblast to monocyte) >= 20% of nonerythroid cells and peripheral blood monocyte component > 5 x 10⁹/l OR
• bone marrow monocyte component (from monoblast to monocyte) >= 20% of nonerythroid cells, confirmed by cytochemistry or increase in lysozyme concentration in serum or urine OR
• bone marrow as in AML M2 but monocyte component in peripheral blood > 5 x 10⁹/l  and confirmation by cytochemistry or increase in lysozyme concentration in serum or urine
• cytogenetics : t(1;3)(p36;q21), t(1;7)(q10;p10), t(1;11)(q21;q23), inv(3)(q21;q26), ins(3;3)(q26;q21q26), t(3;3)(q21;q26), +4, t(6;9)(p23;q34), t(6;11)(q27;q23), +8, +9, +11, t(11;17)(q23;q25), t(11;19)(q23;p23) or +22
• immunophenotype : CD10^-11⁺13⁺14⁺15⁺20^-32⁺33⁺68⁺ HLA-DR⁺
Variants :
• M4 with cytophagocytosis
• M5 / acute monocytic (or monoblastic) leukemia / Schilling's leukemia / histiocytic leukemia (10%) : the predominating cells are identified as monocytes. A few myelocytes may be present, but not as many as in acute myelomonocytic leukemia.

Epidemiology : can affect any age group
Symptoms & signs : extramedullary localizations
Laboratory examinations :
• cytomorphology :
• blasts >= 30% of bone marrow cells
• blasts >= 30% of nonerythroid bone marrow cells
• bone marrow monocyte component >= 80% of nonerythroid cells
• M5a or undifferentiated (t(9;11)(p21-22;q23) or t/del(11q23)): monoblast > 80 % of all cells in monocytic lineage
• M5b or differentiated (t(8;16)(p11;p13)): monoblast < 80 % of all cells in monocytic lineage
• cytogenetics : t(1;11)(q21;q23), t(6;11)(q27;q23), +8, +9, t(10;11)(p11-15;q13-23), t(11;17)(q23;q25), t(11;19)(q23;p23); mucosal infiltrations
• immunophenotype : CD10^-11⁺13⁺14⁺20^-33⁺68⁺ HLA-DR⁺
Variants :
• M5 with cytophagocytosis (mainly erythrophagocytosis)
• M5c (?) with circulating macrophage-like cells
• myelodendritic acute leukemia
• M6 / erythroleukemia / Di Guglielmo syndrome (DG syndrome) : n oligoblastic proliferation of both erythroid and myeloid precursors described firstly by Copelli in 1912 (Copelli M. Di una emoptia sistemizzata rappresentata da una iperplasia eritroblastica eritromatosi. Pathologica 4:460-465 (1912)). Di Guglielmo named it erythroleukemia (Di Guglielmo G. Ricerche di ematologia. Folia Medica 3:386-396 (1917)) and Dameshek proposed the name DG-syndrome. It is the acute counterpart of polycythemia vera

The erythroleukemia developed by spi-1/PU.1 transgenic mice is a multistage process characterized by an early arrest of the proerythroblast differentiation followed later on by malignant transformation. Acquired mutations in the SCF receptor gene (Kit) occur in 86% of tumors isolated during the late stage of the disease. Kit mutations affect codon 814 or 818. Ectopic expression of Kit mutants in nonmalignant proerythroblasts confers erythropoietin independence and tumorigenicity to cells. Using PP1, PP2, and imatinib mesylate, Kit mutants are responsible for the autonomous expansion of malignant cells via Erk1/2 and PI3K/Akt activations^ref.
Laboratory examinations :
• cytomorphology :
• erythroblasts > 50% of nucleated bone marrow cells
• blasts >= 30% of nonerythroid bone marrow cells
• immunophenotype : CD10^-20^- HLA-DR⁺ glycophorin⁺ spectrin⁺ ABH antigens⁺ carbonic anhydrase I⁺
• cytogenetics : inv(3)(q21;q26), ins(3;3)(q26;q21q26),  t(3;3)(q21;q26) or t(3;5)(q21-25;q31-35)
• M7 / acute megakaryocytic (or megakaryoblastic) leukemia (AMKL) :

Epidemiology : can affect any age group
Risk factor : Down's syndrome (overexpression of a gene on chromosome 21 might stimulate abnormal proliferation of HSC, which would then be able to acquire mutations, among which GATA-1 on X chromosome)
Laboratory examinations :
• thrombocytosis
• punctio sicca or bone marrow fibrosis
• cytomorphology :
• blasts >= 30% of nucleated bone marrow cells
• blasts recognized as megakaryoblasts by immunophenotype, ultrastructural or cytochemistry
• cytogenetics : t(1;22)(p13;q13)
• immunophenotype : CD10^-20^-34⁺41⁺42a⁺42b⁺61⁺vWF⁺ HLA-DR⁺
• Rieder's cell leukemia : a form of AML in which the blood contains Rieder's cells, asynchronously developed lymphocytes that have immature cytoplasm and a lobulated, indented, comparatively more mature nucleus.

Laboratory examinations : Rieder's cell or lymphocyte (a myeloblast whose nucleus with wide and deep indentations suggesting lobulation, which may represent asynchronism of nuclear and cytoplasmic maturation)
• mixed lineage leukemias
• acute undifferentiated leukemia (AUL) / stem cell leukemia / blast cell leukemia / hemoblastic leukemia / hemocytoblastic leukemia / undifferentiated cell leukemia : predominating cell is so immature and primitive that it cannot be classified

Laboratory examinations :
• immunophenotype : CD7⁺34⁺45⁺HLA-DR⁺TdT⁺
• cytochemistry : negative SBB, MPO, esterase and PAS
• acute myeloblastic leukemia : a common kind of AML, in which myeloblasts predominate

Epidemiology : it usually occurs in infants and middle-aged to older adults. 2 types are distinguished :
• those that have minimal cell differentiation or maturation
• those that have more advanced differentiation.
Symptoms & signs : Mosler's sign (sternal tenderness in acute myeloblastic leukemia)
Laboratory examinations :
• myeloblastemia : the presence of myeloblasts in the blood, as in AML
• myeloblastosis : the presence of an excess of myeloblasts in the blood, as in AML
• micromyeloblastic leukemia : a form of myelogenous leukemia in which the immature, nucleoli-containing cells are small and are distinguishable from lymphocytes only by supravital staining.
• transient myeloproliferative disorder (TMD) / transient leukemia (TL) occurs in 10% of patients with Down syndrome

Laboratory examinations : pancytopenia, hepatosplenomegaly, and circulating immature WBCs
Prognosis : although most cases of TMD resolve without therapy in 1 to 3 months ( by 1 year of age), data suggest that true acute leukemia may develop in 20-30% of patients with TMD. There is an increased risk of leukemia with an equal incidence of lymphoid and myeloid leukemia. AML-M7 / acute megakaryocytic leukemia (AMKL) is the most common form of acute myeloid leukemia (AML) in this setting, and is uncommon in children without DS. Somatic mutations of the gene encoding the hematopoetic growth factor GATA1 have been shown to be specific for TMD and AMKL in children with DS. Myelodysplastic syndrome can precede AML. Although the risk for leukemia is higher in individuals with DS, these patients have a lower risk of developing solid tumors, with the exception of germ cell tumors, and perhaps retinoblastoma and lymphoma^ref. A prospective Children's Oncology Group (COG) study by Massey and colleagues of 47 patients with Down syndrome with a diagnosis of transient leukemia (more commonly known as transient myeloproliferative disorder) revealed early deaths in 8 patients due to liver failure and disseminated intravascular coagulation, and the subsequent development of acute megakaryoblastic leukemia in 8 patients and B-cell–precursor ALL in another patient at a mean of 20 months from diagnosis. As one might expect from past reports^ref, all but one of these patients, a child with acute megakaryoblastic leukemia, responded well to chemotherapy and remained in continuous complete remission. Early death was associated with a high presenting leukocyte count, increased levels of bilirubin and hepatic enzymes, and failure to regain normal blood counts. By contrast, the presenting leukocyte count, blast cell percentage, and time to clearance of blasts or normalization of blood counts did not predict the emergence of frank leukemia. The presence of karyotypic abnormalities in addition to trisomy 21 was only weakly correlated with leukemia development; that is, 4 of 7 such patients, compared with 5 of 35 lacking additional karyotypic abnormalities, subsequently developed leukemia. Interestingly, patients with overt leukemia were less likely than the unaffected group to be symptomatic at diagnosis of transient leukemia (one of 9 patients vs 26 of 39 patients). The COG study was not intended to evaluate therapy, so patients were treated according to the discretion of their physicians. Only one child received standard AML therapy, and 2 patients got low doses of cytarabine shortly after diagnosis. All three of these patients remained well. Two additional patients received low-dose cytarabine for progressive symptoms and died 1 or 2 days later. On the strength of those observations and limited experience by others,4 the authors recommend early use of low-dose cytarabine as prophylactic chemotherapy in patients with progressive organomegaly and evidence of liver dysfunction or in boys with cytogenetic abnormalities other than trisomy 21. However, for a prospective trial of low-dose cytarabine in all Down syndrome patients with transient leukemia because of the lack of factors that can be used with certainty to predict leukemia development or early death. Indeed, as mentioned, asymptomatic patients were more likely than symptomatic patients to develop leukemia. While 6 of 7 boys with additional cytogenetic abnormalities had a subsequent adverse event (4 leukemias and 2 early deaths), 3 of the remaining 22 boys also converted to overt leukemia, and 4 died early. Finally, low-dose cytarabine appears to be an effective and relatively nontoxic agent in infants with Down syndrome and transient leukemia^ref. It should be emphasized that the true incidence of transient leukemia is currently unknown, since blood counts are not routinely performed in Down syndrome patients. Conceivably, some of these leukemias resolve permanently without being diagnosed in the first place. Thus, an immediate challenge is to learn the exact prevalence and history of this disorder by prospectively studying all neonates with Down syndrome^ref.
Therapy : children with DS and leukemia are more sensitive to some chemotherapeutic agents such as methotrexate than other children which requires careful monitoring for toxicity.
WHO classification, 2001 : during the nearly 3 decades that the FAB system was used for classifying AML, it was discovered that many cases of AML are associated with recurring genetic abnormalities that affect cellular pathways of myeloid maturation and proliferation. The FAB classification provided a consistent morphologic and cytochemical framework in which the significance of the genetic lesions could be appreciated. In some instances, such as APL and acute myelomonocytic leukemia with abnormal eosinophils (M4Eo), the morphologic characteristics predict the genetic abnormalities. However, morphologic-genetic correlations are not always perfect, and the genetic findings may predict the prognosis and biologic properties of the leukemia more consistently than does morphology. Furthermore, in many cases either there is no correlation between morphology and genetic defects or the underlying genetic and molecular defects cannot be identified. Thus, although the FAB classification recognizes the morphologic heterogeneity of AML, it does not always reflect the genetic or clinical diversity of the disease. Some investigators suggest that a more relevant classification of AML can be achieved if 2 distinctive subgroups with different biologic features are recognized:
• AML that evolves from MDS or has features similar to those found in MDS
• AML that arises de novo without significant myelodysplastic features^{ref1, ref2}
The characteristics associated with these 2 groups indicate that they have fundamentally different mechanisms of leukemogenesis. MDS-related leukemia is associated with multilineage dysplasia, poor-risk cytogenetic findings that often include loss of genetic material, and a poor response to therapy. The incidence of this type increases with age, consistent with the hypothesis that MDS and MDS-related leukemia arise through multiple insults to the genetic constitution of the hematopoietic stem cell that occur over time. In contrast, de novo AML usually lacks significant multilineage dysplasia, is often associated with good-risk cytogenetic abnormalities, particularly certain recurring chromosomal translocations and inversions, and often has a favorable response to appropriate therapy, with good failure-free and overall survival times^{ref1, ref2}. This type of leukemia has a relatively constant incidence throughout life and is the type most likely to be observed in children and young adults^ref. It is probable that specific genetic events associated with these leukemias, which often involve transcription factors, are a major, rate-limiting step in their pathogenesis^ref. The concept that these and other subgroups of AML can be recognized and classified as unique diseases through correlation of morphologic, genetic, and clinical data is a major theme of the WHO classification and serves as the basis for the 2 most significant differences between it and the FAB classification: (1) a lower blast threshold for the diagnosis of AML in the WHO classification and (2) the categorization of cases of AML into unique clinical and biologic subgroups in the WHO classification. In the WHO classification, the blast threshold for the diagnosis of AML is reduced from 30% to 20% blasts in the blood or marrow. In addition, patients with the clonal, recurring cytogenetic abnormalities t(8;21)(q22;q22), inv(16)(p13q22) or t(16;16)(p13;q22), and t(15;17)(q22;q12) should be considered to have AML regardless of the blast percentage. A number of studies indicate that patients with 20% to 29% blasts in their blood or bone marrow often have similar clinical featuresincluding response to therapy and survival times as those with 30% or more blasts. According to the FAB criteria for MDS, patients with 20% to 29% blasts in the blood or marrow are classified in the MDS subgroup of refractory anemia with excess of blasts in transformation (RAEBT)^ref. In the WHO proposal, most patients with 20% to 29% blasts and myelodysplasia will be classified as AML with multilineage dysplasiaa subgroup that includes patients with a prior history of MDS as well as patients who present initially with AML and dysplasia in multiple cell lines. AML with multilineage dysplasia can be considered the most advanced manifestation of MDS. Numerous reports indicate that a significant number of patients with RAEBT and AML with myelodysplastic-related features share several important biologic and clinical features. According to some studies, myeloid cells from patients with RAEBT and MDS-related AML have nearly identical profiles of proliferation and apoptosis that differ from those in refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), and refractory anemia with excess blasts (RAEB)^ref. Poor-risk cytogenetic abnormalities, including abnormalities of chromosome 7 and complex abnormalities, increased expression of multidrug-resistance glycoproteins, and poor response to chemotherapy, are also common in RAEBT and in MDS-related AML^{ref1, ref2}. Some investigators have also reported that, when matched for similar disease features, such as white blood cell count or cytogenetic abnormalities, patients with RAEBT and AML have similar survival times if treated with identical therapy^{ref1, ref2, ref3}. In addition, data from the International MDS Risk Analysis Workshop indicate that RAEBT is not an indolent disease. In that study, 25% of patients with 20% to 30% blasts evolved to AML in 2 to 3 months, 50% in 3 months, and > 60% developed AML within 1 year. The median survival time for patients with RAEBT was less than 1 year^ref. The sum of these data indicates that patients with 20% to 29% blasts in the blood and/or bone marrow accompanied by multilineage myelodysplasia have essentially the same disease as do those with AML with multilineage dysplasia and 30% or more blasts and should be classified in the same category. It is important to emphasize that therapeutic decisions for patients with MDS-related AML should be based not only on the percentage of blasts but also on clinical findings, the rate of disease progression, and genetic data. The effect on the blast percentage of any previous therapy, such as growth factor therapy, must also be taken into account. These cautionary notes apply regardless of whether the blast count is 20%, 30%, or more in a patient with myelodysplastic-related disease. The lower blast percentage required for a diagnosis of AML also addresses another issue: the classification of patients who have no evidence of multilineage myelodysplasiathat is, patients with true de novo AMLas RAEBT because their blood or bone marrow specimens have fewer than 30% blasts on the initial evaluation. If the leukemia manifests no evidence that it is myelodysplastic related, it does not seem justified to categorize it as a myelodysplastic syndrome. Such a designation could result in inappropriate stratification for risk-determined therapy. Patients with the specific recurring cytogenetic abnormalities t(8;21)(q22;q22), inv(16)(p13q22) or t(16;16)(p13;q22), and t(15;17)(q22;q12) should be classified as having AML regardless of the blast percentage.
• acute myeloid leukemia with recurrent genetic abnormalities (30%^{ref1, ref2, ref3}) : in cases of AML with t(15;17), t(8;21), and inv(16) or t(16;16), there is such a strong correlation between the genetic findings and the morphology that the genetic abnormality can usually be predicted from the microscopic evaluation of the blood and marrow specimens. Furthermore, because AMLs associated with these abnormalities have distinctive clinical findings and a favorable response to appropriate therapy, they can be considered as truly distinct clinicopathologicgenetic entities^{ref1, ref2, ref3, ref4}. Although abnormalities of 11q23 are often associated with myelomonocytic or monocytic differentiation, AML associated with this abnormality cannot always be predicted with any degree of confidence from the morphology alone. The leukemias associated with these recurrent genetic abnormalities generally have the features of de novo AML. However, all of the genes affected in this group are apparently vulnerable to damage caused by certain types of chemotherapy, specifically with topoisomerase II inhibitors, and can be involved in some cases of therapy-related leukemia^{ref1, ref2}. In these instances, the unique clinical background indicates that the case should be classified as therapy-related AML. It is anticipated that the list of entities included in this subgroup will expand in the future. Some members of the WHO committees suggested that other recurring genetic abnormalities, such as t(8;16), t(6;9), or t(3;3), should be included in the current listing. Although these latter genetic abnormalities are often associated with unique morphologic and/or clinical features, it is not yet clear whether they define a unique disease or are mainly of prognostic significance within other subgroups. Furthermore, at least some of the recurring genetic abnormalities (for example, t(3;3)) are more frequently associated with myelodysplastic-related disease than with de novo AML and would be better incorporated into the subgroup of AML with multilineage dysplasia. It has been suggested that the WHO classification cannot realistically be used for AML because genetic information is not always available in a timely manner^ref. Alternatively, a "realistic" classification has been proposed that permits cases to be classified if the morphologic findings are "suggestive" of a specific genetic abnormality even without adequate knowledge that such a genetic defect is actually present^ref. The lack of complete information is a problem, but because the genetic data often predict response to treatment and prognosis and often drive treatment decisions, hematologists and pathologists are appropriately under pressure to obtain this information in a timely manner. Furthermore, although the recurring genetic abnormalities are often associated with distinctive morphologic findings, identification of the genetic defect provides a more objective, reproducible means of identifying a specific lesion. For example, the detection of the CBF/MYH11 (inv(16) or t(16;16)) by molecular and/or cytogenetic techniques is reported to correlate with the morphologic diagnosis of M4Eo in 30-100% of cases^{ref1, ref2, ref3}. Although the reason for this reported variability is unclear, what is clear is that if the inv(16) is present, the "real" classification is AML with inv(16). In daily practice one often cannot put a given specimen into a precise disease category without more information than is available at the timea problem that is by no means confined to the diagnosis of a particular category of AML. We believe that in a case of AML with morphologic features suggestive of a specific genetic abnormality, but for which complete information is not yet available, the pathologist should issue a report that indicates the case may belong to a particular genetic category but that more data are required to prove it. The report should indicate what data are needed and whether the studies are in progress or if a new specimen is necessary. However, we strongly believe that a classification that includes categories that are suggestive of a specific genetic entity for cases that are "not-quite-yet-classifiable" is not what is necessary to solve the problem of the lack of timely information. What is needed instead is a carefully worded report that informs the clinician of what more needs to be done to accurately complete the diagnosis.
• acute myeloid leukemia with t(8;21)(q22;q22), (AML1/ETO) : AML M2
• acute myeloid leukemia with abnormal bone marrow eosinophils and inv(16)(p13q22) or t(16;16)(p13;q22), (CBF/MYH11) : AML M4 or AML M4Eo
• acute promyelocytic leukemia with t(15;17)(q22;q12), (PML/RAR) and variants
• acute myeloid leukemia with 11q23 (MLL) abnormalities (5%) : AML M2, AML M4, AML M5, AML M7
• acute myeloid leukemia with multilineage dysplasia : the WHO classification "AML with multilineage dysplasia" recognizes the biologic and clinical importance of MDS-related AML
• following MDS or MDS/MPD that has been present for at least 6 months prior to the onset of overt AML. However, the definition of MDS-related AML is more difficult for those cases that present initially as acute leukemia. Whether morphologic, genetic, biologic, clinical features, or some combination of these should be used as defining characteristics was a point of controversy among members of the WHO committees. For practical purposes and worldwide usage, however, it was agreed that morphologic evidence of multilineage dysplasia would be the most universally available marker for its recognition.
• without antecedent MDS or MDS/MPD, but with dysplasia in > 50% of cells in >= 2 myeloid lineages in a pretreatment sample and blasts constitute > 20% of the blood or marrow cells. Although the 50% figure may appear high, it is derived from a number of studies that indicate that a lower threshold of dysplasia may not consistently identify AML with MDS-like features^{ref1, ref2, ref3, ref4}.
In some studies, multilineage dysplasia is an independent prognostic factor only in patients who have favorable cytogenetics but has no additional adverse impact in patients with poor-risk genetics^ref. A combination of genetic and morphologic studies may ultimately be used to further characterize this type of leukemia.
• acute myeloid leukemia and myelodysplastic syndromes, therapy related : 2 types of t-AML and t-MDS are recognized in the WHO classification, depending on the causative therapy:
• alkylating agent/radiation-related t-AML and t-MDS. This disorder usually appears 4 to 7 years after exposure to the mutagenic agent. Approximately two thirds of cases present with MDS and the remainder as AML with myelodysplastic features^{ref1, ref2, ref3, ref4, ref5}. It could be disputed whether a distinct category is needed for alkylating agent-related AML, because it is similar to AML with multilineage dysplasia and could be placed in that category^ref. However, the identification of a known, predisposing etiologic agent is but one major difference between these 2 groups; there is also a higher incidence of abnormalities involving chromosomes 5 and/or 7 and a worse clinical outcome in the therapy-related group^{ref1, ref2, ref3, ref4}
• topoisomerase II inhibitor-related AML (some may be lymphoid). In contrast to alkylating agent-related t-AML and t-MDS, acute leukemia secondary to topoisomerase II inhibitors often does not have a preceding myelodysplastic phase, and it most frequently presents as overt acute leukemia, often with a prominent monocytic component^{ref1, ref2, ref3, ref4, ref5, ref6}.27,28,41-44 The latency period between the initiation of treatment with topoisomerase II inhibitors and the onset of leukemia is short, ranging from 6 months to 5 years, with median times of 2 to 3 years usually reported^{ref1, ref2, ref3, ref4, ref5}. Most often, this type of t-AML is associated with balanced translocations involving chromosome bands 11q23 or 21q22^{ref1, ref2, ref3, ref4, ref5}. However, other translocations including inv(16)(p13q22) or t(15;17)(q22;q12) have been reported, and in these latter instances, as with 11q23 and 21q22 abnormalities, the morphologic and clinical findings are similar to those observed in patients with these translocations and no history of prior cytotoxic therapy^{ref1, ref2}. The initial response to therapy of topoisomerase II inhibitor-related AML as well as the overall survival are reported to be similar to cases of de novo AML with the corresponding genetic abnormality^ref. Acute lymphoblastic leukemia associated with 11q23 abnormalities, for example, t(4;11)(q21;q23), may also result from topoisomerase II therapy^{ref1, ref2, ref3}.
• others
In summary, it is important to identify cases of therapy-related acute leukemia and MDS. These disorders have proved to be models for a better understanding of the pathogenesis of leukemias that arise without preceding therapy, and knowledge gained from their study may lead to more specific and directed therapies for de novo as well as therapy-related cases.
• acute myeloid leukemia, not otherwise categorized (AML-NOC) : cases that do not satisfy the criteria for any of these subgroups, or for which no genetic data can be obtained. The group is intended to provide a framework for classification of cases that do not satisfy criteria for one of the other major categories. One of the primary considerations for the WHO classification of AML is the universality of application. The resources of hematology laboratories worldwide vary substantially. Although the ideal evaluation of patients with acute leukemia includes cytogenetic and, if necessary, molecular genetic studies, we recognize that these resources are not universally available at the present time. Careful morphologic evaluation can provide very significant information, such as multilineage dysplasia, and will result in appropriate classification in many cases. Furthermore, although direct clinical-genetic correlations are not presently recognized for many of the cases classified as AML, NOC,  future studies may uncover relevant clinical, genetic, and morphologic profiles. In most respects, the entities included in this group are defined almost identically as the corresponding entity in the previous FAB classification^ref, and the criteria for their recognition are based principally on identification of the major cell lineage(s) involved and the degree of maturation.
• acute myeloid leukemia, minimally differentiated
• acute myeloid leukemia without maturation (ex M1 FAB)
• acute myeloid leukemia with maturation (ex M2 FAB)
• acute myelomonocytic leukemia (ex M4 FAB)
• acute monoblastic/acute monocytic leukemia (ex M5 FAB)
• acute erythroid leukemia (ex M6 FAB) (erythroid/myeloid and pure erythroleukemia) is characterized by a predominant erythroid proliferation in the bone marrow. Recently, attention has been focused on the heterogeneity of neoplasms of erythroid cells and, in particular, on those that are composed mainly of primitive erythroid precursors with a minimal, if any, myeloblastic component^{ref1, ref2, ref3, ref4, ref5}. These latter cases may not meet the requirements of the FAB classification for a diagnosis of acute leukemia. The WHO classification recognizes 2 subtypes of acute erythroid leukemia, based on the presence or absence of a significant myeloblastic component. The first type, acute erythroid/myeloid leukemia, is defined as having at least 50% erythroid precursors in the entire marrow nucleated cell population and myeloblasts that account for at least 20% of the nonerythroid cell population. It corresponds to AML M6 in the FAB classification. All stages of erythroid maturation are found with a variable shift toward erythroid immaturity. Some cases that meet the criteria for acute erythroid/myeloid leukemia will also meet the criteria for AML with multilineage dysplasia. If that is the case, we would suggest that a diagnosis of "AML with multilineage dysplasia, acute erythroid/myeloid type" be made. The second subtype of acute erythroid leukemia recognized by the WHO is "pure erythroid leukemia," characterized by the proliferation of immature cells committed exclusively to the erythroid lineage. In this disease, 80% or more of all marrow cells are immature erythroid precursors with minimal differentiation, and there is no significant myeloblastic component. Although the blast cell morphology usually suggests their erythroid origin, in some cases they may be so primitive that proving their lineage is difficult^{ref1, ref2, ref3}. This erythroid neoplasm has been referred to previously as DiGuglielmo disease, acute erythremic myelosis, true erythroleukemia, and minimally differentiated erythroleukemia^{ref1, ref2, ref3, ref4, ref5} (DiGuglielmo G. La Malattie Eritremiche ed Eritroleucemiche. Rome: II Pensiero Scientifico Editore; 1962)
• acute megakaryoblastic leukemia (ex M4 FAB)
• acute basophilic leukemia
• acute panmyelosis with myelofibrosis (APMF) is an acute myeloid disorder with an unfavorable prognosis^ref. Although it is uncommon (< 1-2% of all cases of acute leukemia), when APMF is encountered the fibrosis may cause considerable difficulty in arriving at a correct diagnosis^ref. The critical point is to recognize that it is an acute process that has sufficient numbers of blasts to be considered AML. It is often associated with dysplasia and immaturity in multiple cell lines, with a prominent megakaryoblastic and megakaryocytic population. Te diseases known as acute myelosclerosis, acute myelofibrosis, acute myelodysplasia with myelofibrosis, and malignant myelosclerosis^{ref1, ref2, ref3, ref4} (Lewis SM, Szur MB. Malignant myelosclerosis. Br Med J. 1963;ii:472-473) are synonymous with APMF. It is necessary to distinguish this entity from chronic idiopathic myelofibrosis (CIMF) as well as from lower grades of MDS associated with myelofibrosis. Some morphologic features of APMF overlap AML with multilineage dysplasia with the addition of myelofibrosis; whether these are 2 manifestations of the same process is unclear. At present, we suggest that they be recognized as individual entities. There is also some controversy about the relationship of APMF to acute megakaryoblastic leukemia^{ref1, ref2}. The WHO committee recognized this problem. If the leukemia is predominantly megakaryoblastic with myelofibrosis, we suggest the term "acute megakaryoblastic leukemia with myelofibrosis." If the process is a panmyelopathy with myelofibrosis, we suggest the term APMF
• myeloid sarcoma
Symptoms & signs : anemia, fatigue, weight loss (influenza-like illness), fever without chills, defervescence without sweating, easy bruising, thrombocytopenia, and granulocytopenia that leads to persistent bacterial infections. Extramedullary infiltrates in AML-M4 and AML-M5 (20-40%) are associated with CD56 expression, 11q23 abnormalities and inferior clinical outcome^ref :
• tumour nodules (myeloid sarcomas)
• infiltrates :
• maculopapular skin infiltrates
• meningeal infiltrates
• gingival infiltration
• pulmonary and liver/spleen dysfunction > renal dysfunction
Laboratory examinations :
• chemistry : CBC, creatininemia, azotemia, SGOT, SGPT, gGT, alkaline phosphatase, ESR, PCR, electrophoresis, IgG, IgA, IgM, alkaline phosphatase, LDH, sideremia, ferritin, total cholesterol, direct and indirect Coombs test, urinalysis
• [bone marrow aspiration biopsy => morphological-immunological-cytogenetic and molecular (MIC-M) classification :
• molecular biology for PML/RARa, AML1/ETO, CBFB/MYH11, FLT3 mutations
• cytomorphology :
• hiatus leukemicus of Naegeli : a condition observed in AML in which there are numerous myeloblasts and a number of mature neutrophils in the peripheral blood, with few or no intermediate forms
• pancytopenia (rare)
• bone marrow blasts > 20%
• Bennett I
• Bennett II
• Bennett III
• number of erythroblasts
• maturating myeloid component
• monocytic component
• bone marrow
• peripheral blood
• cytogenetics
• serum and urine lysozyme
• cytochemistry
• granulocytes :
• myeloperoxidase (MPO) : negative in M0 and M7
• Sudan black B (SBB) : positive in M1, M2, M3, and M4
• chloracetate esterase (CAE) : positive in M2, M3, and M4
• nonspecific esterase (NSE) : positive in M4, M5
• acid phosphatase : positive in M5, M6
• periodic acid-Schiff (PAS) : positive in M5 (+/-) and M6
• monocytes : a-naphthyl-acetate esterase (ANAE)
• immunophenotype (including Pgp and BCRP) : flow cytometry is nowadays the preferred method for immunophenotypic identification, enumeration and characterization of blast cells at diagnosis. Despite widespread application of standardized protocols, inter-laboratory reproducibility has still not been achieved. Multiparametric immunophenotyping is a powerful method for achieving a clear discrimination between normal and pathologic cells. The specific identification of leukemic cells by immunologic gating forms the basis for immunophenotypic diagnosis, classification as well as prognostic evaluation of patients with acute leukemias. The performance of the procedure with regards to the panels of reagents and the analytic processes, is necessarily different in lymphoblastic and myeloblastic leukemias, since the diagnostic questions are different. Phenotypic information should be specifically provided for the blast cells and antigen expression should preferably be reported in quantitative units and CV. This would allow a standardized cross evaluation of immunophenotypic results between different investigators and laboratories^ref.
• to differentiate
• peroxidase^- AML vs. ALL
• clinically relevant subtypes
• MRD definition
• (AML vs. MDS)
• CD45 is useful for blast cell identification^ref.
• immaturity-associated antigens :
• lineage-unrelated
• panmyeloid : CD117
Antigen panels :
• first panel : EGIL
• second panel for follow-up
The immunodiagnosis report must contain clear statements about^ref :
• demographic identification of patient
• identification of the hospital or division sending the sample
• type of specimen (bone marrow aspirate, peripheral blood, other biological fluids)
• timing of observation (first diagnosis or follow-up)
• diagnostic hypothesis made by the sender
• list of antigens and type of immunofluorescence analysis carried out
• absolute number of cells in the sample
• quality of the sample, in terms of viability
• general description of the gating procedure
• immunophenotype of blast cells
• description of cells surrounding blasts
• diagnostic conclusions
• definition of an antigen panel (when applicable) for the detection of minimal residual disease.
Intracytoplasmic analysis.
Recent reports indicate that phenotypic aberrations reflect genetic abnormalities of leukemic cells and therefore their definition and identification is of clinical relevance not only for minimal residual disease monitoring but also for subclassifying acute myeloid and lymphocytic leukemias.
• AML blasts CD9^-11b^-13^-15⁺33⁺34^-38⁺41^-64⁺MPO⁺ had superior overall survival. This effect was associated with a significantly higher complete remission rate (CD13, CD34, CD41, and CD64) or a longer complete remission duration (CD9, CD11b, and CD64). Cox-regression analysis, including cytogenetic, morphologic, and biologic parameters showed CD9, CD13, CD34, and CD64 as independent factors for overall survival^ref.
• AML blasts CD19⁺34⁺ => highly likelihood of AML1-ETO+ (pink salmon anomalies due to t(18;21))
• AML M2 and M5^ref blasts CD56⁺ has worse prognosis. CD56 is a NK cell marker that has been identified in approximately 15-20% of AML cases, where it has been associated with monocytic morphology and chromosomal abnormalities such as trisomy 8, t(8;21), t(15;17), and 11q23 rearrangements
• AML M4Eo : CBFb-MYH11
• AML M5 : MLL/AF9; many false negatives due to fixed buffer in kits

	cell precursor markers			myeloid markers			monocyte markers
	TdT	HLA-DR	CD34	CD13	CD33	CD117	CD15	CD11b	CD14
AML M0	+/-	+	+	usually +	+/-	often +	usually -	usually -	usually -
AML M1	+/-	+	usually +	usually +	+	often +	usually -	+/-	usually -
AML M2	-	+	usually -	+	+	+	+	+/-	usually -
AML M3	-	-	-	+	+	+/-	+/-	usually -	usually -
AML M4	usually -	+	+/-	usually +	+	+/-	+	+	often +
AML M5	usually -	+	+/-	+/-	+	+/-	+	+	often +
AML M6	-	+/-	+/-	+/-	+/-	+	usually -	+/-	normally -
AML M7	-	usually +	usually +	usually -	+/-	often +	usually -	-	-
overall AML	+ in 10-20%	+ in 70%	+ in 30-40%	+ in 60-90%	in 70-90%	+ in 60-70%	+ in 40-70%	+ in 50-60%	+ in 15-40%

	AML M4	AML M5
ANAE	100%	83%
ANBE	60%	17%
CD13	30%	100%
CD33	30%	100%

Aberrant phenotypes in AML : co-expression of lymphoid markers : CD2⁺7⁺10⁺19⁺
Prognostic factors in newly diagnosed AML :
• better prognosis than average of all patients :
• age < 45 years
• de novo AML
• leukemic cells contain t(8;21) (AML1-ETO), t(15;17) (AML M3), inv(16) (AML M4 Eo), trisomy 21, del(16q) (10%)
• absence of exaggerated dysmyelopoiesis
• residual normal metaphases admixed with clonal cytogenetic abnormalities
• high telomerase activity levels
• high levels of caspase 3
• nucleophosmin
• WBC < 25,000/ml
• no CNS involvement
• Auer bodies
• eosinophilia
• no dysplasia
• no fibrosis
• low CD13, CD14, CD34
• high labeling index (LI)
• intermediate karyotype (45-60%) :
• normal karyotype or without low or high risk-specific abnormalities
• + 8
• +6
• 11q23
• +21
• +22
• del(12p)
• -Y
• poorer prognosis than average of all patients
• older age (threshold = 60 yrs ?) : age at the time of diagnosis has the greatest impact on the probability of remission and on the duration of survival. Children in the first 15 years of life, exclusive of the neonatal period, have the highest rate of remission and longest relapse-free remission; patients over 60 years of age have only half the chance of a young adult to enter remission and less likelihood of a long relapse-free remission. There is a gradient of poor reponse to treatment through adulthood, with the largest decrease after the sixth decade of life.
• t-AML
• unfavorable karyotypes (66% have unfavorable anomalies, 45-60% intermediate anomalies, 10% favorable anomalies) : the cytogenetic pattern of leukemic blast cells influence outcome, but the relationship is complex
• 5-
• 7-
• 5q-
• 7q-
• t(3;3)
• t(6;9)
• t(9;11)
• t(9;22)
• 3q, 9q, 11q, 20q, 21q, 17p
• exaggerated hyperdiploidy (< 47 chromosomes)
• trisomy 8
• trisomy 11
• trisomy 13
• trisomy 21
• > 3 independent aberrations
• multidrug resistance phenotype (40% at diagnosis) : leukemic cells expressing P-glycoprotein, a unidirectional drug-efflux pump, encoded by the MDR1. Expression of this gene product can result in decreased accumulation of anthracyclines, amsacrine, mitoxantrone, and etoposide. Expression of P-glycoprotein does not influence outcome of treatment, but if rhodamine 123 effux also is increased, relapse is more common. Frequently, observed in AML cells after relapse. Associated with CD34 expression and chromosome 7 abnormalities. Alternative non-MDR1-mediated drug efflux mechanisms are important also. MDR1 expression is low in favorable prognosis subtypes of AML.
• relapse within 6 months since completion of therapy
• AML secondary to MDS after > 6 months
• CNS involvement
• prior clonal hemopathy : chemotherapy or radiotherapy remission rates are 33-50% that of de novo AML in the same age group and remission duration is shorter with remissions over 3 years very uncommon. AML developing from the clonal hemopathy may relapse as a smoldering leukemia. It then reverts to AML but can be treated with remissions lasting several years.
• WBC > 30,000/ml (> 100,000 cells at diagnosis ??) or a blast cell count > 15,000/ml. Anyway cases with < 30% blast cells did not have a 5-year OS significantly different from cases with > 30% blast cells, and survival was similar for the low blast cell count group and cases with multilineage dysplasia and > 30% blasts. These findings suggest that the detection of recurring cytogenetic abnormalities and multilineage dysplasia are the most significant features of current AML classification^ref
• thrombocytopenia < 30,000/ml
• high LDH
• another medical disorder : extreme obesity, diabetes mellitus, chronic renal disease
• low serum albumin or prealbumin
• need for intubation or ventilator support during induction therapy
• autonomous clonal growth of leukemic blast cells
• low labeling index
• high Bcl-2 expression
• high Mcl-2 expresion : elevated at the time of leukemic relapse. Either suggests prognostic importance or that chemotherapeutic regimen selects for leukemia cels with elevated levels of apoptosis inhibitors
• low expression of Rb gene
• high levels of WAF/Cip1 protein : this is a regulator at the G1 checkpoint of cell cycle
• high CD34 expression : high CD34 antigen expression often in AML subtypes M0, M1, and M4. Remission rate of 61 compared to 88% in AML not expressing CD34. The correlation is stronger between high-intensity expression of CD34 and lower remission rate.
• GATA-1 expression
• CD7, CD11b, CD13, CD14, CD56 MDR
• elevated soluble L-selectin : seen expecially in extramedullary disease
• higher expression of IL-1b gene
• low FMS expression
• expression of TPO-R / c-MPL mRNA
• FLT3 tyrosine kinase gene length (or ITD) mutation (repeat of 3-30 amino acids in the juxtamembrane region^ref, leading to constitutive activation)
• duplication of the MLL gene on the long arm of chromosome 11^ref
• treatment-related AML require special consideration because, in the absence of balanced translocations known to carry a favorable prognostic impact, such patients fare poorly with standard approaches
• severe splenomegaly ?
• MRD ?
• Ph* ?
• absence of Auer rods has been associated with a poor prognosis
• detection of WT1 (Wilms tumor) transcript
• myelodysplasia
• bone marrow fibrosis
• lung resistance protein : functional test is needed in order to assess activity. Expression may predict poor outcome in de novo AML
A modified Realistic Pathologic Classification joins subtypes with the same prognosis :
• de novo AML
• AML with recurrent cytogenetic abnormalities of t(15;17), t(8;21), inv(16)/t(16;16), and 11q23
• AML M3 / promyelocytic leukemia
Therapy for non-acute promyelocytic leukemia (APL) AML : the approach to adults aged 18-60 years with AML classically involves separate treatment phases :
• induction chemotherapy in which the goal of myelosuppressive chemotherapy is to "empty" the bone marrow of all hematopoietic elements (both benign and malignant) and to allow repopulation of the marrow with normal cells, thereby yielding remission (< 5% marrow blasts : a state in which there is no visible evidence of disease and blood counts are normal). The primacy of the standard regimen of 3 days of an anthracycline (idarubicin 12 mg/m_BSA² > daunorubicin 45 mg/m_BSA² > doxorubicin 60 mg/m_BSA², which has higher gastrointestinal toxicity) and 7 days of cytarabine (3+7) has not been definitely altered despite clinical trials that have added or substituted high-dose cytarabine, or added thioguanine/etoposide.

regimen	nr. of patients	CR	year
mitoxantrone 12 mg/m2 days 1 to 5  etoposide 100 mg/m2 days 1 to 5	61	43%	1988
mitoxantrone 12 mg/m2 days 1 to 3  etoposide 40 mg/m2 days 1 to 3  cytarabine 1 g/m2 days 1 to 3  +/- PSC-833	37	32%	1999
mitoxantrone 12 mg/m2 days 1 to 3  etoposide 200 mg/m2 days 8 to 10  cytarabine 500 mg/m2 days 1 to 3, 8 to 10  GM-CSF 5 mg/kg days 4 to 8	22	30%	1993
idarubicin 12 mg/m2 days 1, 3, 5  cytarabine 500 mg/m2 every 12 h, days 1 to 7	21	52%	1996
fludarabine 30 mg/m2 days 1 to 5  cytarabine 2 g/m2 days 1 to 5 +/- idarubicin 12 mg/m2 days 1 to 3  G-CSF 400 mg/m2 daily until complete remission	85	66%	1995
aclarubicin 60 mg/m2 days 1 to 5  etoposide 100 mg/m2 days 1 to 5	34	29%	1998
idarubicin 12 mg/m2 days 1 to 3  cytrabine 1 g/m2 every 12 h, days 1 to 4	23 refractory  38 relapsed	52%  68%	1997
2-chlorodeoxyadenosine 0.1 mg/kg per day x 7 days continuous infusion +/- daunorubicin 50 mg/m2 days 5, 6, 7	19	0% (100% of treated patients had improvement)	1998
topotecan 4.75 mg/m2 days 1 to 5  cytarabine 1 g/m2 days 1 to 5	53 refractory	4% (39% of treated patients had improvement)	1997
carboplatin 300 mg/m2 per day for 5 days continous infusion  cytarabine 500 mg/m2 for 3 days	31	29%	1999
cyclophosphamide 1 g/m2 days 1 to 3  etoposide 200 mg/m2 days 1 to 3  carboplatin 150 mg/m2 continuous infusion days 1 to 3  cytarabine 1 g/m2 days 1 to 3	25	12%	1998

Recent trials by the Cancer and Leukemia Group B (CALGB) have employed high doses of daunorubicin and etoposide^ref. While tolerable, whether such higher doses offer disease-free or overall survival benefits remains to be proven by ongoing clinical trials). Given the high (75-80%) complete remission (CR) rate typically achieved in younger adults with standard chemotherapy, an extremely large trial or a highly effective agent would be required to show superiority over conventional chemotherapy. Most of the trials that have reported an improvement with a new chemotherapeutic agent or with a higher dose of a standard drug during induction have documented a disease-free survival benefit, implying that such remissions yield a lower disease burden. However, the inability to translate such benefits into overall survival improvements suggests a limited ability to affect a major difference in the depth of remission. Promising results have been published in recent years using fludarabine-containing combination therapy for AML, most commonly
• fludarabine + cytarabine + G-CSF [FLAG] : fludarabie 30 mg/m2/day IV over 30 minutes days 1-5, cytarabine 2000 mg/m²/day IV over 4 hours after completion of fludarabine days 1-5, G-CSF 5 mg/kg/day IV 0 (24 hours prior to starting chemotherapy) until ANC recovery. Grade 4 neutropenia (100%), documented infections > grade 3 (44%), bacterial infections (34%), FUO (44%), fungal infections (10%), mucositis > grade 3 (10%), diarrhea > grade 3 (8%), hepatic > grade 3 (8%), neurologic > grade 3 (2%) => median time to ANC > 500 was 21 days, mediant time to platelet levels > 20,000 was 26 days. Emetogenic potential : days 1-5 level 4^ref.
• FLAG + mitoxantrone (FLANG)
• FLAG + idarubicin (FLAG-Ida).
Such combinations maximize favorable cytotoxic interactions between cytarabine and G-CSF, and between cytarabine and fludarabine. In small studies, such combinations used as second-line therapy have resulted in complete response (CR) rates of 36-59%. Early retrospective analyses suggested higher CR rates in patients with refractory AML than in those with relapsed AML, but this observation has not been confirmed in recent prospective trials. Fludarabine-containing combinations have also been evaluated as first-line therapy in high-risk patients and resulted in CR rates of 34-70%, with median survival from 7 to 16 months. The current large MRC randomized high-risk study will provide further data on the use of fludarabine-containing regimens in patients with poor prognosis AML^ref
• FLAI^ref
• ICE
• G-CSF + cytarabine
• fludarabine administered before cytarabine reduces araC catabolism
• cyclophosphamide, etoposide, carboplatin, cytarabine^ref : cyclophosphamide 1000 mg/m²/day IV over 2 hours days 1-3, etoposide 200 mg/m²/day IV over 3 hours days 1-3, carboplatin 150 mg/m²/day IV continuous infusion days 1-3, cytarabine 1000 mg/m²/day IV over 2 hours days 1-3. Agents administered sequentially, with cyclophosphamide first, then etopoiside, and finally cytarabine. Neutropenic fever (76%), grade 3 mucositis (4%), grade 3 cardiac toxicity (4%), grade 3 diarrhea (4%), grade 4 hemorrhagic cystitis (4%), grade 4 hyperbilirubinemia (4%). Emetogenic potential : days 1-3 level 5
• cytarabine^ref:100 mg/m²/day IV continuous infusion days 1-5 (for patients > 60 years of age). Repeat cycle every 28 days or 1 week after marrow recovery. Emetogenic potential : days 1-5 level 2. 1% of patients died while in remission. 76% of patients tolerated full-dose therapy for 4 courses. Patients requiring hospitalization due to neutropenic fever 16%, patients requiring transfusions due to thrombocytopenia 28%
• cytarabine + daunorubicin (5+2) for reinduction^ref : cytarabine 100 mg/m²/day IV continuous infusion days 1-5, daunorubicin 45 mg/m²/day IV 1, 2. Grade 3-4 toxicities : infection (51%), hemorrhage (20%), hepatic (7%), renal (5%), cardiac (4%). Emetogenic potential : days 1, 2 level 4, days 3-5 level 2. Toxic mortality 32%, sepsis most common cause of death.
• cytarabine + daunorubicin (7+3)^ref : cytarabine 100 mg/m²/day IV continuous infusion days 1-7, daunorubicin 45 mg/m²/day IV days 1-3. Grade 3-4 toxicites : neutropenic infection (54%), hemorrhage (12%), hepatic (2%), gastrointestinal (1%) => toxic mortality (23%), sepsis most common cause of death. Emetogenic potential : days 1-3 level 4, days 4-7 level 2
• HiDAC^ref : cytarabine 3000 mg/m² IV over 3 hours every 12 hours days 1, 3, 5 (total 6 doses). Administer with saline, methylcellulose, or steroid eye drops OU, every 6 hours beginning with cytarabine and continuing 48-72 hours after the last cytarabine dose. Repeat cycle every 28 days or 1 week after marrow recovery. Grade 3-4 toxicities : grade 4 thrombocytopenia (86%), CNS toxicities (12%; 32% with age > 60 yrs) => 5% of patients died while in remission, patients hospitalized for neutropenic fever/infection 71%, 56% of patients tolerated 4 courses of therapy at full dose.
• risk-adapted postremission, continuation or consolidation therapy : it is definitely clear that once remission is achieved, additional therapy is required to reduce the undetectable burden of leukemic cells to a level low enough that long-term disease-free survival (i.e., cure) might be possible
• for those with high-risk cytogenetics (age > 55 years, no CR, secondary AML, MDR, alterations in chromosomes 3, 5, 7, or 8 or complex karyotype) the most effective anti-leukemic approach is allogeneic hematopoietic stem cell transplantation (TRM = 30%; 2-yr OS = 5%, 5-yr OS = 0%; 5% in Ph⁺ AML). However, this technique carries a high degree of initial mortality and a significant degree of long-term morbidity in the form of cGVHD, tending to offset the low likelihood of disease relapse. Chemotherapy-based approaches with or without autologous stem cell rescue can be performed relatively safely, but there remains a high chance for disease recurrence. Some of the patients who relapse after chemotherapy or even high-dose chemotherapy with autologous stem cell rescue can be salvaged with an allogeneic transplant performed in early relapse or second remission. Obstacles to broad applicability of HCT therapy for the majority of patients with AML have included inability to control leukemia with primary induction therapy, lack of suitable hematopoietic cell donors, toxicities of HCT conditioning regimens and long-term complications of the transplant procedure. Autologous transplant procedures had transitioned from marrow to cytokine-primed (G-CSF and/or GM-CSF) peripheral blood stem cells (PBSC) for ease of collection and rapid engraftment by the early 1990s. However, expectation of higher rates of GVHD due to the 10-fold increase in T cells in PBSC products delayed their use in allogeneic settings until the latter-half of the 1990s. In 2003, National Marrow Donor Program (NMDP) statistics showed 60% of unrelated donor products collected for transplants for AML were PBSC. Collection via apheresis is physically easier for many donors. The 4- to 5-fold higher number of CD34⁺ cells in a PBSC product facilitates more rapid engraftment in comparison with a marrow product (median of 17 days versus 24 days for granulocytes and median of 28 days versus 47 days for platelets, respectively), which decreases the risk for bacterial infections in the early post-transplant period^ref. Despite initial concerns that a 10-fold increase in the number of CD3⁺ cells in the PBSC graft would lead to higher incidence of severe GVHD, this has not been demonstrated in clinical trials. Several studies have shown no significant difference in the incidence of acute GVHD; however, higher rates of late onset chronic GVHD (beyond 6 months) have been reported^ref. In both a large randomized trial and a retrospective review of International Bone Marrow Transplant Registry (IBMTR) data, PBSC grafts were associated with improved relapse-free survival in patients with leukemia beyond first remission (CR1)^{ref1, ref2, ref3}. In addition to producing a HPC product that results in more rapid engraftment, G-CSF exposure also shifts donor T cells to Type 2 cytokine secretion (IL-4 and IL-10) and downregulates Type 1 (IL-2 and IFN-g) cytokine production, phenomena which may be associated with less severe acute GVHD. Improvements in post-transplant supportive care and the development of newer immunosuppressive agents have also had an impact on transplant-related toxicities. More refined PCR-based screening for CMV reactivation allows pre-emptive therapy tailored to viral load. Newer less toxic antifungal agents such as voriconazole and caspofungin have decreased early mycotic infections and make it feasible to provide long-term fungal prophylaxis in patients at high risk due to chronic GVHD. Newer immunosuppressive agents, such as sirolimus and mycophenolate mofetil (MMF) are being incorporated into GVHD prophylactic regimens to decrease the incidence of acute and chronic GVHD in high-risk populations including unrelated donor recipients and older (> 50 years) patients^{ref1, ref2}. However, the change that may have the greatest impact on AML treatment is the advent of reduced intensity or nonmyeloablative HCT (NMSCT; TRM = 20%). Reduced-intensity HCT relies upon the graft-versus-leukemia (GVL) effect of the allograft rather than the direct tumoricidal activity of the conditioning regimen. Because of the paucity of any direct antileukemic effect of the conditioning regimens, the truly nonmyeloablative (NMABT) regimens can only be used in patients with low volumes of disease, whereas the reduced-intensity conditioning regimens, which usually contain fludarabine and some alkylating agent, do have direct antileukemia activity and can be used with more extensive disease. The shortened duration of cytopenias and the minimal mucosal toxicity of the newer RIC regimens provide a reasonably safe transplant option for patients 2 decades older than the population that was treated with traditional fully ablative high-dose chemoradiotherapy regimens.

The possible need for HCT as a component of future therapy should be acknowledged at diagnosis in AML. For all patients under age 56 who do not have significant co-morbid conditions, HLA typing should be part of the patient's initial evaluation, and family typing should be initiated when the cytogenetic information is available. Only a minority of patients will have a favorable karotype that would obviate consideration of allogeneic HCT either as part of consolidation or for salvage of resistant disease. For patients with poor-risk cytogenetics, antecedent myelodysplasia or therapy-related AML, an unrelated donor search should be promptly instituted in patients lacking a family donor. Early donor identification allows optimal timing for HCT as consolidation or salvage therapy. In older patients (56-70 years), however, it is reasonable to delay HLA typing of family members until remission is achieved and the patient has recovered a good performance status since there is substantial attrition in the rank of candidates for the investigational use of reduced-intensity or nonmyeloablative HCT as consolidation due to failure to achieve a remission or decline of functional status as a consequence of induction therapy. The decision to proceed to either autologous or allogeneic HCT is strongly influenced by the following factors: expectations of outcome with the prevailing non-HCT conventional therapy, the effectiveness of salvage options for disease relapse, the toxicities of transplant including long-term complication of infertility and second malignancy, and the individual patient's co-morbid conditions. The patient's cytogenetic risk group and time to achieve remission are currently the major disease-specific factors to be considered in opting for HCT as consolidation therapy, whereas donor availability and the impact of recipient performance status on the ability to tolerate the rigors of even a reduced-intensity HCT are important factors to consider at relapse. While there are several studies with long-term outcome data comparing myeloablative conditioning regimens followed by either autologous or allogeneic HCT, the follow-up data on reduced intensity HCT is much shorter and the patient populations are not comparable.
Autologous HSCT has been used primarily as a component of consolidation following CR1; less commonly, it can be used as a component of salvage therapy for patients in CR2. To minimize both the total body leukemic burden and the contamination of the PBSC product, current practice is to administer 1-2 cycles of a high-dose cytarabine-based consolidation to achieve "in vivo purging" prior to collection of G-CSF-mobilized PBSC. To assure that an adequate hematopoietic progenitor cell product of at least 5 x 10⁶ CD34⁺ cells/kg recipient body weight can be collected, it is recommended that < 2 cycles of consolidation occur before PBSC collection. While the nonhematologic toxicities of the conditioning regimens used for autologous HCT are significantly greater than those seen with high-dose cytarabine consolidation, the hematologic recovery is quite rapid (8-11 days) following infusion of autologous G-CSF-mobilized PBSC. The combined treatment-related mortality (TRM) for one cycle of high-dose cytarabine-based consolidation followed by 1200 cGy fractionated total body irradiation (FTBI), etoposide and cyclophosphamide (Cy) and autologous HCT is 3-6% in 3 sequential trials inclusive of patients up to age 60. Attrition between consolidation and autologous transplant is 18-24% due to relapse, inadequate HPC collection or treatment-related toxicities such as invasive fungal infection and cytarabine-induced neurotoxicity, making this a reasonable consolidation option for most patients up to age 60^ref. Over the last 15 years, the upper age limit considered to be acceptable for a fully ablative allogeneic HCT has advanced from 40 to "robust" 60 year olds (perhaps in response to the maturing age of the transplanters themselves as much as to improvements in supportive care and GVHD prophylaxis)^ref. If an HLA-matched donor has been identified while the patient was receiving induction therapy, there is no advantage to administering postremission chemotherapy prior to allogeneic HCT^ref. GVHD and transplant-related toxicity remain the major obstacles to successful outcome with allogeneic HCT.
• the introduction of 2 new immunomodulatory drugs for GVHD prophylaxis may have an important impact on both these problems
• investigators from the Dana-Farber recently reported a Phase II trial in which sirolimus was substituted for methotrexate (MTX) in a tacrolimus-based GVHD prophylaxis regimen for PBSC HCT utilizing a fully myeloablative conditioning regimen^ref. Engraftment was prompt and only 3/30 (10%) patients developed acute GVHD (all grade II) compared to the expected incidence of 35-40% acute GVHD in sibling-donor HCT with tacrolimus/MTX. TRM was only 6% at 1 year compared to 25-30% in most traditional allogeneic studies. Chronic GVHD developed in 40% of the study patients. Toxicities included significant hyperlipidemia, thrombotic microangiopathy and a 10% incidence of veno-occlusive disease primarily in patients with recent exposure to gemtuzumab ozogamicin
• other studies of the substitution of mycophenolate mofetil (MMF) for MTX in GVHD prophylaxis regimens have demonstrated decreased regimen-related toxicities of severe mucositis and interstitial pneumonias, as well as more rapid engraftment compared to a MTX-containing regimen (14-16 days versus 19-22 days for neutrophil engraftment)^{ref1, ref2}.
Improvements in molecular matching for unrelated donor-recipient pairs have significantly improved outcomes for this group, although there continues to be an approximately 10% higher incidence of TRM when sibling allogeneic HCT is compared to allogeneic HCT from molecularly matched unrelated donors for patients in CR1. Because of the perception of higher TRM, the profile of patients who undergo unrelated donor HCT in CR1 is skewed toward those with poorer risk cytogenetics, prolonged time to achieve a remission, or a history of antecedent myelodysplasia or therapy-related AML. The majority of unrelated donor transplants are still being performed for AML patients beyond CR1. The foremost advantage of the nonmyeloablative or reduced-intensity HCT is the reduction of TRM. Mucositis in the immediate post-HCT period is much reduced due to the low antiproliferative activity of the conditioning regimen and the substitution of MMF for MTX in the GVHD prophylaxis regimen. The reduction in mucosal disruption led to a decreased incidence of acute (12%) and chronic (19%) GVHD for reduced-intensity fludarabine/melphalan versus 36% acute and 40% chronic GVHD for patients receiving ablative sibling HCT in an MD Anderson Cancer Center study^ref. Infectious complications are also reduced, with a 9% incidence of bacteremia at day 100 for nonmyeloablative conditioning versus 27% for standard HCT and a difference in day 100 survivals of 93% versus 81%, respectively, in a case-matched control series from Seattle^ref. However, other investigators have reported higher rates of CMV reactivation before day 100 and persistent risk of bacteremia and fungal infections late post-HCT with nonmyeloablative regimens because of the prolonged immunosuppressive effects of fludarabine even in the absence of GVHD. Treatment outcomes and toxicity for autologous and allogeneic hematopoietic cell transplantation (HCT) for consolidation or salvage therapy :

		autologous HSCT	alloSHCT (Sib)	alloHSCT/MUD	reduced-intensity alloHSCT + MUD
consolidation (CR1)	cytogenetic risk t(15;17)	no role	no role	no role	no role
	t(8;21)inv(16)	5-yr DFS 60-80% (because of high salvage rate and long-term sequelae, many would reserve autologous HCT for relapse for patients with favorable cytogenetics)  TRM 4-8%	5-yr DFS 65%  TRM 18% = No Role	no role	no role
	intermediate	5-yr DFS 42-55%  TRM 4-6%	5-yr DFS 48-62%  TRM 16-20%		insufficient data for  nonmyeloablative
	poor	5-yr DFS 18-25%  TRM 4-8%	5-yr DFS 35-45%  TRM 18-20%	5-yr DFS 30-40%  TRM 30%	reduced intensity 5-yr DFS 50%  for older AML CR1 at 2 yr
salvage	CR2	5-yr DFS 30% overall  5-yr DFS 60-80%  for t(15;17)	5-yr DFS 40%	Pediatric 40%  Adult 5-yr DFS 30%  TRM 30%	2-yr DFS 40-50%reduced intensity
	relapse	not an option unless  "back-up" product from  CR1 available	5-yr DFS 20-30%	Pediatric 5-yr DFS 20%  10-15%	2-yr DFS 10-30%  Adult 5-yr DFS depending on the volume  of residual disease
	induction failure	no role	3-yr DFS 30-40% (3 yrs)  (20% for untreated  secondary AML)	5-yr DFS 20-30% (most of these patients represent patients with high grade myelodysplastic syndrome (MDS) in which an unrelated donor search was initiated prior to leukemic evolution)	1-yr DFS 15-30% (sibling)

A caveat for interpretation of all consolidation trials in which HCT is a component is the existence of selection bias. There have been several large national or co-operative group trials conducted between 1986 and 1995 that tried to answer the question of the role of transplantation in consolidation in younger patients with AML^{ref1, ref2, ref3}. Patients with sibling donors were assigned to the allogeneic marrow transplantation and the remainder were randomized, between a variety of consolidation (2-4 cycles) chemotherapy versus autologous marrow transplant after 1-4 cycles of consolidation. The conclusions ranged from no survival advantage to either form of HCT compared with consolidation chemotherapy in the initial analysis of the US National Trial involving the Eastern Cooperative Oncology Group (ECOG), Southwest Oncology Group (SWOG) and CALGB to a significant difference in disease-free survival for both allogeneic (55%) and autologous HCT (48%) compared to chemotherapy (30%) in the European Organisation for Research and Treatment of Cancer (EORTC) trial (albeit with no overall survival advantage as a high percentage [58%] of chemotherapy relapses were salvaged with transplant). When the US National Trial was reanalyzed using cytogentic risk groupings there were clear differences in favor of HCT (allo and auto) for favorable risk and for allogeneic in patients with poor risk cytogenetics^ref. In all these trials as well as the MRC10 trial, there was considerable patient attrition from achieving remission to initiating any form of consolidation^ref. Fully one third of younger (< 55 yr) patients received no documented form of consolidation; 50-80% of patients actually received the designated HCT treatment. While it is appropriate to compare treatment strategies on an intent-to-treat basis from the time CR is achieved, the interposition of multiple nontransplant therapies before HCT may cause high attrition rates that provide a negative bias, while analyses that start only at time of transplant may give an overly optimistic prediction of outcome.
• consolidation therapy :
• good-risk cytogenetics : given the high curability of APL with conventional chemotherapy, there is no role for autologous or allogeneic HCT for patients in CR1 who achieve a molecular remission by the completion of consolidation. Several series have demonstrated that autologous HCT in patients without HLA-matched donors can produce durable CR2 in 75-100% of the patients in whom a molecularly negative product can be collected^ref. However, for patients with persistence of the PML/RAR gene product, allogeneic transplantation should be considered since the relapse rate following infusion of a molecularly positive product exceeds 85%. Relapse free-survivals of 60-83% have been reported for patients with t(8;21) or inv(16) using ITT analysis from the start of high-dose cytarabine consolidation through autologous HCT with relapse rates of 15-20% post-HCT and TRM of 4-8% overall^{ref1, ref2}. While these DFS rates are higher than the DFS seen with multiple cycles of high-dose cytarabine-based consolidation and the time to completion of treatment may be shorter, long-term toxicities associated with autologous HCT need to be factored into the decision to pursue autologous HCT in individual patients. Many with expertise in AML would reserve autologous HCT for relapse in patients with good risk cytogenetics. In patients who opt for conventional chemotherapy, one should consider cryopreserving autologous PBSC as a "back-up" for salvage therapy in the event of relapse in patients who lack a histocompatible sibling. Autologous HCT can provide long-term salvage for 40% of patients who achieve a second remission overall; factors that correlate with better outcome include cytogenetics and duration of first remission^ref. Sibling allogeneic HCT in CR1 in the group with favorable cytogenetics had only 62% DFS with a 17% TRM in the EORTC/GIMEMA (Gruppo Italiano Malattie Ematologiche dell'Adulto) trial based on ITT from time of remission that compared autologous to allogeneic transplant during the time period of 1993-1999. The high TRM associated with allogeneic HCT indicates that allogeneic HCT has no advantage over conventional chemotherapy or autologous HCT until the toxicity profile improves^ref. 3-yr LFS = 40-50%; 5-yr LFS = 43%, OS = 51%, RI = 53%, TRM = 9%
• intermediate cytogenetics : the European Bone Marrow Transplant (EBMT) group reported an overall 2-year leukemia-free survival of 49% for 1040 patients who received autologous unpurged marrow transplant during the period of 1986-1994^ref. The TRM was 11%, with a median time to granulocyte and platelet recovery of 29 days and 42 days, respectively. Factors that influenced outcomes were time to achieve remission (1 vs 2 inductions) for leukemia-free survival and older age (> 35 years) for TRM. Karyotypic analysis was not included in that study. In the EORTC/GIMEMA study, patients with normal karyotype who underwent autologous HCT had a 48% DFS with a 5% TRM compared to 45% DFS and 20% TRM in patients transplanted from a sibling donor^ref. In a consortium study that included 128 patients up to age 65 receiving autologous HCT in CR1, Linker et al reported a 51% DFS for patients with intermediate risk cytogenetics, with a combined TRM of 3% for both consolidation and transplant^ref. Since TRM in allogeneic recipients increases significantly with age, autologous transplant may offer equivalent DFS with less morbidity for older patients (50 years) than standard myeloablative allogeneic transplant. Younger (40 years) patients with a sibling donor can expect a better relapse-free survival of 62% with a 15% relapse rate and TRM of 15-20%. Since TRM is 10-18% higher for unrelated-donor HCT using myeloablative conditioning, a donor search is rarely initiated during CR1 for intermediate-risk patients in the absence of other risk factors such as antecedent MDS.
• poor-risk cytogenetics : a donor search for a related or unrelated donor should be initiated as soon as the patient is identified as having poor-risk cytogenetics as this group may not achieve remission and may require allogeneic HCT for early salvage. Patients with poor-risk cytogenetics are proportionally underrepresented in autologous transplant series, accounting for less than 10% of patients because remissions are more difficult to achieve and the recovering marrow may have underlying myelodysplasia, which impairs the ability to collect adequate numbers of stem cells with normal proliferative capacity. In most series, the results of autologous HCT for patients in this group are poor, with DFS of 20% or less^ref. In contrast, sibling allogeneic transplant for this group results in a 40-45% DFS for patients  45 years of age^ref. Most unrelated donor transplants in CR1 are performed in patients with poor-risk cytogenetics. Data from the NMDP demonstrated an overall 40% 5-year relapse-free survival for all AML patients in CR1 transplanted between 1987 and 2001, which is comparable to the 44% reported by the EORTC for sibling HCT of patients with poor-risk karyotypes. Reported outcomes for reduced intensity transplants in patients with AML in CR1 are based on small samples. The group from the University of Michigan treated 21 patients over age 55 (median 61 years) with MDS or AML who had HLA-matched sibling donors with the reduced intensity regimen of fludarabine (Flu) and busulfan (Bu) with tacrolimus and MMF for GVHD prophylaxis^ref. 7 of 12 patients who were in CR at time of transplant remain in remission with a median follow-up of 1 year (range 210-733 days). There were 3 relapses and 2 treatment-related deaths. All the patients with active disease died (5 relapse and 4 TRM) with a median survival of 108 days. Overall, the incidence of acute GVHD was 38% with a maximum severity of grade 2. TRM at day 100 for the whole group was 19% and was only 8% in the patients who did not have active disease at transplant. The German Cooperative Transplant Study Group reported on 113 AML patients treated with Bu/Flu (93 patients) or Flu/TBI (20 patients) with a radiation dose of either 400 or 800 cGy. The reduced intensity regimens were chosen for these patients because of age > 50, co-morbid condition or prior HCT; the median age was 51 and more than half the patients received unrelated donor allografts^ref. The minority of patients (22%) were in CR1, and half the patients were in relapse at HCT. Five patients with relapse did not clear their disease and 1 did not engraft. Acute GVHD (grade 2-4) occurred in 42% of patients and chronic GVHD occurred in 35% of patients at risk after day 100 with the majority of these cases being of limited extent. The 2-year DFS was 50% for those in CR1, 40% for those in CR2 or 3, and 15% for those with relapsed disease. Causes of death were evenly divided between relapse and treatment-related toxicity (29 patients each). In addition to remission status, performance status (70%) and donor type (sibling versus unrelated donor) were predictive factors for event-free survival.
• salvage therapy : the majority of allogeneic transplants are performed as salvage for patients who have relapsed after conventional chemotherapy or autologous HCT. The decision regarding reinduction prior to salvage transplant is influenced by (1) the availability of an identified donor and (2) the likeliness of achieving a remission, which in turn is based on cytogenetic risk group, duration of CR1 and comorbid conditions. Data from EBMT, IBMTR and the NMDP show DFS of 44% with sibling allografts and 30% with matched unrelated donor allografts at 5 years for patients transplanted in second remission and DFS of 35-40% in sibling transplants and 10% in unrelated donor transplants for patients with induction failures or HCT in relapse^ref. It should be noted that the majority of these patients received marrow rather than PBSC. In the Seattle study comparing sibling marrow versus PBSC in patients with more advanced disease, there was a significant difference in DFS (57% vs 33%) using PBSC compared to marrow^ref. Additional data from the IBMTR also showed an advantage for PBSC compared to marrow for patients transplanted in second remission^ref. The reduced intensity regimens have had a tremendous impact on TRM in the setting of second transplants or secondary MDS/AML following prior autologous HCT, increasing the DFS from 16% with standard Bu + Cy conditioning to 40% with Flu/Bu or Flu/Melphalan conditioning^ref. The recent formation of the Blood and Marrow Transplant Clinical Trials Network (CTN) to facilitate the conduct of large scale comparative transplant trials should enable the transplant community to expeditiously define the role of newer reduced-intensity conditioning regimens and GVHD prophylaxis regimens in exploiting the antileukemic effect of donor alloreactivity, minimizing transplant-related toxicities and broadening the applicability of HCT as a curative treatment strategy for patients with AML.
• for patients with age < 55 years and favorable prognosis chromosome abnormalities (15%) [e.g., t(8:21); or inv(16)]^ref : autologous HSCT (TRM < 5%; TRM for age < 65 years < 1% !) or chemotherapy with 2-4 cycles of high-dose cytarabine (3 g/m² bid at days 1-6) + idarubicin or etoposide. While the optimal number of such cycles remains to be defined, at least 3 are probably required^ref. Whether the high-dose cytarabine or repetitive cycles of intensive chemotherapy is the critical factor remains debatable; however, a cure rate of 60-70% (52-mo CR = 44%) with chemotherapy suggests that the more risky strategy of allogeneic transplant should be reserved for early relapse or second complete remission in this subset of patients.
• for the remainder intermediate risk (other RFs : FLT3-TDK, FLT3-ITD): either intensive chemotherapy, high-dose chemotherapy with autologous stem cell rescue or sibling-matched allogeneic transplant
• salvage therapy : fludarabine 10 mg daily for 10 days + allopurinol 300 g daily
That a "standard approach" to the treatment of AML in the 18-60 year old patient can be described is still compatible with the notion that all should be referred for a clinical trial. Many questions need to be answered, even for those with so-called 'favorable' prognoses, that usually involve the addition of a new agent onto a backbone of "standard" therapy. The ideal candidates for investigational therapy, many of whom are described subsequently, with a single agent are those likely to fare poorly with induction chemotherapy, including older adults and those with disease relapse less than a year from diagnosis. While many different chemotherapy regimens exist for AML in relapse, the choice of regimen is less important than the duration of first remission. Those who relapse more than one year after diagnosis have good chance to achieve a second remission after administration of the original induction regimen, or at least one of similar intensity. However a second remission is achieved, the only post-remission therapy with significant utility is allogeneic transplant if possible, or high-dose chemotherapy with autologous stem cell rescue.
Protocols :
• age < 60 years : GIMEMA/EORTC AML12
• low-risk OR intermediate-risk with wild-type FLT3 : autologous HSCT
• intermediate risk with mutated FLT3 OR high-risk : allogeneic HSCT
• age < 45 years : myeloablative conditioning (sibling, MUD or haploidentical HSCT)
• age > 45 years OR contraindications to myeloablative conditioning : nonmyeloablative or reduced-intensity conditioning
• age 60-75 years : induction with 3+7 (idarubicin) => reevaluation at day +30
• CR or good PR (blasts in bone marrow 5-20%) :
• no previous infections : allogeneic HSCT with nonmyeloablative or reduced-intensity conditioning IF
• previous infections or not eligible : maintenance therapy with ara-C and idarubicin once a month alternated to VP16
• PR (blasts in bone marrow > 20%) : idarubicin 10 mg once a month alternated to VP16 100 mg/day 3 days a month + ara-C 2 mg/day for 5 days a month
• age > 75 years : GIMEMA AML19 (eligible only if previously treated with <= 14 days of HU and/or GR agonists, no concurrent neoplasia or CNS involvement, severe systemic infections, cardiovascular or pulmonary diseases). At diagnosis :
• if WBC < 30,000/ml no pretreatment
• if WBC > 30,000/ml pretreatment with hydroxyurea <= 14 days
• phase II :
• arm A : Mylotarg 6 mg/m² day 1 and 3 mg/m² day 8 => if no PD continuation therapy with 8 x 2 mg/m² (q4wk)
• arm B : Mylotarg 6 mg/m² day 1, 3 and 5 mg/m² => if no PD continuation therapy with 8 x 2 mg/m² (q4wk)
• arm C : supportive care
• phase III :
• arm 1 (best of arm A or B selected from phase II) : Mylotarg induction => if no PD continuation therapy with 8 x 2 mg/m² (q4wk)
• arm 2 : supportive care
Selected clinical trials in AML :

US Intergroup	APL: ATRA/daunorubicin/ara-C induction, followed by As2O3 + daunorubicin/ATRA x 2 vs daunorubicin/  ATRA x 2 consolidation, followed by ATRA/6MP/methotrexate vs ATRA x 1 yr for maintenance.  AML (age > 70 years and not a candidate for chemotherapy): Oral tipifarnib (R115777) at 2 doses and 2  schedules
CALGB	AML (age > 60): dauno/ara-C vs dauno/ara-C/oblimersen  AML (age < 60): dauno/ara-C/etoposide, followed by post-chemotherapy/PBSCT, followed by IL-2 vs observation
ECOG	AML (age > 60): dauno/ara-C ± MDR modulator (LY335979)  AML (age < 60): daunorubicin (45 mg/m2 x 3 d)/ara-C vs daunorubicin (90 mg/m2 x 3 d)/ara-C, followed by  ± gemtuzumab ozogamicin (GO) prior to autoPBSCT (if no sib donor)
SWOG	AML (age > 55): continuous infusion daunorubicin/ara-C ± cyclosporine A followed by assignment to mini-allo BMT  (if HLA-matched sibling donor)  AML (age  55): daunorubicin/ara-C vs daunorubicin/ara-C + GO
EORTC	AML (age > 60): ida/ara-C vs ida/ara-C/GO  AML (age < 60): ida/ara-C vs ida/high dose ara-C, followed by intensive consolidation/allogeneic BMT,  followed by IL-2
HOVON	AML (age > 60): dauno (45 mg/m2)/ara-C vs dauno (90 mg/m2)/ara-C f/b intermediate dose ara-C,  followed by GO x 4 vs observation  AML (age < 60): ida/ara-C ± G-CSF vs ida/high-dose ara-C ± G-CSF, followed by mitoxantrone/etoposide (good risk) or Mito/etop vs autoBMT or alloBMT (if sibling donor)
MRC	APL: ATRA/dauno/high-dose ara-C/6-thioguanine (MRC) vs ATRA/ida (Spanish), followed by MRC or Spanish  chemo ± GO  AML (age > 60): intensive chemo (dauno/ara-c at various doses) vs non-intensive chemo (hydroxyurea/low dose ara-C ± ATRA)  AML (age < 60): dauno/high dose ara-C/6-thioguanine vs fludarabine/ara-C/G-CSF/ida +/- GO, followed by  allo BMT (if sibling; and will be nonmyeloablative if > 50, "full" if under 35 years old)

Categories of novel therapies for AML :
• drug-resistant modulator (DRM) : the notion that blasts from AML patients, and particularly from older individuals, are likely to express genes capable of mediating drug resistance, most notably the ABCB1 / MDR1 / GP170 drug efflux pump, has prompted clinical trials with so-called reversing agents that inhibit this function. Since many of these agents also inhibit the metabolism of chemotherapeutic drugs like anthracyclines, pharmacokinetic considerations are important. Most of the trials that have compared chemotherapy with or without a DRM have shown no benefit or have been terminated early due to excess toxicity^ref. However, a randomized clinical trial involving cyclosporine A as a reversing agent has been positive^ref, prompting the SWOG to treat older adults with chemotherapy (using a novel regimen including continuous infusion daunorubicin) with and without cyclosporine A. Secondly, trials with newer DRMs that do not have pharmacological effects on chemotherapy are also underway. A problem with these drugs as well as with many novel approaches is that the mechanism of resistance is likely to be pleiotropic. The inability to initiate cell death in response to chemotherapy is another potential mechanism of resistance. Overexpression of the bcl-2 anti-apoptotic gene has been associated with poor prognosis in AML patients. Preclinical data documenting synergism between chemotherapy and the antisense 18-mer oligonucleotide oblimersen (G3139) have prompted Phase I clinical trials documenting the safety of induction chemotherapy with this agent^ref. The CALGB has begun a randomized a trial in older adults that compares chemotherapy alone to chemotherapy administered with oblimersen. Synergistic antileukemic activity between chemotherapy agents and the proteosome inhibitor bortezomib^ref has spurred the clinical development of this agent in conjunction with chemotherapy in AML. The use of hematopoietic growth factors (HGFs) in AML has received much attention. Once concerns diminished that growth factors would enhance disease resistance via their known ability to stimulate leukemia cells to enter S-phase, a host of clinical trials examined the ability of HGFs to shorten the period of neutropenia and reduce infectious mortality. While the duration of neutropenia was reduced by several days when G-CSF or GM-CSF was used in the postchemotherapy period, the inability of these agents to alter the nadir, allay mucositis, or decrease the death rate diminished their utility. Perhaps more interesting is the use of these agents before or during chemotherapy in an effort to increase the S-phase fraction of cells, thereby using them as chemosensitizing agents. Although most of the trials using HGFs in this fashion have been disappointing, a recent randomized study in which G-CSF was used before and during chemotherapy, yielding a disease free survival benefit^ref, has prompted renewed interest in this strategy. At the present time the use of G- or GM-CSF can be considered optional using supportive care guidelines for neutropenic fever while their use as chemotherapy-enhancing agents needs to be explored further before routine use is warranted.
• proteasome inhibitors
• agents that promote differentiation : almost a quarter century has elapsed since clinical trials suggested that low-dose cytarabine's effect in AML and myelodysplasia resulted from hematopoietic cell differentiation. In the ensuing years, the enthusiasm for low-dose cytarabine as an effective therapy that induces maturation of AML cells has waned. However, an increased understanding of how the transcription of differentiation-associated genes is regulated has prompted the exploration of new and potentially effective therapies. Among the many biochemical events that must occur as a cell changes from a stem cell to a mature functional blood cell are removal of methyl groups from DNA bases and addition of acetyl groups to the histone DNA protein coat. Such effects change the conformation of the DNA and allow transcription of certain genes. The DNA hypomethylating agent azacitidine^ref was recently approved by the US Food and Drug Administration (FDA) for use in all subtypes of myelodysplastic syndrome. Many clinical trials are now underway with so-called histone deacetylase inhibitors, although outcome data are sparse. Given the general hypothesis that AML results from a combination of over proliferation of the stem cell compartment as well as failure to differentiate, it is rational to consider the future use of these differentiating agents in combination with agents that inhibit mitogenic signals.
• pro-apoptotic approaches
• signal transduction inhibitors : the mechanism by which cells receive and transmit mitogenic signals is complex and involves the coordinated action of many different proteins, commonly represented by so-called signal transduction cascades. Insofar as such cascades might be overactive in leukemia cells compared to normal hematopoietic cells, a therapeutic opportunity is provided. Moreover, an activating mutation in one of the proteins responsible for transmitting proliferative signals in AML defines a potential target.
• "RAS"-targeted (e.g., farnesyl transferase inhibitors (FTI)) : mutations in one of the RAS family proteins, 21-KD guanine-nucleotide binding proteins, have been described in 10-50% of AML patients. Such activating mutations are thought to allow autonomous growth. RAS proteins require several post-translational modification steps including addition of a farnesyl lipid moiety that allows translocation to the plasma membrane and activation. FTIs have been shown to inhibit the growth of mutant RAS-transformed cell lines. The FTI in furthest development in AML, tipifarnib (R115777), was associated with responses in advanced-stage patients in a Phase I trial^ref. Despite disappointing results in an international Phase II trial in refractory/relapsed patients (although RAS mutations were not required), the drug has been administered to over 100 chemotherapy-naïve AML patients. A preliminary report documented a 20% complete remission rate in this group of poor prognosis, largely older patients who received this oral agent as their initial therapy (Lancet JE, Gojo I, Gotlib J, et al. Tipifarnib (ZARNESTRA) in previously untreated poor-risk AML and MDS: interim results of a phase 2 trial [abstract]. Blood. 2003;102:176a). These exciting results, if confirmed, might provide a new and less toxic therapy compared to chemotherapy for older adults with AML. FTIs are also being evaluated in younger patients in earlier disease states and in combination with chemotherapy
• tyrosine kinase targeted (e.g., FLT3, c-kit) : an active area of current research in AML is the development of agents known as FLT3 inhibitors. FLT3 is a transmembrane tyrosine kinase. Approximately 30% of AML patients can be shown to have an activating mutation that generally carries a poor prognosis^ref. The activating mutation may be a duplication of between 3 and 60 amino acids in the juxtamembrane region or, less commonly, a point mutation in the activation loop.2 Activating mutations of the FLT3 tyrosine kinase transform leukemic cell lines into growth factor independence and cause a fatal myeloproliferative syndrome in a murine bone marrow transplant model. Small molecules capable of inhibiting FLT3 enzyme activity can selectively kill such transformed cell lines and improve survival in the murine model^ref. Thus, the preclinical data supporting the development of these drugs as therapeutic agents in AML is at least as strong as that used to support clinical trials with imatinib in patients with CML. There are two major differences between imatinib's development and that of the FLT3 inhibitors: 1) CML in chronic phase is probably based solely on the activation of bcr-abl, whereas AML is almost certainly a "multi-hit" disease; 2) Multiple agents and drug companies are developing FLT3 inhibitors. However, just as was the case for imatinib, these drugs have a spectrum of activity beyond FLT3 inhibition alone. For example, PKC-412 (N-benzoylstaurosporine) inhibits FLT3 as well as protein kinase C and the vascular endothelial growth factor receptor. Such a wide spectrum of activity could have positive or negative consequences. SU5416, which inhibits FLT3 reasonably potently, was developed as a c-kit inhibitor. The drug demonstrated modest activity in AML, but no activity was observed in the 7 patients who retrospectively were found to have an activating mutation of FLT3 in their myeloblasts^ref. Minor responses have been observed in patients without known FLT3 mutations who have received PKC-412. Biological responses, demonstrated by a major reduction in peripheral blast count, have occurred with PKC-412,25 CEP-701,26 and MLN-518,27. However, they have minimal ability to reduce the bone marrow blast count, and therefore, complete remissions have occurred in 1/42 reported patients. Moreover, the duration of response has been brief. Although much more work with these oral agents needs to be done, initial impressions suggest that (1) the multiplicity of genetic lesions in the typical AML cell may be problematic for expecting these agents to work alone; (2) pharmacokinetic issues require prolonged therapeutic drug levels and the ability to get to the target leukemia progenitor cell; and (3) the contribution of alternative enzymes and pathways are important^ref. FLT3 inhibitors in clinical development^ref :

tyrosine kinase inhibitor	class	receptor inhibitor activity (in descending order of potency)	FLT3 autophosphorylation IC50 in vitro	clinical trials/comments	toxicity
PKC-412	benzoylstaurosporine	PKC  PDGFR  KDR  KIT  FLT3  ABL	528 nM	phase II: AML with/without FLT3-ITD  In FLT 3 mut pts (n = 20), 35%  significant reduction in blast  countref	nausea,  emesis, fatigue
CEP-701	indolocarbazole	FLT3  TRKA  KDR  PKC  PDGFR  EGFR	2-3 nM	phase II: AML with FLT3-ITD  Several pts had reduced blast  counts, autophosphorylation  inhibitedref	nausea,  emesis, fatigue
MLN-518	piperazinyl quinazoline	KIT  PDGFR  FLT3  FMS	170-220 nM	phase I: AML/MDS with/without  FLT3-ITD phase II: AML with FLT3-ITD A few pts with FLT3 ITD treated  at higher doses had biological  response (DeAngelo D, Stone RM, Bruner RJ, et al. Phase I clincal results with MLN518, a novel FLT3 antagonist: tolerability. Pharmacokinetics and pharmacodynamics [abstract]. Blood. 2003;102:65a)	generalized  weakness,  fatigue, nausea  and vomiting
SU5416	indolinone	FLT3  KDR  KIT	250 nM	phase II: refractory  AML/MDS/MPD/MM  Phase II: Refractory AML (c-KIT  positive). No responses in  FLT3 ITD pts.ref	fatigue,  nausea, sepsis  and bone pain

Nonetheless, further development of these drugs at different doses and schedules and in combination with other signal transduction inhibitors and/or chemotherapy is warranted. Other tyrosine kinase inhibitors, including those that inhibit VEGFR, are also in clinical trials in AML^ref.
• downstream signal inhibitors
• immunotherapeutic approaches
• TAAs :
• anti-CD33 immunotoxin conjugate (gemtuzumab ozogamicin) (Mylotarg^®) was approved based on a 30% response rate (half of whom were patients who never fully recovered their platelet count) in patients with untreated first relapse who had, in most cases, a first remission > 6 months^ref. Disappointment with this drug due to its low single-agent efficacy in treatment of patients with refractory AML as well as its association with veno-occlusive liver disease (VOLD) when administered proximal to or following a bone marrow transplant^ref has dampened enthusiasm. Nonetheless, based on the apparent safety of combining this agent with chemotherapy in newly diagnosed patients (De Angelo DJ, Stone RM, Durrant S, Liu D, Baccarani M, Schiffer CA, Amrein PC, Sherman ML. Gemtuzumab ozogamicin in combination with induction chemotherapy for the treatment of patients with de novo acute myeloid leukemia: two age-specific phase II trials [abstract]. Blood. 2003;102:100a : MY-FLANG) important studies are now underway to determine if gemtuzumab ozogamicin combined with induction or consolidation chemotherapy, or as a single agent in the post-remission setting in older adults might lead to a better outcome. Gemtuzumab 9 mg/m2 IV for up to 3 doses with at least 14 days, but not greater than 28 days between doses. Appropriate premedication. Grade 3-4 neutropenia (97%), infection (28%), sepsis (16%), fever (15%), pneumonia (7%), chills (13%), thrombocytopenia (99%), mucositis (4%), hyperbilirubinemia (24%), transaminases (17%), nausea/vomiting (11%), dyspnea (9%), hypertension (9%), hypotension (8%), asthenia (7%) => grade 3-4 bleeding (15%), 19 patients (13%) died during the treatment period. Incidence of infusion-related symptoms was significantly lower after the second dose compared with the first dose (grade 3-4 34% vs. 12%). Hepatotoxicity, including severe veno-occlusive disease (VOD) has been reported. Median time to onset of grade 3-4 hyperbilirubinemia was 8 days; median duration was 20 days^ref.
• anti-GM-CSF receptor
• antigens unknown :
• stimulate immune system (IL-2, GM-CSF)
• present tumor antigens effectively
• dendritic cell vaccines : immunotherapeutic approaches which induce a patient’s immune system to recognize and eliminate malignant cells at the minimal residual disease (MRD) stage are considered to be the most effective strategy. By generating CD8⁺ T and CD4⁺ T responses^ref, primary T-cell responses or even maintenance of tolerance is controlled by DC. DC pulsed with peptides eluted from patients’ AML cells could stimulate in vitro autologous peripheral blood mononuclear cells to elicit specific T_h1 responses and generate CD4⁺ and CD8⁺ T cells^ref. This is the first report on acid stripped peptides inducing anti-AML T-cell responses, presumably by activating pre-existing tumor specific precursor T cells. This strategy is relatively simple and potent; it only requires that autologous tumor cells express MHC class I and II complexed tumor-specific peptides. In fact, this approach could also be utilized for cloning and characterization of novel potential antigens for immunotherapy of AML. Taken together with similar findings for other malignancies, the data indicate the feasibility of the approach for the induction of anti-AML responses in patients with MRD. However, future translation to the clinic should be approached with caution given the fact that DC have been shown to elicit severe autoimmune responses^ref
• dendritic cell fusion :
• transfer hematopoietic growth factor genes
• a gene expression-based high thoughput screening (GE-HTS) identified 8 compounds that induce AML differentiation signature and can induce at least one hallmark of differentiation assayed by conventional methods^ref
Characteristics of older and younger adults with AML : AML is a disease of older adults. In the US, the median age is 68 years and the age-adjusted population incidence is 17.6 per 100,000 for people 65 years of age or older, compared with an incidence of 1.8 per 100,000 for people under the age of 65 years^ref. Therefore, of the estimated 11,900 new AML diagnoses in the US in 2004, over half will affect patients 60 years of age or older, a population considered "elderly" in the leukemia literature^{ref1, ref2, ref3, ref4}. This number will only increase as the population of US citizens 65 years or older, estimated to be approximately 35 million in the year 2000, is expected to double by the year 2030. Older adults with AML, when compared to younger patients with the same disease, have a poor prognosis and represent a discrete population in terms of disease biology, treatment-related complications, and overall outcome. As a result, older patients require distinctive management approaches to determine whether standard treatment, investigational treatment, or low-dose therapy or palliative care is most appropriate. Particularly in this population, the tradeoff between potential for cure or survival prolongation and quality of life must be weighed carefully.

characteristic		older AML  patients (in general, older AML patients are defined as  60 years of age; younger AML patients as < 60 years of age.)	younger AML  patients
population incidence (new diagnoses, per 100,000 US citizens per year. Older/younger division occurs at 65 years)		17.6	1.8
favorable cytogenetics (percentages rounded to nearest whole number)	t(8;21)	2%	9%
	inv 16 or t(16;16)	1-3%	10%
	t(15;17)	4%	6-12%
unfavorable cytogenetics (percentages rounded to nearest whole number)	-7	8-9%	3%
	+8	6-10%	4%
	complex	18%	7%
ABCB1 / MDR1 expression		71%	35%
secondary AML		24-56%	8%
treatment-related mortality (rates following remission-induction therapy with an anthracycline- or anthracenedione-based regimen)		25-30%	5-10%
complete remission (rates following remission-induction therapy with an anthracycline- or anthracenedione-based regimen)		38-62%	65-73%
long-term survival (percentages rounded to nearest whole number)		5-15%	30%

• disease biology :
• cytogenetics : older adults with AML have a lower incidence of favorable chromosomal abnormalities [including the core binding factor abnormalities, such as t(8;21) or abnormalities of chromosome 16, or the t(15;17) associated with APL] and a higher incidence of unfavorable abnormalities (including complex cytogenetics or abnormalities of chromosomes 5, 7, or 8) compared to younger adults with AML^{ref1, ref2, ref3}. The rare older patient fortunate enough to have good-risk cytogenetic abnormalities may have a survival advantage over other older patients^{ref1, ref2}, though it is not clear that this finding holds for patients older than 65 years.
• bone marrow biology : in older adults, AML is more likely to arise from a proximal bone marrow stem cell disorder, such as myelodysplastic syndrome (MDS), and with leukemia-specific abnormalities in more than one hematopoietic cell lineage. This may explain the different disease behavior in this group, as well as prolonged neutropenia following chemotherapy^ref. Older adults with AML also are more likely to have reduced proliferative capacities in normal hematopoietic stem cells, which may affect blood count recovery following intensive chemotherapy.
• drug-resistance genes : the expression of genes that mediate drug resistance occurs with increased frequency in this age cohort. MDR1, the so-called P-glycoprotein (gp170) chemotherapy efflux pump, was found in 71% of leukemic blasts in subjects in a SWOG study of AML patients over the age of 55 years, compared to a prevalence of 35% in younger AML patients^ref. MDR1/P-glycoprotein expression is associated with lower complete remission (CR) rates and more chemo-resistant disease.
• prior stem cell insult : older adults with AML are more likely to have a secondary leukemia arising from an antecedent MDS or from prior treatment with chemotherapy or radiation therapy for another cancer. Patients with this type of AML are predisposed to having abnormalities in chromosome 5 and/or 7^ref. Secondary AML (AML that arose after MDS, myeloproliferative disorders, and therapies of malignancy) comprises 24-56% of AML diagnosis in older patients^{ref1, ref2}, compared to a prevalence of approximately 8% in younger AML patients in the Medical Research Council (MRC) AML 10 trial^ref.7 AML arising from prior bone marrow stem cell disorders or antecedent hematologic disorders, particularly when the process is > 10 months in duration prior to the development of AML, is less responsive to chemotherapy, resulting in shorter event-free survival, a lower CR rate, and conferring a worse prognosis^ref.
• response to therapy : older adults are not as tolerant of or responsive to remission induction and consolidation chemotherapy compared to their younger counterparts. High mortality rates likely result from inherent disease biology, an increased prevalence of comorbid disease, and a differential metabolism of induction regimen drugs, particularly cytarabine, resulting in supratherapeutic drug levels^ref.4 Concern over potential treatment-related toxicities may result in undertreatment of disease. Paradoxically, administration of full-dose daunorubicin, for example, may result in a reduction in early deaths by effecting a more rapid CR. The outcome of older adults with AML is also worse. Adults under the age of 60 years treated with an induction regimen consisting of an anthracycline combined with cytarabine have a 65-73% chance of attaining a complete remission (CR), while those over 60 years of age have a 38-62% chance of a CR^{ref1, ref2, ref3, ref4, ref5, ref6}. Patients who fall into the "very elderly" category (80 years or older) can attain a CR with intensive therapy, but their chance of doing so is 30%, and only 7% of treated patients are alive at one year. Similar numbers hold for patients 70 years of age or older^ref. Moreover, long-term survival occurs in approximately 30% of younger adults (or 45% of those entering a CR), compared to only 5-15% long-term DFS in adults over the age of 60 years^{ref1, ref2, ref3, ref4}
• indications for treatment : for 85-95% of older AML patients, any therapy ultimately will be purely palliative. Treatment options range from supportive care (blood and platelet transfusions when needed, antibiotics to treat infections, and growth factor support) to low-dose chemotherapy (e.g., hydrodyurea or low-dose cytarabine) or investigational agents as part of clinical trials, and high-dose chemotherapy (anthracycline- or anthracenedione-based remission induction therapy). 2 trials have randomized older patients to receive either immediate remission induction chemotherapy with an anthracycline-based regimen versus a less aggressive or palliative approach^{ref1, ref2}. Only one of these studies16 demonstrated a survival advantage for patients receiving induction chemotherapy (21 vs 11 weeks, P = 0.015), for a median survival time only 16 days longer than the median amount of time they spent hospitalized to receive therapy. Thus, the decision of whether or not to offer remission induction therapy (on the part of physicians) or to receive it (on the part of patients) is not straightforward. In making this decision, older patients overestimate the potential benefit they may derive from intensive chemotherapy and may not recall all treatment options. One study^ref found that 74% of older patients estimated their chance to be cured by remission induction therapy to be  50%, and almost 90% estimated their chance of being alive in 1 year to be 50%. In contradistinction, physicians caring for these patients estimated the chance of cure to be  10% almost 89% of the time. Nearly two-thirds of patients did not recall being offered treatments other than the one they chose, despite physician documentation of alternatives in all cases. Patients choosing to receive remission induction therapy spent 79% of their days during the first 6 weeks of the study period either hospitalized or being seen in clinic, compared to 14% of days for patients choosing less aggressive therapy or best supportive care. Thus, treatment decisions should be based on individual patient preferences after an informed discussion has taken place that incorporates risk estimates modified to a patient's performance status, comorbidities, and leukemia-specific risk factors, without using absolute cutoffs for prognostic factors or chronological age. For example, an active, "younger" older adult with de novo AML and favorable cytogenetics should be presented with different prognostic information than a bed-bound septuagenarian with secondary disease and complex cytogenetics. Ideally, this discussion should include specifics about prognosis and treatment-related complications, and the potential impact therapy will have on a patient's quality of life. When available and clinically appropriate, older patients should always be offered the opportunity to participate in clinical trials for up-front therapy.
• treatment options :
• remission induction therapy : as with younger AML patients, the backbone of remission induction in older adults consists of an anthracycline (daunorubicin or idarubicin) or anthracenedione (mitoxantrone) and cytosine arabinoside (Ara-C), a regimen that has not changed in over 2 decades. Typically, daunorubicin is given at a dose of 45 mg/m²/d x 3 days, or mitoxantrone or idarubicin are given at doses of 12 mg/m²/d x 3 days, in combination with Ara-C, which is administered as a continuous infusion at 100 or 200 mg/m²/d x 7 days (frequently referred to as 7+3 chemotherapy). While studies have compared different anthracyclines and anthracenediones, varied doses and schedules, and added additional agents with some improvement in CR rates, they have not demonstrated an improvement in overall survival (OS) rates^{ref1, ref2, ref3, ref4, ref5, ref6} (Buchner T, Hiddemann W, Wormann B, et al. Daunorubicin 60 instead of 30 mg/sqm improves response and survival in elderly patients with AML [abstract]. Blood. 1997;90:583a). For example, a recent study from the ECOG randomized older AML patients to remission induction therapy with either daunorubicin, idarubicin, or mitoxantrone along with a standard dose of Ara-C^ref. The outcome was not significantly different, with CR rates of 40%, 43%, and 46% and median survivals of 7.7, 7.5, and 7.2 months, respectively. Once a decision has been made to initiate intensive chemotherapy, it should not be delayed, as this may impact outcome^{ref1, ref2}. Selected randomized studies defining remission induction therapy in older adults :

goal		remission-induction agents	complete remission	overall survial	comments
compare anthracyclines and anthracenediones	Lowenberg, 1998ref	mitoxantrone (8 mg/m2/d) + Ara-C (cytosine arabinoside) (100 mg/m2/d)  vs. daunorubicin (30 mg/m2/d) +  Ara-C (cytosine arabinoside) (100 mg/m2/d)	47%  vs.  38% P = 0.07	39 weeks (median)  9% (5 years)  vs.  36 weeks (median) 6% (5 years)  P = 0.23	Early and post-induction death rates  were similar for both arms. Patients  receiving mitoxantrone had a significantly  higher rate of severe infections (25.1%  vs 18.6%) and a trend toward a longer  duration of aplasia (22 vs 19 days).
	AML Collaborative  Group, 1998ref	idarubicin (8-20 mg/m2/d) +  Ara-C (cytosine arabinoside) (100-200 mg/m2/d)  vs.  daunorubicin (45-50 mg/m2/d) +  Ara-C (cytosine arabinoside) (100-200 mg/m2/d)	51%  vs.  46% P = not done	33.6 weeks (median)  vs.  29.9 weeks (median) P = 0.58	Early induction failure tended to be higher  with idarubicin, while late induction failure  was lower. Myelosuppression was greater in patients receiving idarubicin
	Archimbaud, 1999ref	idarubicin (8 mg/m2/d) +  Ara-C (cytosine arabinoside) (100 mg/m2/d) +  etoposide (100 mg/m2/d)  vs.  mitoxantrone (7 mg/m2/d) +  Ara-C (cytosine arabinoside) (100-200 mg/m2/d)+  E (100 mg/m2/d)	45%  vs.  50%  P = 0.52	7 months (median)  21% (2 years)  vs.  7 months (median)  21% (2 years)  P = not done	No difference between groups in degree  of myelosuppression or in early death  rates.
vary 7+3 dose	Buchner T, Hiddemann W, Wormann B, et al. Daunorubicin 60 instead of 30 mg/sqm improves response and survival in elderly patients with AML [abstract]. Blood. 1997;90:583a	daunorubicin (60 mg/m2/d) +  Ara-C (cytosine arabinoside) (100 mg/m2/d)  vs.  daunorubicin (30 mg/m2/d) +  Ara-C (cytosine arabinoside) (100 mg/m2/d)	54%  vs.  42%  P = 0.038	16% (5 years) vs.  10% (5 years)  P = 0.11	The 30 mg daunorubicin arm was closed  prematurely due to higher response rates  in the 60 mg arm, resulting in 42 patients  receiving lower dose daunorubicin, and  130 patients receiving the higher dose.
	Dillman, 1991ref	daunorubicin (45 mg/m2/d) + Ara-C (cytosine arabinoside) (100 mg/m2/d)  vs.  daunorubicin (45 mg/m2/d) +  Ara-C (cytosine arabinoside) (200 mg/m2/d)	44%  vs.  38%  P = 0.68	11.0 weeks (median)  vs.  9.6 weeks (median)  P = 0.23	This study has short overall survival times  compared to other studies in this patient  population
add agents	Goldstone, 2001ref	daunorubicin (50 mg/m2/d) +  Ara-C (cytosine arabinoside) (100 mg/m2 q 12 hours)+  thioguanine (100 mg/m2 q 12 hours)  vs.  daunorubicin (50 mg/m2/d) + Ara-C (cytosine arabinoside) (100 mg/m2 q 12 hours) +  etoposide (100 mg/m2/d)  vs.  mitoxantrone (12 mg/m2/d) +  Ara-C (cytosine arabinoside) (100 mg/m2/d	62% vs. 50%  P = 0.002 (for comparison of DAT to ADE)  vs. 55%  P = 0.04 (for comparison of DAT to MAC)	12% (5 years)  vs.  8% (5 years)  P = 0.02 (for comparison of DAT to ADE)  vs.  10% (5 years)  P = 0.1 (for comparison of DAT to MAC, 0.2 (for comparison of ADE to MAC	There were no significant differences in  myelosuppression or other toxicities,  neutrophils were slower to recover in the  mitoxantrone arm. Patients receiving ADE  had higher rates of induction death (26%  compared to 16% for DAT and 17% for MAC)
	Baer, 2002ref	daunorubicin (60 mg/m2/d) +  Ara-C (cytosine arabinoside) (100 mg/m2/d)+  etoposide (100 mg/m2/d)  vs.  daunorubicin (40 mg/m2/d) +  Ara-C (cytosine arabinoside) (100 mg/m2/d) +  etoposide (60 mg/m2/d) +  PSC-833 (10 mg/kg/d)	46%  vs.  39%  P = 0.008	7 months (median)  vs.  2 months (median)  P = 0.48	Because of concern about excessive  mortality on the ADEP arm (25 deaths vs  12 on the ADE arm), it was closed early to  to further accrual. Survival between the  2 arms was similar at 1 year
use hematopoietic growth factors	Stone, 1995ref	daunorubicin (45 mg/m2/d) +  Ara-C (cytosine arabinoside) (200 mg/m2/d)  vs.  daunorubicin (45 mg/m2/d) +  Ara-C (cytosine arabinoside) (200 mg/m2/d) + GM-CSF (5 µg/kg/d)	54%  vs.  51%  P = 0.61	10.8 months  (median)  vs.  8.4 months  (median)  P = 0.10	Median duration of neutropenia was 15  days in the GM-CSF arm and 17 days in  placebo arm (P = 0.02). The duration of  hospitalization did not differ between the  arms; nor did the rates of life-threatening  infection, or persistent leukemia.
	Godwin, 1998ref	daunorubicin (45 mg/m2/d) +  Ara-C (cytosine arabinoside) (200 mg/m2/d)  vs.  daunorubicin (45 mg/m2/d) +  Ara-C (cytosine arabinoside) (200 mg/m2/d) +  G-CSF	50%  vs.  41%  P = 0.89	9 months (median)  vs.  6 months  (median)  P = 0.71	The duration of neutropenia was 15%  shorter in the G-CSF arm compared to the  placebo arm (P = 0.14). The duration of  hospitalization did not differ between the  arms; nor did the rates of life-threatening  infection, or persistent leukemia

• hematopoietic growth factors : in the majority of AML patients, death results from bleeding or infectious complications. This is particularly true in older adults with AML. The utility of hematopoietic growth factors (HGF) for ameliorating the myelosuppressive complications of AML therapy in older adults has been studied extensively^{ref1, ref2}. These trials were also designed to determine whether or not HGF had detrimental effects due to inappropriate stimulation of leukemic cell proliferation and thus resistance, or whether they had beneficial effects in "priming" leukemic cells to proliferate prior to the administration of S-phase specific chemotherapy agents such as Ara-C^{ref1, ref2}. With the exception of one ECOG study that demonstrated a CR rate and overall survival benefit in patients randomized to the GM-CSF arm (compared to patients receiving no growth factor support), these trials found that while HGF are safe, reduce the duration of neutropenia (by a range of 2-6 days), and do not support leukemia cell proliferation, they also do not reliably improve the CR rate, the length of hospitalization, or the induction death rate or prolong survival.
• post-remission chemotherapy : no randomized trial has ever demonstrated that any amount of post-remission therapy in older AML patients provides better outcomes than no post-remission therapy. That being said, the only studies demonstrating that long-term DFS is possible in older AML patients have included remission induction and post-remission therapy. It is reasonable, then, to administer post-remission therapy consisting of a repeat of remission induction therapy, single-agent Ara-C, or 2 days of an anthracycline or anthracenedione (the same type of drug given at the same doses as with remission induction therapy) combined with 5 days of Ara-C, again given at the same dose as with remission induction therapy (frequently referred to as post-remission therapy). There does not appear to be any additional survival benefit attained from administering > 1-2 cycles of post-remission therapy or in treating older AML patients with maintenance therapy. In the Medical Research Council (MRC) AML 11 trial, 371 patients who entered a complete remission following anthracycline- or anthracenedione-based remission induction therapy were randomized to receive either 1 cycle of daunorubicin, Ara-C, and thioguanine (DAT) consolidation therapy, or DAT along with 3 additional cycles of Ara-C-based consolidation therapy (for a total of 4 cycles of post-remission therapy)^ref. Of those randomized to the long consolidation course, 61% were able to complete all 4 cycles. Survival was similar at 5 years for patients randomized to the short and long consolidation arms.
• postremission bone marrow transplantation : an even more aggressive approach than induction therapy followed by consolidation consists of bone marrow transplantation. Nonmyeloablative allogeneic bone marrow transplants take advantage of a GvL effect using a less-intensive preparative regimen with lower up-front mortality. The reduced TRM and ability to perform these transplants in the outpatient setting make them an appealing option for the older AML patient with few comorbidities and an adequate performance status. Preliminary studies that include older AML patients have demonstrated that durable complete remissions are attainable with this treatment, though with limited follow-up. One study of 19 patients with myeloid malignancies (17 of whom had advanced MDS or AML) and a median age of 64 years (range, 60-70 years) demonstrated a 68% survival at a median follow-up of 825 days following nonmyeloablative transplantation^ref. These early data have prompted cooperative groups to explore the role of bone marrow transplantation in older AML patients in first CR.
• newer approaches : remission induction therapy for older adults with AML is no panacea, with median and 5-year survival rates resembling those of patients with advanced lung cancer^ref. It is thus reasonable to consider investigational agents for older AML patients as initial therapy, particularly those that may be associated with less TRM. Potential targets for antileukemia therapy include specific signaling molecules required for the maintenance of the leukemic state, such as tyrosine kinases; overexpression of bcl-2, an anti-apoptosis signal; DNA methylation, associated with suppression of regulatory genes and with disease progression; indirect pathways that maintain leukemogenesis, including angiogenesis and drug resistance; and investigational agents with mechanisms of action that differ from anthracyclines, anthracenediones, and Ara-C, such as nucleoside analogs, farnesyl transferase inhibitors (FTIs), and MDR modulators, alone or in combination with standard therapies^{ref1, ref2, ref3, ref4} (Stone R, Klimek V, DeAngelo D, et al. PKC412, an oral FLT3 inhibitor, has activity in mutant FLT3 AML: a phase II clinical trial [abstract]. Blood. 2002;100:316a). The efficacy of the FTI Zarnestra will be examined in newly diagnosed AML patients over the age of 70 years in the US cooperative group setting (SWOG 0432). One recent trial from the Dutch-Belgian Hemato-Onocology Cooperative Group (HOVON) randomized predominantly older patients, most of whom had a diagnosis of advanced MDS or AML, to Ara-C and G-CSF or the same regimen + fludarabine for 2 cycles followed by Ara-C and daunorubicin for 1 cycle^ref. This study is typical of many novel combination trials in older AML patients in that, while the CR rate was improved in AML patients receiving fludarabine (95% vs 71%, P = 0.046), survival was not impacted.
• aggressive supportive/palliative care : intensive chemotherapy provides only marginal, if any, survival benefit to older AML patients, so non-intensive (or non-chemotherapy-based) approaches are reasonable. Blood and platelet transfusions should be administered to alleviate symptoms stemming from anemia and thrombocytopenia, and antibiotics started when appropriate. Low-dose chemotherapy should only be used in the setting of leukocytosis and/or associated symptoms. Any recommendations for the institution of neutropenic precautions (i.e., avoiding crowds, refraining from ingestions of raw foods) must be balanced with the lack of evidence supporting the benefit of these maneuvers and the impact such restrictions will have on a patient's quality of life. Hospice services should be instituted within 6 months of anticipated demise. While some hospice organizations prohibit blood product transfusions, we consider these to be palliative in this population as they may result in improved quality of life in terminal cancer patient populations.
Prognosis :
 

	CR	5-yr OS
age < 60 yrs	75%	35-40%
age > 60 yrs	45-55%	< 20%

• 20-60% will relapse (median : 4 months; higher risk in the first 2 years) : 10-20% are primarily refractary to chemotherapy (< 10% survive), patients should not be retreated after a relapse. The duration of the primary CR is related to the likelihood to achieve a second CR
• 50-mo DFS
• allogeneic HSCT : 33.7%
• autologous HSCT : 58.3%
• chemotherapy only : 20%
• allogeneic BMT :
• during disease : TRM = 30-50%
• after first remission : TRM = 6%
• after second remission : TRM = 20-30%
• chronic myeloproliferative disorders (CMPDs) : polycythemia vera (PV), essential thrombocythemia (ET), chronic idiopathic myelofibrosis (IMF), and chronic myelogenous leukemia (CML) were grouped into spectrum of pathogenetically related disorders of varying clinical manifestations by Dameshek in 1951^ref. Recently, the WHO classification of the chronic myeloproliferative diseases (CMPDs) recognizes 7 entities. One, CML, is defined not only by its classic morphology and clinical features but also by the genetic abnormality with which it is always associated, the Philadelphia chromosome or BCR/ABL fusion gene. Unfortunately, specific genetic abnormalities have not yet been identified for any of the other CMPDs. Therefore, the classification of the CMPDs that lack the BCR/ABL fusion gene requires careful correlation of morphologic findings in the blood and bone marrow with clinical and laboratory findings. In particular, the morphologic findings of the bone marrow biopsy figure prominently in the recognition of these diseases. Although the WHO criteria for the CMPDs incorporate recent knowledge and newer disease markers whenever possible, the criteria established by the PVSG remain integral components for the diagnosis of some diseases. To date, no major issues have been raised regarding the WHO proposals for the CMPDs.

Pathogenesis : thrombocytosis => PDGF => activation of bone marrow fibroblasts => collagen (various grade of myelofibrosis) and TGF-b (inhibition of HSC => chronic anemia). Although the origin of CML has been traced to dysregulated protein kinase, the product of the BCR/ABL oncogene^ref, and that of SMCD to of dysregulated receptor tyrosine kinases, c-kit, or PDGFRA^ref, the molecular basis of the 3 classic disorders, PV, ET, and IMF, which as group share more similarities with each other than with CML, has been more recalcitrant to solution. However, a major insight into the molecular basis for the enhanced myeloproliferation and clonal dominance that characterizes these disorders has now been reported; the answer is another kinase, and, best of all, it all makes sense! In recent issues of the Lancet^ref, Cancer Cell^ref, Nature^ref, and the New England Journal of Medicine^ref, a single, somatic mutation in the protein tyrosine kinase Janus kinase 2 (JAK2) appears responsible for many of the features of PV, ET, and IMF, a finding that promises to impact the diagnosis and treatment of patients with these disorders and to spur additional research into the origins of dysregulated cell growth and function. CMPDs share many features: the marrow is hypercellular and overproduces one or more of the formed elements of the blood in the absence of any apparent appropriate or pathologic stimulus; the exuberant hematopoiesis often also extends to one or more extramedullary sites. Based on studies using polymorphic genes, these disorders were shown to be clonal (excepted for some cases of ET)^ref, thought to arise in a single, multipotent hematopoietic progenitor or stem cell, which comes to dominate the marrow and blood. Hematopoietic progenitor cells from the marrow or peripheral blood display altered growth properties, proliferating in serum containing cultures in the absence of exogenous hematopoietic growth factors^ref. Marrow megakaryocytes are hyperplastic and variably dysplasia and are responsible for the myelofibrosis of these disorders, patients often display tendency toward thrombotic and hemorrhagic complications in addition to their signs and symptoms related to expansion of each hematopoietic lineage. However, compared with patients with CML, transformation to acute leukemia is far less common, especially in the absence of therapy with known mutagenic agents. But despite these clinical and pathologic features, the diagnosis of an individual patient with isolated erythrocytosis or thrombocytosis is often difficult, and their treatment is usually non-specific suppression of hematopoiesis. Although consistent chromosomal rearrangements have been the vital roadmap pointing investigators toward the molecular pathogenesis of CML and multiple types of acute myelogenous and lymphoid leukemia, these same clues have been neither abundant nor particularly informative in the study of CMPDs; without such signs, progress in understanding PV, ET, and IMF has been slow. Nevertheless, some successes have been reported. Instances of inherited, isolated erythrocytosis and pure thrombocytosis have been solved, traced to the loss of negative regulatory domain of the EPO-R^ref, to an abnormality in an inhibitory regulator (VHL protein) of the key transcription factor for EPO, hypoxia inducible factor 1^ref, to abnormal splicing of thrombopoietin (TPO) mRNA resulting in enhanced translational efficiency and high levels of the primary humoral regulator of platelet production^ref, or to 2 separate point mutations in the TPO receptor, c-Mpl^{ref1, ref2}. Nevertheless, none of these clinical instances are characterized by the clonal, panmyeloid myeloproliferation that characterizes patients with CMPDs. One of the most intriguing characteristics of PV has been endogenous erythroid colony (EEC) formation, in which erythroid progenitor cells obtained from the marrow or peripheral blood proliferate in semisolid, serum-containing cultures in the absence of exogenous EPO. Although initially described for patients with PV^ref, similar results can be demonstrated in significant proportion of patients with ET and IMFor in patients suffering from thrombotic diseases, such as the Budd Chiari syndrome but no evidence of an MPD, but never in healthy individuals. Thus, EEC formation has become a relatively useful, albeit cumbersome diagnostic test for myeloproliferative disorders. Unfortunately, the assay is technically demanding and is positive in only about 35% to 80% of patients with these diseases. Although initially believed to represent "EPO-independent" growth, subsequent studies revealed the phenomenon of EEC to represent hypersensitivity to the EPO present in the serum used in the cultures^ref. These findings suggested that abnormalities of the EPO receptor might underlie PV. Unfortunately, except for the description of familial erythrocytosis noted earlier, the EPO receptor has been found to be completely normal in all patients with bona fide PV^ref. Moreover, subsequent studies revealed that marrow and blood cells from patients with PV were hypersensitive not only to EPO but also to several other hematopoietic growth factors, including IL-3, SCF, GM-CSF, IGF-1, and TPO^{ref1, ref2, ref3, ref4}. These findings suggested that events downstream from receptor engagement might be responsible for EEC formation. Protein tyrosine phosphorylation is vital for transduction of growth-promoting signals for wide range of cellular mitogens. However, with the cloning of the EPO-R in 1989^ref, a conundrum emerged; unlike the receptors for well-characterized mitogens such as EGF, insulin, and PDGF, which all contain cytoplasmic tyrosine kinase domains, the cloned EPO receptor failed to display any recognizable intracellular signaling motifs. With the cloning of the GM-CSF, G-CSF, TPO, and multiple interleukin receptors, family of receptors, termed the type I cytokine receptors, was born, and mechanism for signaling was soon identified. Based on similarities with the interferon receptors, which use novel kinase/signal transduction system termed the JAK/STAT pathway^ref, it was quickly realized that the type I hematopoietic cytokine receptors also used JAKs and STATs to initiate signaling^ref. First cloned (1989) based on homology to other kinase domains^ref and playfully named JAK for "just another kinase," within years 60 publications appeared on the 4 members of the JAK family, number which has grown to > 2000 at present, attesting to the importance of these kinases in cytokine and growth factor signaling pathways. Certainly, proteins of such importance must have equally important names, leading to their being termed Janus kinases, for the Roman God of gates and passages. Structurally, JAKs are characterized by 3 critical domains, JH1, the kinase domain, resides near the carboxyl terminus of the protein. Immediately upstream of this lies the JH2 domain, which resembles the JH1 domain but is not an active kinase. Toward the amino terminus of JAKs lies FERM (4-point-one, Ezrin, Radixin, Moesin) homology domain, which is responsible for noncovalent binding to the "box1/motifs" present in the juxtamembrane cytoplasmic region of type I cytokine receptors. It is not hyperbole to state that for this type of cell surface receptor, all signaling begins with JAK. The first target of kinase action is the initiating receptor itself, which once tyrosine phosphorylated provides docking site for src homology (SH2)-bearing secondary signaling molecules, such as nascent transcription factors (STATs) adaptor molecules (Shc, Gab/IRS [Grb2 associated binder/insulin receptor substrate]), and kinase regulatory subunits (p85 phosphoinositol-3-kinase) among others. Much is known of the mechanism of JAK activation and regulation. Important insights into the effects of binding of cognate ligand to type I cytokine receptor came with elucidation of the tertiary structure of the EPOR in the absence and presence of an activating ligand^ref. In the unliganded state, the EPOR exists as dimer, with the transmembrane domains separated by 73 Å. Upon EPO binding, or that of an EPO mimetic peptide, large conformational shift ensues, bringing the cytoplasmic domains into closer juxtaposition, separated by 39 Å. It is believed that this close apposition of 2 prebound JAK2 molecules is responsible for their mutual activation by cross-phosphorylation. Numerous molecular mechanisms have been identified that regulate the activity of JAK kinases. In the unliganded state, perhaps the most important is the presence of the adjacent JH2 domain of the kinase. Expression of an isolated JAK2 JH1 kinase domain leads to its constitutive activity; addition of the pseudokinase JH2 domain greatly reduces the level of autoactivation^ref. Molecular modeling of the JH1and JH2 domains of JAK2, based on the dimeric structure of the fibroblast growth factor receptor kinase, indicates that a region of JH2 interacts with the activation loop of the kinase domain. Subsequent mutagenesis experiments have more directly shown that the region of amino acids 619 to 670 is required for JH2-induced inhibition of JH1 domain kinase activity^ref. Moreover, mutation of 2 separate residues in this region of the JH2 domain, Glu⁶⁶⁵ to Lys and Tyr⁵⁷⁰ to Phe, results in constitutive activity of the kinase^{ref1, ref2}. Additional levels of regulation of cytokine receptor activation exist, all of which operate at the postligand binding stage. Upon ligand binding, growth factor/receptor complexes are rapidly internalized through clathrinand ubiquitin-mediated processes^{ref1, ref2}; down-modulation of receptor expression thus reduces additional signaling. Another mechanism of reduced receptor signaling is removal of critical phosphotyrosine residues, mediated by receptor-associated phosphatases^ref. Of considerable interest for the field, genetic elimination of one such phosphatase, the hematopoietic cell phosphatase, now termed SHP1, results in a myeloproliferative disorder in the mouse, termed motheaten^ref. Finally, activation of receptor-bound JAKs invariably leads to induction of STAT activation; one group of genes rapidly induced by the transcriptional activity of STATs is the suppressor of cytokine signaling (SOCS) family. Upon transcription and translation, SOCS proteins bind to either JAKs or to the phosphotyrosine residues of the activated cytokine receptor, blocking further signaling^ref. Based on a growing understanding of the molecular mechanisms that initiate and regulate cytokine action, multiple investigators hypothesized that the chronic myeloproliferative diseases might represent disorders of cytokine signal transduction. Moreover, a strong link exists between activated signaling kinases and hematologic malignancies; Bcr-abl is dysregulated hematopoietic kinase wholly responsible for CML^ref, an internal tandem duplication in the FLT-3 receptor tyrosine kinase is seen in approximately 30% of blasts from patients with AML and contributes to the disease^ref, JAK2 contributes to the pathogenesis of the AML associated with t(9;12)(p24; p13), in which the transcription factor Tel is linked to JAK2, leading to its constitutive activation^ref, and the same partner is linked to the receptor tyrosine kinase PDGF-Rß in t(5;12)-associated CMML^ref. Because so many clues pointed toward a signaling defect in the CMPDs, and altered kinases contribute to a growing number of other myeloproliferative diseases, JAK2 became an attractive candidate gene for PV, ET and/or IMF. Among the 3 main subtypes of Bcr-Abl negative MPDs, PV is certainly the most straightforward model to utilize in order to define the underlying molecular pathogenesis since cytokine-independent terminal erythroid differentiation only depends on Epo signaling. In contrast, the main abnormality in ET and IMF involves MK proliferation and differentiation which are dependent upon not only Tpo, but also other cytokine signaling events. EPO and the EPOR are crucial for mature red blood cells formation. In the absence of EPO or EPOR, committed erythroid progenitors undergo apoptosis at the CFU-E and pro-erythroblast level^ref. Equally important is the Janus kinase JAK2 bound to EPOR and activated upon EPO binding to the receptor^ref. The knock out of JAK2 in mouse embryos is lethal at embryonic day 12.5 due to the absence of mature red cell formation in the fetal liver. Thus, EPO, EPOR and JAK2 are absolutely required for red blood cell formation. The number of EpoRs on the cell surface of erythroid precursor cells is low (typically 500-2000 receptors/cell) and the EPOR has apparently evolved to be poorly expressed at the cell surface. Thus, the EpoR must be efficiently activated by Epo in conditions where receptor numbers at the cell surface are low. One strategy by which this may be achieved is the ligand-independent dimerization of the EpoR on the cell surface^ref. Upon EPO binding, the dimer conformation changes in such a way that a rotation in the transmembrane and cytosolic juxtamembrane -helix is required to activate JAK2^ref. JAK2 is bound to the EPOR cytoplasmic domain in the absence of ligand activation. In fact, binding of the N-terminus FERM domain of JAK2 to sequences encompassing Box1 and Box2 of the EPOR cytoplasmic domain is required for trafficking the EPOR to the cell surface^ref. The presence of JAK2 promotes receptor trafficking to the Golgi and acquisition of a mature glycosylation pattern. Signaling by EPOR starts with JAK2 activation. 3 EPOR cytoplasmic hydrophobic residues located before Box1 seem to be crucial for activating JAK2^ref. These residues however are not required for the chaperone effect of JAK2 on EpoR trafficking. Upon receptor activation, JAK2 becomes phosphorylated at the activation loop Y1007, Y1008 and at many other Y residues, such as at Y570 (with inhibitory effect) and at Y221 and Y813 (with positive effects on signaling)^{ref1, ref2}. JAK2 also phosphorylates the EpoR cytoplasmic Y residues. All these p-Y residues in EpoR and JAK2 become recruitment sites for STAT5, STAT3, STAT1, SH-PTP1, CIS and shc/grb2. As a consequence, EpoR signaling activates STAT, MAP-kinase, PI3K and AKT. The biological consequences of this cascade of events are survival, proliferation and differentiation of erythroid progenitors.
• spontaneous activation of STAT3 was found in the granulocytes of 30% of PV patients^ref and an increased AKT phosphorylation was observed in erythroid progenitors after cytokine stimulation^ref. Blood cells of patients with CMPDs display constitutive activation of STAT3^ref. In erythroblasts derived from EEC, the anti-apoptotic mitochondrial protein Bcl-x_L regulated by Epo signaling through STAT5 activation was shown to be increased^ref. Moreover, different kinase inhibitors, including a JAK2 inhibitor are known to block EEC formation^ref. Similarly in IMF, spontaneous MK growth was blocked by a STAT5 dominant negative^ref
• JAK2 is involved in EpoR15 and MPL^ref trafficking from the endoplasmic reticulum to the cell membrane, and the processes involving MPL trafficking/maturation are altered in PV and IMF^ref.
• although not recurrent, a common cytogenetic abnormality in PV is a gain in 9p where the JAK2 gene is localized. In addition, loss of heterozygosity (LOH) of chromosome 9p was found in 30% of PV patients identifying a large genomic region of DNA as a target for the search for a potential candidate disease associated genetic defect^ref, which is due to a mitotic recombination and leads to a duplication of the mutated allele with a deletion of the normal allele^{ref1, ref2}, which correlates with the percentage of PV patients being homozygous for the V617F mutation. This implies that the cells homozygous for the mutation have a clonal advantage over the heterozygous and non-mutated cells. Usually, LOH is a mechanism of oncogenesis by which tumor suppressor genes are inactivated. To our knowledge, LOH has never been associated with gain of function mutations
A recurrent unique acquired clonal mutation in JAK2 was recently found by 5 different research teams. This mutation was observed in a majority of PV patients and a significant proportion of patients with other MPDs, including ET, IMF and some rare MPDs. Our group worked on the mechanisms leading to EEC formation and identified JAK2 as a potential candidate gene because JAK2 kinase inhibitors as well as a JAK2 siRNA inhibited EEC^ref. 3 other teams sequenced JAK2 as a consequence of kinase gene sequencing in MPDs^{ref1, ref2, ref3}. Finally, one team mapped the minimal region on chromosome 9p to a 6.2 Mbp interval that contains several genes including JAK2^ref. A unique valine to phenylalanine substitution at position 617 (V617F) in the pseudokinase JAK2 domain has been identified. The incidence of this mutation in PV patients is very high (78% of 506 patients), ranging from 65% to 97% depending on the study. These variations in percentage of patients involved is likely due to the clinical criteria used for diagnosis, either the Polycythemia Study Group (PSVG) or World Health Organization (WHO) classifications, and also the sensitivity of the assay used to detect JAK2 V617F. The JAK2 V617F mutation was, however, not found in some patients who were shown to have both monoclonal hematopoiesis and to form EEC, demonstrating that some cases of true PV are related to other molecular defects. Sequencing of genomic DNA from granulocytes was used in most studies to detect the mutation^{ref1, ref2, ref3, ref4, ref5}. The sensitivity of this technique is difficult to assess precisely, but it is likely that a threshold of 5% to 15% cells harboring the mutation is required for such an analysis to be positive. In one study, the mutation was detected in 97% of the PV samples (71/73) with a sensitive PCR assay^ref. The variable prevalence estimates of the JAK2 V617F in MPD patients therefore may be related to the fact that in some patients with JAK2 V617F, < 10% of granulocyte JAK2 alleles are JAK2 V617F alleles, suggesting that MPD patients could retain normal stem cell clones. These data are surprising since PV is considered to be a clonal disease with the majority of myeloid cells, including granulocytes, belonging to the clone. Further studies are, therefore, required to confirm these data. The JAK2 mutation is somatic and occurs at the level of a HSC. In all studies, the mutated JAK2 was found in myeloid cells, i.e., bone marrow cells, granulocytes, platelets and erythroblasts derived from CD34⁺ cells, but not in T cells. In addition, mutant JAK2 was found in hematopoietic colonies derived from hematopoietic progenitor cells^ref. In contrast, the mutation was not present in non-hematopoietic cells in most patients^{ref1, ref2}. Since familial MPDs have been reported, it was important to know if this mutation can be germ-line. Linkage analysis did not show co-segregation with the 9p locus suggesting that the V617F substitution occurred as a somatic mutation even in these families. The similar frequency of the V617F mutation in familial and sporadic PV is in favor of this assumption^ref. In one study, however, the mutation was detected in buccal swabs of 4% of the patients^ref. This finding may be due to contamination by granulocytes because in another study two patients positive for the mutation after analysis of buccal swabs were negative in cells within hair follicles^ref. The JAK2 V617F mutation has been found in other Bcr-Abl negative MPDs with a frequency of about 50% in IMF and more variably in ET (from 25% to 57%)^{ref1, ref2, ref3, ref4}. In many cases, the mutation was detected only at the threshold of the sequencing technique in ET. This may signify that hematopoiesis is not entirely clonal in many ET patients and that the mutation must be searched by more sensitive techniques or directly in platelets. The JAK2 mutation was recently found in other MPDs with a frequency of about 20% in atypical CML and unclassified MDS and at a lower frequency (2-3%) in HES, CMML or SM^ref. By contrast, the mutation was not detected in AML, except for AML secondary to an MPD or megakaryocytic leukemia^{ref1, ref2}. Thus, the presence of JAK2 V617F mutation provides a means of developing a new classification of MPDs that includes the majority of PV cases, a large fraction of cases of IMF and some cases of ET (the precise frequency remains to be determined) and some atypical MPDs. This mutation opens new avenues both for the diagnosis and the classification of MPDs. JAK kinases are composed of 4 conserved domains which include a N-terminal FERM domain required for interaction with cytokine receptors, an SH2 domain followed by a pseudokinase (JH2, from JAK-homology 2) and a C-terminal kinase (JH1, from JAK homology 1) domain. The V617F mutation is located in the JH2 pseudokinase domain of JAK2, which is involved in inhibition of the kinase activity^{ref1, ref2}. Several point mutations in the JH2 domain, like Y570F and E665K lead to constitutive JAK2 kinase activity^{ref1, ref2, ref3}. A model structure of JAK2 attributes an important function to amino acid residues around position 618 in the interaction between JH1 and JH2 and thus in the auto-inhibitory function of JH2^ref. It is likely that the V617F substitution leads to a gain of function as confirmed by two observations: when transfected into factor-dependent cell lines, the mutant JAK2 is auto-phosphorylated and STAT5, ERK/MAP kinase and PI3K/AKT are activated in the absence of any cytokines^ref. As a result, factor-dependent cells become hypersensitive to cytokine, with only a minority of cells acquiring cytokine independence^{ref1, ref2, ref3}. The effects of V617F JAK2 are even more striking in cell lines expressing the EpoR suggesting that this mutation has a more dramatic effect on the Epo/EpoR signaling^ref. At present, it is unknown whether the mutant JAK2 requires a cytokine receptor to scaffold and signal or whether it signals independently of receptor association. In JAK2-deficient cells, the mutant JAK2 could initiate a signal alone but it also transduces a signal from the EpoR upon Epo addition^ref, demonstrating that the mutant JAK2 binds to the EpoR cytoplasmic domain and transduces a ligand-dependent signal. Since both the mutant JAK2 and EpoR are expressed normally at low levels, it is likely that the mutant JAK2 uses and amplifies the signal normally triggered by EpoR. In the same cells or in retrovirally transduced Ba/F3 cells, the mutant JAK2 was able to promote trafficking of the EpoR to the cell-surface, albeit at a lower efficiency than a wild-type JAK2. Further experiments are still required to establish whether the mutant JAK2 also promotes down-regulation of activated EpoRs and whether the lower enhancement of trafficking is not due to this effect. The model would predict that the mutant JAK2 binds to EpoR while the receptor is in the endoplasmic compartment, the Golgi apparatus and the cell-surface. This binding promotes EpoR maturation but may also lead to a novel mechanism of cytokine receptor activation from the cytoplasmic domain (inside out signaling), which could occur before the EpoR reaches the surface. Bound JAK2 V617F would thus phosphorylate discrete tyrosine residues and would be autophosphorylated. These events would attract SH2-containing proteins to the EpoR-JAK2 complex and continuously activate STAT5, MAP-kinase, PI-3-kinase and AKT pathways. The dimeric conformation of the receptor might be switched on by this intracellular-to-extracellular activation. Additional experiments are needed to explore the ligand-independent EpoR tyrosine phosphorylation in cells that express the mutant JAK2. It remains possible that receptor signaling and phosphorylation might not require the "switch" residues of the cytoplasmic juxtamembrane domain, which are required for ligand-dependent activation of JAK2^ref. The present evidence suggests that the JAK2 V617F binds to the cytoplasmic domain of the intracellular and cell-surface EpoR and that this event promotes ligand-independent signaling. Since constitutively active EpoR mutants have been shown to produce in vivo not only erythrocytosis but also thrombocytosis^ref, it is tempting to speculate that the interaction between EpoR and the mutant JAK2 may contribute not only to the pathobiology of PV but also to ET. These data raise the intriguing question of how a single mutation might give rise to at least three different diseases (PV, ET and IMF) and also to some other atypical MPDs. Evidence from clinical, biological and pathology data indicates that ET, PV and IMF are related disorders. The evolution of ET to PV has been well documented, although some authors have stated these ET patients have been falsely diagnosed and represent in reality a forme fruste of PV. About 15% of ET patients progress to myelofibrosis and approximately 20% of PV progress to IMF (spent phase)^ref. The boundaries between IMF and ET are not well defined during the so-called pre-fibrotic form of IMF^ref. In addition, biological features of PV, i.e., EEC, high PRV1 mRNA in granulocytes are also found in ET and IMF^ref, while diminished membrane expression of Mpl (TpoR) by platelets has also been described in ET and IMF^ref. Recent results suggest that these criteria except Mpl expression correlate with the JAK2 V617F mutation. 3 different events may explain how a single mutation might give rise to different phenotypes: transformation of different hematopoietic stem cells/progenitor cells, different genetic backgrounds and additional somatic mutations. It is unlikely that PV, ET and IMF reflect the transformation of hematopoietic progenitors at different stages of differentiation. Indeed, there are many similarities between the biological properties induced by Bcr-Abl and V617F JAK2 abnormalities, both of which mimic cytokine signaling. There is evidence that Bcr-Abl exclusively transforms HSC and not later progenitor cells, because, in contrast to other oncogenes, it does not confer self-renewal properties to hematopoietic progenitors^ref. It is expected that the situation will be identical for the mutant JAK2, although it has been demonstrated that a constitutively active JAK2 can cooperate with Kit signaling to enhance self-renewal of multipotent progenitors^ref. The absence of the JAK2 mutation in T cells from PV does not rule out that the transformation event can occur in an HSC. Indeed, this observation is reminiscent of the situation in CML, where Bcr-Abl is hardly detected in T cells. The possibility that a spontaneously active JAK2 is deleterious to mature T cells cannot, however, be excluded. Further studies are required to directly demonstrate that the JAK2 mutation occurs at the level of the HSC. It seems likely that some as yet unknown factors might modulate the activity of the mutant JAK2. The V617F substitution is a subtle mutation that only changes the basal JAK2 activation, otherwise all the biological properties of JAK2 are maintained including its binding to cytokine receptors. However, it is not known if the mutant JAK2 requires receptor binding or spontaneously oligomerizes on the membrane or in the cytosol to induce signaling. The highly transforming TEL-JAK2 protein that has lost all the regulatory and cytokine receptor elements of JAK2 induces direct signaling by oligomerization. Thus, it seems likely that slight changes in the V617F JAK2 kinase activity might profoundly change the phenotype of the disease. Gene dosage and the presence of a normal JAK2 may influence the activity of the mutant. Indeed, normal JAK2 is capable of inhibiting STAT5 activation induced by the mutant. Furthermore, expression of normal JAK2 in a cell line made factor-independent by the mutant restores cytokine dependency^ref. The competition between the 2 JAK2s, wild type and mutated, is not directly due to inhibition of the mutant since wild type JAK2 does not inhibit auto-phosphorylation of the mutant^ref but may occur on their substrate(s) by competing for cytokine receptor binding. One might hypothesize that the level of kinase activity modulates the phenotype of the disease. The level of kinase activity could be modified by gene dosage, by polymorphisms of JAK2 or by cooperating genetic events, such as changes in a phosphatase or a SOCS gene, which encode for proteins that normally attenuate JAK2 signaling from cytokine receptors. We hypothesize that a low kinase activity would lead to ET, an intermediate level to PV and a very high level to IMF. Alternatively, the phenotype of the disease may be dependent on genetic events not related to the V617F JAK2 kinase activity. These different hypotheses can now be tested both in patients and in animal models of MPD. In a recent issue of the Lancet a group headed by Anthony Green identified somatic missense mutation in the JAK2 gene in 97% of patients with PV, 57% of patients with ET, and 50% of those with IMF, but in none of their 90 normal control samples^ref. A group headed by Gary Gilliland identified the same point mutation in 74% of patients with PV, 33% of patients with ET, and 35% of patients with IMF^ref. Moreover, those researchers failed to find any mutations in the activation loops or autoinhibitory domains of 85 other kinases in a Herculean sequencing effort. Likewise, 89% of patients with PV, 43% of those with ET, and 43% of patients with IMF bear the JAK2 mutation, but in none of their controls or in patients with secondary erythrocytosis^ref. And using genetic LOH to home in on 9p chromosome, Kralovics et al found that 83 of 128 patients with PV (65%), 13 of 23 with IMF (57%), and 21 of 93 patients with ET (23%) harbored the same JAK2 mutation^ref. Importantly, this group of investigators also found that patients who bore the mutation displayed poorer prognosis than those not carrying it, having a statistically greater likelihood of developing myelofibrosis, hemorrhage, and thrombosis. The majority of individuals with the Val⁶¹⁷Phe mutation in all studies carry one copy of the gene, although approximately one third were reported as homozygous in each of the studies, the result of mitotic cross-over and clonal expansion. Obviously, these findings are of great interest on several levels. Of note, of the growth factors to which hematopoietic progenitors are hypersensitive in CMPDs, EPO, SCF, GM-CSF, IL-3, TPO and IGF-1, all use JAK2 for signaling. More remarkable, precisely the same mutation was identified in all of the patients studied, despite there being very few related individuals in the study cohorts. The mutation, Val⁶¹⁷Phe, was acquired, as it was identified only in short-lived blood cells, not in lymphocytes or in buccal mucosal cells. As noted earlier, this site in JAK2 lies immediately adjacent to a region of the pseudokinase domain found necessary for inhibition of kinase activity, and, in a previously reported model structure of JH1-JH2 interaction, Val⁶¹⁷ appears to make close contact with the activation loop of the kinase domain (presumably stabilizing it) in the inactive conformation^ref. That this mutation contributes to the myeloproliferative state is virtually certain, based on several reported findings :
• in patients with mixed clonal and polyclonal progenitors, only erythroid progenitors carrying the mutation were able to grow in the absence of exogenous EPO^{ref1, ref2, ref3}
• a siRNA reagent that reduces JAK2 expression by 90% greatly reduced EEC formation^ref, a hallmark of these disorders
• expression of the mutant kinase in human kidney (293T) cells leads to its autophosphorylation (activation) and, when expressed in a factor-dependent cell line, BaF3/EPO, expression of the mutant, but not the wild-type, JAK2 kinase leads to EPO hypersensitivity and growth factor-independent cell survival^{ref1, ref2, ref3}
• the occurrence of individuals homozygous for the mutation further suggests that the cellular crossover events that lead to loss of heterozygosity result in a proliferative advantage, allowing their replacement of the clone of cells carrying a single mutant JAK2 locus. This supposition was confirmed by finding that, compared with cells expressing only the mutant JAK2, coexpression of both wild-type and mutant JAK2 reduced the total cellular autonomous JAK2 activity and led to reduced growth factor independent behavior^ref
• expression of Val⁶¹⁷Phe JAK2, but not the wild-type kinase, along with the EPO-R in a cell line led to EPO-independent STAT5 activation^ref
• introduction of Val⁶¹⁷Phe JAK2 into murine marrow cells, but not the wild-type kinase, leads to substantial erythrocytosis (hematocrit [Hct] ~60%) with splenomegaly, but not significant increases in granulocyte or platelet counts, following their transplantation into lethally irradiated recipients^ref
• the mutation induces IGF-1 hypersensitivity in cell lines and a defect in Mpl trafficking
Just as these reports are of great pathophysiologic interest, they carry tremendous potential for the clinical assessment and management of patients with chronic myeloproliferative disorders. 2 initial questions that arise include the following:
• do patients with the Val⁶¹⁷Phe JAK2 mutation differ from those without the mutation ?
• do patients with homozygous expression of the mutant kinase display more aggressive disease?
Although in the 3 cited studies^{ref1, ref2, ref3} patients with Val⁶¹⁷Phe were not reported to differ from patients with PV, ET, or IMF not having the mutation, as the number of such patients in those studies was relatively small, the presence or absence of EEC, or additional chromosomal abnormalities, or whether complications of the disease such as marrow fibrosis, bleeding or thrombosis, or transformation to acute leukemia are more common in patients with Val⁶¹⁷Phe, or in patients with copies of the mutant gene, must await larger studies. The finding of a consistent activating mutation in JAK2 in patients with CMPDs raises a number of additional questions. For example, how can one mutation give rise to disorders? Perhaps this reflects the accumulation of additional genetic alterations not yet detected, or is due to the mutation arising in different subpopulations of primitive hematopoietic cells or to a particular biochemical property of the mutant kinase that only allows its activity in cells of specific lineage(s). Does this finding presage explanations for other disorders whereby JAK kinases are of central signaling importance? Disorders of fat metabolism (the leptin receptor uses JAK2), of body growth (the human growth hormone receptor uses JAK2), or of the immune system (numerous interleukin receptors use JAK1 and JAK3) are candidate diseases that should garner future research attention in this context. What is the cause of familial myeloproliferative syndromes, or the cases of the disorder that do not carry the Val⁶¹⁷Phe mutation? Is the thrombotic diathesis in ET and the other disorders related to JAK2 activation? Recent evidence suggests that TPO, hormone that promotes the activation of JAK2^ref, acts to increase platelet aggregation and adhesion^{ref1, ref2}. And why do the counterregulatory measures that normally shut down signaling from hematopoietic growth factor receptors not operate in JAK2 mutant-bearing patients? Does this observation provide important insights into the molecular mechanism of SOCS inhibition of JAK signaling, or of the phosphatases that inactivate JAK? Although these and many additional questions arise from the finding of a single genetic alteration in the JAK2 kinase in patients with PV, ET, and IMF, the finding nevertheless represents a remarkable step forward in both the molecular pathogenesis and clinical evaluation of patients with the myeloproliferative disorders. Salient features stand out :
• the results have important implications for the diagnosis of patients with PV, ET, and IMF, allowing for a rapid, reliable, and unambiguous diagnosis to be made in a large number of patients
• the new information should spur development of targeted therapy against the mutant form of the kinase
• research can now focus on MPDs that are negative for the V617F mutation, which might constitute a distinctive entity
• further work is also needed to determine how a single mutation in JAK2 gives rise to three different diseases.
It is gratifying that a large number of observations, beginning with Dameshek^ref in 1951, expanded upon by Prchal and Axelrad^ref in 1974, by Adamson et al^ref in 1976, by Zanjani et al^ref in 1977, and by Krantz (Dai et al^{ref1, ref2}) and by Axelrad (Correa et al^ref) in the 1990s, are now explicable at the molecular level. Simply put, it all makes sense, and the results of future investigation in the field almost certainly hold many satisfying clinical and pathogenetic insights into the CMPDs.
A total of 168 of 190 female patients were informative for a clonality assay at the HUMARA locus; 80% of MMM, 75% of PV, and 67% of ET patients demonstrated clonal granulopoiesis. The JAK2V617F allele was detected by quantitative RT-PCR in 99% of PV, 72% of ET, and 39% of MMM. A correlation between clonality and JAK2V617F allelic ratio was demonstrated for PV (P < .001) but not for ET or MMM (both P > .6). These data suggest that acquisition of the JAK2V617F mutation may be sufficient for the development of PV, but additional genetic events are necessary in ET and MMM. In addition, some ET and MMM patients with clonal granulopoiesis have somatic mutations other than JAK2V617F^ref.
Varieties :
• polycythemia (rubra) vera (PV) / myelopathic, primary or splenomegalic polycythemia / Vaquez-Osler disease / erythremia (Vaquez H. Sur une forme spéciale de cyanose s'accompagnant d'hyperglobulie excessive et persistante. C.Rend.Soc.Biol.44:384-388 (1892))
• Ayerza's disease : a form of PV associated with sclerosis of the pulmonary artery, marked by chronic cyanosis, dyspnea, bronchitis, bronchiectasis, hepatomegaly and splenomegaly, and hyperplasia of bone marrow.
Epidemiology : incidence = 0.8-1 case every 100,000 inhabitants per year (rangin from 2 cases every 1,000,000 inhabitants in Japan to 13 per 1,000,000 in Australia); male-to-female ratio 2:1, age 15-90 years (median 60 years)
Aetiology : familial PV : LOH of chromosomes 20, 9, 10 and 11 in granulocytes and HSC
Pathogenesis : Janus kinase 2 (JAK2)^V617F mutation is present in cells belonging to each of the myeloid lineages and was also present in B- and T-lymphocytes in a subpopulation of patients with PV. The proportion of hematopoietic cells expressing the JAK2^V617F mutation decreased after differentiation of CD34⁺ cells in vitro in the presence of optimal concentrations of SCF, IL-3, IL-6 and Epo. These data suggest that the JAK2^V617F mutation may not provide a proliferative and/or survival advantage for the abnormal PV clone. While the JAK2^V617F mutation plays an important role in the biological origins of PV, it is likely not the sole event leading to PV^ref. V617F⁺ BFU-E were more frequent in PV patients compared to ET patients (p=0.022) and, strikingly, V617F-homozygous BFU-E were detected in all 17 PV patients, but in none of the ET patients (p<0.0001). Moreover, mutation-homozygous cells were present in 2 ET patients after polycythemic transformation^ref.
Symptoms & signs :
• due to hypervolemia and hyperviscosity: secondary systemic arterial hypertension (3%), headache, vertigo, tinnitus, blurred vision (scotomas, diplopia) due to ingorged retinal veins at fundus oculi examination
• due to coagulopathy (40% of deaths) :
• due to thrombotic diathesis (thrombocytopathy : platelets produce more TxA₂) :
• venous thrombosis (Budd-Chiari syndrome, portal vein thrombosis, pulmonary thrombosis)
• Mosse's syndrome : PV with cirrhosis of the liver
• arterial thrombosis (15-20% at diagnosis) : coronary thrombosis (5.5 cases every 100 patients per year), cerebral thrombosis (1.40 cases every 100 patients per year), finger thrombosis
Predictive factors are age > 65, previous thrombosis, smoking, hypertension and CHF (see ECLAP study)
• due to hemorrhagic diathesis : epistaxis, capillary fragility => ecchymoses, gastrointestinal hemorrhages
• due to excess of histamine : pruritus worsened after hot shower, peptic ulcer
• due to hyperoxemia :
• erythrosis : reddish or purplish discoloration of the skin and mucous membranes; rubeosis of face and hand palms
• pletoric facies : the skin of the face is often ruddy, swollen, and cyanotic
• hypermetabolism : weight loss and hyperuricemia
• due to myeloid metaplasia => splenomegaly (75%) : splenic infarctions, dysphagia ("small stomach syndrome") => cachexia
Stages :
• asymptomatic stage : erythrocytosis
• erythrocytotic stage : erythrocytosis (thrombocytosis, leukocytosis), splenomegaly, thrombosis, hemorrhage, pruritus
• spent stage : stable hematological parameters which don't require therapy
• myeloid metaplasia : anemia, thrombocytosis/thrombocytopenia, leukocytosis, incremental splenomegaly, fever, weight loss
• acute leukemia : AML, other leukemias
Laboratory examinations :
• first level :
• CBC : HGB = 18-20 g/dl, RBC = 7-10 x 10¹²/ml; RBC mass >= 36 ml/kg in males and > 32 ml/kg in females; WBC = 12,000-25,000/ml with neutrophilia, myelocytes and metamyelocytes in advanced stages; basophilia (66%), normal or mildly increased LAP; thrombocytosis (50% : 400,000-800,000/ml => hyperkalemia; reduction of platelet factor 3, c-MPL (also in 50% of ET and MMM), and TPO); normal bleeding time, INR, aPTT and fibrinogen
• arterial blood gases (PO₂)
• ferritin
• leukocytosis => increase in [transcobalamine-II] => serum vitamin B₁₂> 900 pg/ml and binding capacity unit of vitamin B₁₂ > 2200 pg/ml
• folates
• serum creatinine
• liver function tests (azotemia, SGOT, SGPT, gGT, alkaline phosphatase)
• uricemia
• abdominal echography
• reduced serum and urine EPO
• second level :
• BMAB/BMTB
• cytogenetics : 20% at diagnosis, 45% during myelosuppressive treatment, 80% at 10-yrs (del20q (also in 1-2 of AML and in 20% of MDS), del12q, +8, +9, duplications of 1q mark poor prognosis; -7 and -5 in advanced PV; del20q in ³²P- or radiotherapy-treated patients)
• BFU-E colonies (endogenous erythroid colonies (EEC))
• O₂ dissociation curve
• functional respiratory tests
• chest X-rays
• echocardiography
• EPO-R mutations
• additional :
• ESR, PCR, serum protein electrophoresis, IgG, IgA, IgM, sideremia, LDH, total cholesterol, urinalysis
• clonality assays : a significant minority of patients exhibit polyclonal haematopoiesis with polyclonal patterns in granulocytes/platelets. Female patients with polyclonal haematopoiesis differ from those with clonal haematopoiesis in terms of age and platelet count and possibly in their requirements for treatment^ref.
• increased serum and urinary histamine
• PRV-1 mRNA increased by 8-64-folds in granulocytes (also in ET)
When splenomegaly is absent, leukocytosis or thrombocytosis is a surrogate criterion for diagnosis. Pearson criteria :
• major criteria :
• A1 : raised red cell mass with ⁵¹Cr release assay (> 25% above predicted, or hematocrit > 0.60 in males or > 0.56 in females) (these hematocrit values are invariably associated with a raised red cell mass in an adult population)
• A2 : absence of causes of secondary erythrocytosis (arterial oxygen saturation > 92% and no elevation of serum erythropoietin) (note that it is possible in rare cases for PV to coexist with a cause of secondary erythrocytosis)
• A3 : palpable splenomegaly
• A4 : presence of Janus kinase 2 (JAK2) V617F mutation or other cytogenetic abnormality (excluding BCR-ABL) in hemopoietic cells
• minor criteria :
• B1 : thrombocytosis (platelets > 400,000/mL)
• B2 : neutrophilia (neutrophils > 10,000/mL; > 12,500/mL in smokers)
• B3 : radiological splenomegaly / high LAP
• B4 : endogenous erythroid colonies or low serum erythropoietin / increased serum vitamin B₁₂ or BCUB
A1 + A2 + either another A or 2 B criteria is required for a diagnosis of PV.
The Polycythemia Vera Study Group (PVSG) first developed a set of rigorous criteria for the diagnosis of polycythemia vera (PV) in the 1970s. A number of the original criteria have been superceded by the development of newer tests^ref. The JAK2 mutation is present in most patients with PV, but it is also present in half of those with ET or idiopathic myelofibrosis (IMF). Establishing the presence of an absolute erythrocytosis (usually by demonstrating a raised red cell mass) therefore remains important for distinguishing PV from other MPDs positive for the JAK2 mutation. It is likely that, with the establishment of PCR-based methods for detecting the JAK2 V617F mutation, this tool will become routinely available in laboratories offering molecular diagnostic services. This will allow most patients to be diagnosed on the basis of a raised red cell mass, normal O2 saturation (or absence of a raised serum erythropoietin) and positive allele-specific PCR for JAK2 V617F. It is also important to emphasize that the V617F mutation has been identified in a small proportion of other hematological malignancies (for example, acute myeloid leukemia, chronic myelomonocytic leukemia and myelodysplasia). However, none of these disorders is associated with a raised red cell mass and clinical distinction from PV is rarely an issue. The reported prevalence of PV patients lacking the JAK2 mutation has varied from 70% to nearly 100%. The lower end of this range is likely to reflect the use of relatively insensitive diagnostic methods and/or less rigorous diagnostic criteria. When investigating V617F^- patients who appear to have PV, it is therefore particularly important to exclude secondary causes of erythrocytosis by looking for a suggestive clinical history, reduced arterial O₂ saturation, raised serum erythropoietin levels or renal abnormalities on ultrasound. However, the existence of even a small number of V617F^- PV patients justifies the continued use of the B criteria for the time being. Although marrow histology has been included as a minor criterion in the WHO classification (Pierre R, Imbert M, Thiele J, Vardiman JW, Brunning RD, Flandrin G. Polycythaemia vera. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. WHO Classification of Tumours, Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2001:32-34), the role of bone marrow biopsy in the evaluation of PV remains controversial because of inter-observer variation in quantifying erythroid and megakaryocytic proliferation. It has recently been demonstrated that V617F⁺ patients with essential thrombocythemia (ET) exhibit many similarities with PV^ref. These results suggest that the 2 disorders may be better viewed as a continuum with the degree of erythrocytosis determined by physiological or genetic modifiers. This model suggests that, in patients at the thrombocythemia end of the continuum, the effects of the V617F mutation on erythropoiesis are constrained by physiological mechanisms, including erythropoietin suppression and depleted iron stores, or by genetic modifiers, either acquired or constitutional. Acquisition of homozygosity for the V617F mutation may favor development of a polycythemic phenotype since homozygosity for mutant JAK2 occurs in approximately 30% of patients with PV, but is rare in ET^{ref1, ref2, ref3, ref4}. Gender may influence presentation of V617F⁺ disease, since PV is more common in men^{ref1, ref2}, whereas V617F-positive thrombocythemia is more common in women. Other genetic modifiers are likely to influence disease phenotype in a manner analogous to polygenic predisposition to many common diseases, a concept which may also account for the variability in platelet count, white cell count and marrow fibrosis in V617F⁺ patients. Over the past few years several potentially useful markers have been described^{ref1, ref2, ref3}, including increased expression of PRV1 and BCL-X_L, and diminished platelet MPL expression, but none are available outside specialized centers and all are likely to become redundant with the advent of testing for the JAK2 mutation.
Prognosis of untreated PV in the first half of the 20th century was dismal with a median survival of approximately 18 months. However, the advent of venesection, together with antithrombotic and cytoreductive agents, has greatly improved the outlook. Patients with PV have a 1.6-fold increased mortality compared with the general population (15-yr OS = 65%)^ref.
• cardiovascular :
• arterial thrombosis is particularly prevalent in older patients
• venous thrombosis (cerebral thrombosis, embolism, splanchic thrombosis)
• hemorrhage :
• intestinal
• cerebral
• neoplasia may account for reduced survival of younger patients :
• acute myeloid leukemia (11%  if treated with 32P vs. 9% if treated with radiotherapy vs. < 1% in controls)
• breast carcinoma
• colon carcinoma
• lung carcinoma
• MDS
• MMM
The clinical epidemiology of PV has been reviewed recently^{ref1, ref2}. 2 large Italian studies (GISP and ECLAP) have provided important insights into the natural history of PV. In both studies, overall mortality was approximately 3 per 100 patients/year. Compared to age- and sex-matched controls, the risk of death from cardiovascular causes was modestly increased (1.4-fold), whereas the risk of death from hematological transformation was markedly increased (36.1-fold)^ref, and major thrombosis also occurred in approximately 3 per 100 patients/year. Age over 65 years and history of a prior thrombosis were the most important risk factors identified for the development of additional thrombotic events. Duration of disease and age > 70 years were associated with an increased risk of developing myelofibrosis with splenic myeloid metaplasia (20-25%) and erythroleukemia (M6 AML) (1%), respectively.
Therapy :
• phlebotomy / venesection to maintain hematocrit < 0.45 based on the seminal studies by Pearson and Wetherley-Mein^ref
• low-dose ASA (unless contraindicated : avoid aspirin while platelet count > 1500 x 10⁹/L)
• manage reversible thrombotic risk factors aggressively (e.g., smoking, hypertension, hypercholesterolemia, obesity)
• consider cytoreduction if :
• patient intolerant of venesection
• thrombocytosis develops
• symptomatic or progressive splenomegaly
Choice of cytoreductive therapy :
• < 40 years - IFN-a_2b (anagrelide plus venesection may be useful in patients intolerant of or resistant to hydroxyurea and IFN-a)
• > 40 years - hydroxyurea (anagrelide plus venesectionmay be useful in patients intolerant of or resistant to hydroxyurea and IFN-a; consider ³²P or intermittent use of busulfan if patient is very elderly and outpatient attendance is impractical)
• other/supportive :
• H₂ antagonists
• for splenomegaly and thrombocytosis : anagrelide, splenectomy, hydroxyurea and IFN-a_2b
• for erythromelalgia : ASA or as above; combination of ASA, 300 mg 3 times daily, and dipyridamole, 75 mg 3 times daily, failed to reduce the rate of thrombosis and was associated with an increased rate of hemorrhage
• for hyperuricemia: allopurinol if symptomatic or before therapy with hydroxyurea or IFN-a_2b
• surgical prophylaxis : phlebotomy or postpone surgery
• antiplatelet drugs (when hypertension is effectively controlled : do not use diuretics, but rather ACEI) : 100 mg/day reduces thrombotic events by 58% after 6 months. RR for hemorrhages = 1.82 (major hemorrhages = 1.62)
• autologous PBSC collection is feasible in PV several years after diagnosis. Organomegaly and MF are not absolute contraindications for collection. Discontinuing myelosuppresion for 2 weeks before mobilization appears sufficient to collect adequate numbers of CD34⁺ progenitors^ref.
Over the past 25 years, several clinical trials have investigated the optimal management of PV, and these have been reviewed recently^ref.
• in the 1970s, the PVSG-01 study randomized 431 patients to venesection alone, phosphorus 32 (³²P) + venesectionor chlorambucil + venesection. Patients treated with venesection alone had a better median survival (13.9 years) than those in the other 2 arms (11.8 and 8.9 years, respectively). However, patients in the venesection arm showed an excess mortality within the first 2-4 years mainly caused by thrombotic events, whereas patients in the other 2 arms subsequently showed a higher rate of leukemia and other malignancies. The PVSG-05 and 08 trials were designed to try to reduce the major causes of death identified in the PVSG-01 trial. In an attempt to lower the thrombotic events seen in the venesection-only arm, the PVSG-05 study reduced the target hematocrit to 0.45 and compared venesection plus ASA (900 mg/day) and dipyridamole (225 mg/day) with venesection plus ³²P. The study was terminated early (median follow-up 1.2 years) because patients in the venesection/ aspirin/dipyridamole arm exhibited a high incidence of major gastrointestinal hemorrhage (presumably reflecting the high doses of aspirin and dipyridamole) and no reduction in thrombotic events.
• the PVSG-08 study compared 51 patients receiving hydroxyurea for a median period of 8.6 years with historical controls from the venesection-only arm of PVSG-01. Although no significant differences were observed, the hydroxyurea group tended to suffer fewer total deaths (39% vs 55%), less myelofibrosis (8% vs 13%), and more acute leukemia (10% vs 4%). However, the analysis excluded any patient in the venesection-only arm whose disease required cytoreductive therapy, and so this arm was highly selected and likely to represent a good-prognosis subgroup.
• in Europe, the EORTC compared 293 patients randomized to busulfan or ³²P and followed for a median of 8 years. Survival at 10 years was significantly better in the busulfan arm mainly because of a lower incidence of vascular deaths.
• Najean and colleagues compared 292 patients below the age of 65 years randomized to pipobroman or hydroxyurea with a follow-up of 16 years. No significant difference was observed in the incidence of death, thrombosis or acute leukemia. However, patients treated with hydroxyurea exhibited a high rate of developing myelofibrosis, possibly related to the fact that platelet counts were significantly less well controlled
• the French group also compared 461 patients over 65 years of age randomized to ³²P alone or in combination with hydroxyurea and who were followed for up to 16 years. No difference in overall survival was observed, but an increase in hematological and other malignancies was noted in patients receiving both ³²P and hydroxyurea.
• the most recent randomized study (ECLAP) compared 518 patients randomized to receive ASA (100 mg/day) or no aspirin^ref. There was no difference in overall mortality, cardiovascular mortality or major bleeding. However, treatment with aspirin significantly reduced the risk of the combined end point of nonfatal myocardial infarction, nonfatal stroke, major venous thrombosis and death from cardiovascular causes. These results suggest that low-dose aspirin can reduce thrombotic complications in patients with PV
• although there are no randomized data, IFN-a_2b and anagrelide may have a role in subgroups of patients with PV. Interferon is often favored in young patients, since it is not thought to be leukemogenic or teratogenic, and remains the treatment of choice when cytoreduction is required during pregnancy. Its main drawbacks are that it is expensive, requires parenteral administration and produces considerable side effects. However, the development of pegylated interferon allows once-weekly dosage. Anagrelide specifically reduces the platelet count (and the hemoglobin in some patients) without affecting the white cell count. Its efficacy and side effects are discussed below, but there is no suggestion that it is leukemogenic.
The ECLAP study^ref, together with primary and secondary prevention studies in patients without PV^{ref1, ref2}, argue strongly for the use of low-dose aspirin in all patients lacking a clear contraindication. It also seems sensible for other predictors of cardiovascular morbidity (systemic arterial hypertension, diabetes mellitus, smoking, hypercholesterolemia, congestive heart failure) to be managed aggressively^ref. Cytoreductive therapy is clearly indicated if patients are intolerant of venesection or if they develop symptomatic splenomegaly thought to be due to disease progression. The treatment of thrombocytosis is more controversial. It has been suggested that it is unnecessary to control the platelet count in patients with PV, particularly since a clear relationship between platelet number and thrombosis has not been established^ref. However, several lines of evidence suggest that controlling the platelet count is likely to be beneficial for patients with PV:
• the ECLAP study^ref strongly suggests that platelets contribute to thrombosis in patients with PV.
• the beneficial effect of controlling the platelet count has been demonstrated by Barbui and co-workers^ref in the closely related disorder ET.
• treatment with cytoreductive agents reduced thrombotic events in patients with PV in the PVSG-01, -05 and -08 studies. The incidence of thromboses in the venesection only arm of PVSG-01 was 32.8% after 7.5 years, whereas among patients treated with hydroxyurea in PVSG-08 the incidence was 9.8% at the same time point. Although the target hematocrit in the early stages of the PVSG-01 trial would now be viewed as too high, in a separate study in which hematocrits and platelets were more tightly controlled at < 0.45 and < 400 x 10⁹/L, respectively, the thrombosis rate was reduced to 5.6% after 7-8 years^ref
• 2 French trials^{ref1, ref2} have produced data consistent with the idea that a persistently raised platelet count is associated with an increased risk of developing myelofibrosis. It has been suggested that the development of myelofibrosis does not have adverse prognostic implications, but the difficulty with this assertion is the variable definition of myelofibrosis. Some patients with increased reticulin as an isolated finding can have prolonged survival, but it seems probable that the development of clinically overt myelofibrosis in a patient with PV is associated with a poor prognosis.
No one of these arguments is definitive on its own, but the current balance of evidence suggests that it is sensible to control the platelet count in most patients with PV, although in young patients (< 40 years) with a low risk of thrombosis, it may be reasonable to use aspirin alone. In most patients, hydroxyurea represents the first-line cytoreductive agent. Although doubts remain about possible long-term leukemogenicity, there are no robust data which show that, when used as a single agent, it increases the risk of leukemia. Interferon- is useful in young patients and during pregnancy. Anagrelide may be useful for control of the platelet count when hydroxyurea and interferon-a are unsuitable. In very elderly patients for whom regular clinic attendance is impractical, ³²P or intermittent busulfan may still have a place.
Risk-stratified therapy :
• low-risk (age < 60 yrs, PLT < 1,500,000/mL, no thrombosis) : periodical phlebotomy until HCT < 45% in males and 42% in females (adverse events : hyposideremia and increase of thrombocytosis; decreased protection from thrombotic events in the first 3 years of treatment, but same OS = 12-13 yrs)
• intermediate-risk (age < 60 yrs, PLT > 1,500,000/mL or cardiovascular risk factors) : periodical phlebotomy + ASA + platelet-lowering agent (pipobroman : the 10-year risk of acute leukemia is 5%^ref)
• high-risk (age > 60 yrs or thrombosis) : phlebotomy + myelosuppressive agents (hydroxyurea, IFN-a_2b and PUVA: alkylating agents and ³²P radiometabolic therapy is no longer used due to risk of transformation to erythroleukemia (M6 AML))
Protocols :
• erythroapheresis + acetylsalicylic acid 100 mg/day to keep HCT < 45%
• if phlebotomy not tolerated, progression, high risk of thromboses or hemorrhages, increased LDH, splenomegaly > 15 cm in the highest diameter : cytoreductive therapy :
• age < 60 years : IFN-a_2b (1.5 MIU on Monday for 1 week => 3 MIU on Monday-Wednesday-Friday (PEG-IFN-a) adjusting the dose on response (up to 9 MIU))
• age > 60 years : hydroxyurea OR (if untolerant) busulfan
Follow-up :
• sTfR at diagnosis and every 6 months during follow-up
• bone marrow trephine biopsy (BMTB) every 2 years
• true or essential thrombocythemia (ET)

Epidemiology : first described by Epstein and Goedel in 1934; incidence = 1-2.5 cases per 100,000 inhabitants per year; female-male ratio = 1.6:1; age 40-70 years (median = 50 years), although since the introduction of routine assessment of platelet counts, more patients are being diagnosed at an earlier age
Aetiology : ?
Pathogenesis : analysis of 776 patients with ET demonstrated that just over half had the JAK2 V617F mutation, and this divided patients into 2 biologically distinct subgroups^ref.
• V617F⁺ ET patients displayed multiple features resembling PV, with significantly higher hemoglobin levels, neutrophil counts, bone marrow erythropoiesis and granulopoiesis, more venous thromboses and a higher incidence of polycythemic transformation. In addition, V617F⁺ patients had lower serum erythropoietin and ferritin levels than V617F^- patients with ET. These results imply that V617F⁺ thrombocythemia and polycythemia may be better viewed as a continuum, and not as 2 distinct entities.
• V617F^- ET patients do nonetheless exhibit features characteristic of an MPD, including cytogenetic abnormalities, hypercellular bone marrow with abnormal megakaryocyte morphology, PRV1 overexpression, growth of erythropoietin-independent erythroid colonies, and a risk of myelofibrotic or leukemic transformation^ref. These facts suggest that ET should be subclassified as either V617F⁺ or V617F^- ET, both bona fide MPDs, although future studies may prove V617F^- ET to be biologically heterogeneous.
These data are consistent with two models for the relationship between V617F^- and V617F⁺ thrombocythemia :
• the first model postulates that the JAK2 V617F mutation is neither necessary nor sufficient for the development of an MPD phenotype, but occurs in a sub-clone with a preceding unknown mutation^ref. Under such a model, the first, unknown mutation gives rise to V617F^- thrombocythemia, which subsequently evolves into V617F⁺ thrombocythemia with acquisition of the JAK2 mutation. Circumstantial evidence in favor of the model comes from the finding that the JAK2 mutation may be present in some but not all individuals with a familial MPD
• the second model postulates that V617F^- and V617F⁺ ET are different diseases
Data from analysis of a large ET cohort favors the second model, because there were no significant differences between V617F⁺ and V617F^- ET patients in disease duration or in the frequency of features associated with advanced disease^ref. Moreover, transplantation experiments show that expression of mutant JAK2 in bone marrow cells is sufficient to cause short-latency erythrocytosis in mice^ref.
Symptoms & signs : mild splenomegaly (60%), hepatomegaly (40%), splenic infarctions => splenic atrophy (acquired hyposplenism), predisposition to both thromboembolic, and more rarely, haemorrhagic complications as platelet are uneffective (> 1,000,000/ml cause acquired vWD); abortion in 43% of pregnants, erythromelalgia, headache, paresthesia, instable gait, dysarthria, vertigo, scotomas, amaurosis, syncope.
Laboratory examinations :
• A1 : platelet count > 600,000/ml (usually < 1,500,000/ml) for at least 2 months
• A2 : acquired Janus kinase 2 (JAK2) mutation. Using allele-specific PCR on peripheral blood granulocytes the V617F JAK2 mutation is found in approximately 50% of patients with ET^{ref1, ref2}. A lower proportion of mutation-positive patients was identified by sequencing^{ref1, ref2, ref3, ref4}, an observation that reflects the lower sensitivity of the method and the fact that a variable proportion of granulocytes are derived from the malignant clone. In the presence of the JAK2 mutation, a diagnosis of ET still requires excluding other myeloproliferative and related disorders. In the absence of the JAK2 mutation, it is also important to exclude reactive thrombocytosis and iron deficiency.
• B1 : no cause for a reactive thrombocytosis
• e.g., normal inflammatory indices
• B2 : no evidence of iron deficiency
• stainable iron in the marrow or normal ferritinemia and MCV (if these measurements suggest iron deficiency, PV cannot be excluded unless a trial of iron therapy fails to increase the red cell mass into the polycythemic range (patients need to be monitored closely since a rapid rise in hematocrit can precipitate thrombosis))
• B3 : no evidence of PV
• hematocrit < midpoint of normal range (0.46) or normal red cell mass (male < 36 ml/kg, female < 32 ml/kg) in presence of normal iron stores
• B4 : no evidence of chronic myeloid leukemia (rare patients have chronic myeloid leukemia coexisting with ET or other myeloproliferative diseases^ref)
• no Philadelphia chromosome or bcr-abl gene rearrangement
• B5 : no evidence of myelofibrosis
• no collagen fibrosis and <= grade 2 reticulin fibrosis (using 0-4 scale) (collagen fibrosis in the trephine biopsy is < 33% with absence of leucoerythroblastic features on the blood film)
• B6 : no evidence of a myelodysplastic syndrome (approximately 5% of patients with MDS carry the V617F JAK2 mutation)
• no significant dysplasia
• no cytogenetic abnormalities suggestive of myelodysplasia
Diagnosis of ET requires A1+A2+B3-6 (V617F⁺ ET) or A1+B1-6 (V617F^- ET). Nonetheless, in principle, ET remains a diagnosis of exclusion^ref.
A number of attempts to develop other new diagnostic tests have been reviewed recently^ref.
Additional criteria include :
• increased MPV and PDW
• cytogenetics : del(13q22), +8, +9, dup(1), del(20q11), +21
• down-regulation of PGD₂ and adrenergic receptors => defects in ADP- and adrenaline- (but not collagen-)induced platelet aggregation and adhesion => enlengthened bleeding time (50%) and thrombosis/hemorrhage
• neutrophilic leukocytosis (12,000-20,000/ml) => increase in [transcobalamine-II] => serum vitamin B₁₂> 900 pg/ml and binding capacity unit of vitamin B₁₂ > 2200 pg/ml (33%); basophilia and eosinophilia, normal or increased LAP
• bone marrow trephine biopsy (BMTB) histology has also been suggested as a useful positive diagnostic criterion. It has been reported that patients with ET can be divided into histologically distinct subgroups (true ET, prefibrotic myelofibrosis, and early overt myelofibrosis) with different prognoses^ref. However, megakaryocyte morphology is notoriously difficult to assess in a reproducible manner and detailed studies of inter-observer variation are lacking. It is, therefore, not yet clear whether this sort of histological classification is robust enough to be applied widely outside specialized centers, and the inclusion of trephine biopsy histology in the WHO criteria is regarded by many as controversial.
• thrombopoietin (TPO)-independent megakaryocyte colonies or erythropoietin-independent erythroid colonies (EECs) : both assays are laborious, difficult to standardize and only available in a few specialized centers. Moreover, TPO-independent colonies have been reported in patients with reactive thrombocytosis
• serum TPO levels are not helpful since they are normal or raised in a variety of conditions associated with a high platelet count, including reactive thrombocytosis, ET and other MPDs
• reduced expression of MPL, the TPO receptor, has been reported in patients with ET by some groups but not by others
• increased levels of PRV1 mRNA in peripheral bloods granulocytes has been reported in approximately 50% of patients with ET
• clonality assays : X chromosome inactivation patterns are of limited use as a diagnostic test for 2 main reasons. Firstly, a clonal pattern (skewed granulocytes and balanced T cells) is found in a significant proportion of normal elderly women and therefore lacks specificity. Secondly, approximately 50% of patients who fulfill clinical criteria for ET have a balanced pattern in their peripheral blood granulocytes suggesting that the majority of these cells are polyclonal. Therefore, the presence of a polyclonal pattern (balanced granulocytes and T cells) in younger women cannot be used to exclude the diagnosis of ET. A significant minority of patients exhibit polyclonal haematopoiesis with polyclonal patterns in granulocytes/platelets. Female patients with polyclonal haematopoiesis differ from those with clonal haematopoiesis in terms of age and platelet count and possibly in their requirements for treatment^{ref1, ref2, ref3, ref4}. A large proportion of patients with a diagnosis of ET according to PVSG criteria do not have a monoclonal disorder and may be at lower risk of thrombotic complications^ref

Clonality in ET and PV studied by the HUMARA assay :

study	cases	age in years	CD3 separation methods	definition of clonality	cut-off level of clonality	homozygous	treated	clonal	noninformative	clonal controls	complications
El-Kassarref	26	29-81	no separation	allele ratios	80%	10	1/16	3/16	9/16	5/23	NA
Championref	8	60-88	magnetic beads	Asimakopoulos equation	> 50% (Rg < 0.33 and Rt = 1.0)	0	NA	26/41	1/41	15/65	NA
El-Kassarref	17	13-70	magnetic beads	allele ratios	80%	0	11/17	9/17	4/17	4/44	5/9 clonal 2/4 polyclonal
El-Kassarref	53	13-80	magnetic beads	allele ratios	80%	4	18/30	31/49	5/49	NA	not conclusive
Harrisonref	43	11-89	magnetic beads	allele ratios	80%	0	NA	10/43	20/43	0/9	6/10 clonal 2/13 polyclonal
Mitterbauerref	23	27-88	NA	allele ratios	75% (allele ratio > 3:1)	3	NA	17/20	0/20	22/242	NA
Chiusoloref	40	20-63	magnetic beads	allele ratios	75% (allele ratio > 3:1)	0	34/40	17/40	8/40	NA	7/17 clonal 1/15 polyclonal
Shihref	73	18-92	RBC rosetting	Asimakopoulos equation	> 50% (Rg < 0.33 and Rt = 1.0)	9	NA	42/64	10/64	12/43	NA
Shihref	89	15-92	RBC rosetting	Asimakopoulos equation	> 50% (Rg < 0.33 and Rt = 1.0)	10	NA	54/79	10/79	NA	25/45 clonal 1/15 polyclonal
Zamoraref	44	32-91	magnetic beads	allele ratios	75% (allele ratio > 3:1)	6	21/44	11/38	7/38	3/64	3/11 clonal  4/20 polyclonal

Differential diagnosis : reactive thrombocytosis
Prognosis : several cohort studies have reported no significant reduction of life expectancy compared to the general population (15-yr OS = 73%, SMR = 1)^{ref1, ref2}. As with PV, thrombotic events are the major complication encountered during follow-up, with hemorrhagic events occurring less commonly. The reported frequency of thrombosis is variable since different studies have included distinct patient populations and have used varying definitions of major and minor vascular events. A number of risk factors for thrombosis have been identified^ref.
• low risk :
• age < 60 years
• no history of thrombosis
• platelets <= 1,500,000/ml. An increased risk of thrombosis has been suggested with platelet counts > 1,000,000/mL at diagnosis or in patients with a platelet count above the normal range during treatment, but other studies have not found such an association. However, the increased platelet numbers seem likely to be contributing to the thrombotic diathesis in ET patients since aspirin relieves microvascular symptoms in ET, cytoreduction with hydroxyurea reduces the incidence thrombosis in ET^ref and the ECLAP study shows that aspirin reduces the incidence of thrombotic events in PV
• no cardiovascular diseases
• intermediate risk : no low or high risk factors
• high risk of thrombosis :
• age >= 60 years
• previous history of thrombosis
Major hemorrhage is reported to be more common in patients with platelets > 1,500,000/mL. A number of other features have been claimed to correlate with thrombotic complications^ref. These include a clonal pattern of X chromosome inactivation, reduced expression of MPL in bone marrow megakaryocytes and over-expression of PRV1 in peripheral blood granulocytes. An increased risk of thrombosis may also be associated with antiphospholipid antibodies, heterozygosity for factor V Leiden and a number of cardiovascular risk factors, including systemic arterial hypertension, tobacco smoking and hypercholesterolemia.
In the longer term, patients with ET may also develop :
• MMM : the risk is difficult to assess from published data partly because different criteria have been used to define myelofibrotic transformation. In one recent study of 195 patients with a median follow-up of 7 years, the incidence of MMM was 8% at 10 years^ref
• AML/MDS : the reported incidence is very variable because most studies are retrospective and involve small numbers of patients with different lengths of follow-up. However, several points do appear to be clear. First, untreated patients can develop AML. A retrospective study of 2316 Italian patients suggested that AML/MDS occurs in approximately 1% of untreated ET patients^ref. Second, patients treated with hydroxyurea alone (and no other cytotoxic agent) display a low incidence of AML/MDS. Third, patients who require more than one cytotoxic agent have an increased risk of AML/MDS. However, it is not clear whether this reflects the combined effect of these drugs or whether it is a consequence of particularly aggressive disease. Increased incidence of abnormalities involving 17p in patients treated with hydroxyurea^ref
Therapy : there have been only 2 prospective randomized studies of the treatment of patients with ET. In the first, 114 high-risk patients (age > 60 years or prior thrombosis) were randomized to receive hydroxyurea or no cytoreductive agent^ref. Patients with a platelet count > 1500 x 10⁹/L were excluded. During a median follow up period of 27 months, patients on hydroxyurea developed significantly fewer thrombotic events (P = 0.003). This was the first clear demonstration that cytoreductive therapy reduces thrombotic events in patients with ET. The second randomized study was the Medical Research Council (MRC) primary thombocythemia-1 (PT-1) trial^ref, in which high-risk patients (prior thrombosis, age > 60 years or platelets > 1000 x 10⁹/L) were randomized to receive hydroxyurea plus aspirin or anagrelide plus aspirin. With over 800 patients randomized and with central clinical and histological review of end-points, it is the largest and most comprehensive study of essential thrombocythemia performed to date. The results demonstrate several major differences between the two arms. Compared to hydroxyurea plus aspirin, treatment with anagrelide plus aspirin was associated with increased rates of arterial thrombosis, major hemorrhage, myelofibrotic transformation and treatment withdrawal, but a decreased rate of venous thromboembolism. It is informative to compare these results with the previous Italian study^ref. The actuarial rate of first thrombosis at 2 years was 4%, 8% and 26% for patients receiving hydroxyurea +/- aspirin (both studies), anagrelide plus aspirin (PT-1) or no cytoreductive therapy (Italian study). Notwithstanding the difficulties of such comparisons these data suggest that anagrelide plus aspirin provides partial protection against arterial thrombosis. In marked contrast to arterial thrombosis, the rate of venous thrombosis was significantly lower in the anagrelide plus aspirin arm. The incidence of venous thrombosis in untreated patients with high-risk essential thrombocythemia is unknown and so it is not clear whether this rate is increased by hydroxyurea plus aspirin or decreased by anagrelide plus aspirin. The optimal management of a patient with prior venous thrombosis will depend on individual circumstances, remembering that arterial thrombotic events are > 3 fold more common than venous thrombotic events in ET. The different rates of thrombosis in the two arms of PT-1 is intriguing given the equivalent long-term control of the platelet count. These data imply that, in addition to lowering the platelet count, hydroxyurea or anagrelide may modulate thrombosis by other mechanisms (e.g., altered white cell count or function; altered endothelial function). The unexpected increase in major hemorrhage observed in patients receiving anagrelide plus aspirin may reflect an ability of anagrelide to interfere with platelet function in a way that synergizes with low-dose aspirin. Anagrelide blocks platelet phosphodiesterase activity^ref and at high doses (0.5-10 mg/kg) inhibits thrombus formation in animal models^ref. Although most assays of platelet function are normal in patients with essential thrombocythemia receiving anagrelide, some subtle effects on platelet function have been reported^{ref1, ref2}. The PT-1 results suggest that, if anagrelide is used, the decision whether to use concurrent aspirin should depend on the relative risk of arterial thrombosis and hemorrhage in each individual patient. Patients receiving anagrelide plus aspirin experienced an increased rate of transformation to myelofibrosis compared to patients receiving hydroxyurea plus aspirin. It is important to emphasize that a diagnosis of myelofibrotic transformation required not only a trephine biopsy showing  grade 3 fibrosis, but also the development of other clinical or laboratory evidence of transformation, which was absent at trial entry. The higher rate of myelofibrotic transformation was not an artifact of the precise definition used, since making the diagnostic criteria more stringent did not affect the statistical significance. It is possible that the results reflect a chance excess of "prefibrotic form of myelofibrosis" in the anagrelide arm. However, PT-1 is a large randomized trial and the probability that the results can be explained by such a chance occurrence is 1 in a 100 (P value was 0.01). The incidence of myelofibrosis in untreated essential thrombocythemia is unknown, and so it is not clear whether the observed differences reflect a protective effect of hydroxyurea or an acceleration of myelofibrosis by anagrelide. Hydroxyurea has been reported to reduce reticulin fibrosis in a variety of myeloproliferative disorders, including essential thrombocythemia^{ref1, ref2}. On the other hand, anagrelide blocks megakaryocyte differentiation, and the resultant relative increase in immature forms could conceivably result in altered production of profibrotic cytokines^{ref1, ref2}. Interestingly, analysis of the response to therapy and complications in the PT-1 trial by JAK2 mutation status revealed that V617F-positive patients were much more sensitive to hydroxyurea^ref. Compared to V617F-negative patients, they required substantially lower doses of hydroxyurea and yet had greater reductions in platelet counts, white cell counts and hemoglobin levels. No such effect was seen in patients receiving anagrelide. Furthermore, the rate of arterial thrombosis appeared to be lower in V617F-positive patients receiving hydroxyurea compared to those receiving anagrelide, an effect that was not evident in V617F-negative patients. Taken together the results of the PT-1 trial suggest that hydroxyurea plus aspirin should remain first-line therapy for patients with essential thrombocythemia at high risk of developing vascular events. For other patients at a lower risk of thrombosis, the situation is less clear. The decision whether or not to use a cytoreductive agent requires balancing 2 opposing risks, both of which are small: the risk of a thrombotic event and the risk of a significant drug-related side effect. Unfortunately, the frequency of these two types of events is not clear from existing data. Some studies suggest that patients aged < 60 years and with no prior thrombosis do not exhibit an increased frequency of thrombosis compared to controls^ref. However, the number of patients studied was small, the number of events very small and the choice of an appropriate control population is difficult. Moreover, other studies have found that such patients do have a significant risk of thrombosis^ref. With respect to the risks of treatment, these differ for the different agents. The most commonly used drugs are hydroxyurea, anagrelide and interferon.
• hydroxyurea (HU) 15-30 mg/kg has emerged as first-line therapy for high-risk patients because of its efficacy, low cost and rare acute toxicity. The main side effects are leg ulcers, a variety of other skin conditions (including photosensitivity and solar keratosis) and reversible bone marrow suppression. A common concern is whether hydroxyurea might be leukemogenic. It is apparent that some cytotoxic drugs such as ³²P and chlorambucil are leukemogenic. However, hydroxyurea has a distinct mechanism of action and does not increase the frequency of acquired mutation in adults with an MPD or sickle cell disease^ref. Some studies have reported that 5-10% of patients receiving hydroxyurea develop AML/MDS. However, these involved small numbers of patients, many of whom had also received other cytoreductive agents. Patients who receive more than one cytotoxic agent do have a significantly higher risk of developing AML/MDS, although is not clear whether this association reflects an effect of the drugs or a consequence of aggressive disease. By contrast, there are now a number of studies which show that ET patients receiving hydroxyurea alone have a low incidence of AML/MDS (3-4%)^{ref1, ref2, ref3} and there are no data to show that this incidence is significantly different from that observed in untreated patients. Moreover, follow-up of patients receiving hydroxyurea for sickle cell disease over the past 10 years also suggests that its leukemogenic potential is very low.
• pipobroman : the 10-year risk of acute leukemia is 3%, which is only slightly higher than that expected as a natural evolution of the disease^ref
Return to older approaches such as ³²P and AA in patients who fail on HU is to be discouraged. A much more appropriate approach involves the use of :
• anagrelide is an oral imidazoquinazoline derivative originally developed as an inhibitor of platelet aggregation^ref. It was subsequently shown to lower the platelet count in a species-specific manner and at doses lower than that which inhibit platelet aggregation. It is effective at reducing the platelet count (to < 600) in 70-80% of patients, but approximately 10% are completely refractory, perhaps because of an inability to generate an active metabolite. It is a platelet cAMP PDE inhibitor and acts as a vasodilator and a positive inotrope. Anagrelide is extensively metabolised in the liver and its metabolites are primarily excreted in the urine. Acute side effects are primarily caused by the drugs' direct vasodilating and positive inotropic effects and include headaches, palpitations, fluid retention, systemic arterial hypotension, tachycardia and arrhythmias, nausea, abdominal pain and diarrhoea. The only randomised trial to date (Primary Thrombocythaemia 1 (PT-1)) shows that anagrelide is not as effective or as well tolerated as hydroxyurea and should not be used as first-line therapy in high-risk patients. The starting dose of anagrelide ranges from 0.5 mg q.i.d. to 1 mg b.i.d. with a maximum dose of 2.5 mg q.i.d. Adequate responses have been maintained with a median dose of 2-2.5 mg/day. Platelet counts begin to decrease in 7-10 days, however, they return to pre-treatment levels within 4-8 days if therapy is stopped. Anagrelide 2 mg/day for 1 year costs approximately US$6439, and treatment must continue indefinitely^ref. It is indicated in Europe for the reduction of elevated platelet counts in at-risk patients with essential thrombocythaemia who are intolerant of or refractory to their current therapy, and in the US for the reduction of elevated platelet counts and the amelioration of thrombohaemorrhagic events in patients with thrombocythaemia associated with myeloproliferative disorders. Anagrelide is well established as an effective platelet-lowering agent in most patients with essential thrombocythaemia, including both treatment-naive patients and those refractory to other cytoreductive therapy. Results of the PT1 study indicated that the composite primary endpoint (arterial or venous thrombosis, serious haemorrhage or death from vascular causes) occurred more often in recipients of anagrelide + aspirin than in those receiving hydroxyurea + aspirin. This trial also indicated that the incidence of the secondary endpoints transient ischaemic attack and gastrointestinal bleeding favoured hydroxycarbamide plus aspirin, while the incidence of venous thrombosis favoured anagrelide plus aspirin. There were no differences between the groups in the incidence of secondary endpoints myocardial infarction, stroke, unstable angina, pulmonary embolism, hepatic-vein thrombosis, other serious haemorrhage or related deaths. The design of the PT1 study has been queried with respect to the heterogeneous nature of the study population (possible inclusion of patients with early myelofibrotic disease) and the concomitant use of aspirin (interaction with anagrelide causing increased bleeding events). Further data are therefore required before the role of anagrelide in essential thrombocythaemia can be finalized. In the meantime, when considering treatment options for patients with this disorder, anagrelide's positive effects on platelet function, lack of mutagenicity and lack of association with leukaemia or angiogenesis must be balanced against its comparative expense and positive inotropic effects. Thus, the role of anagrelide in the management of high-risk patients with essential thrombocythaemia will ultimately depend on individual patient assessment and future clarification of the potential leukaemogenicity of hydroxyurea^ref.
• IFN-a_2b (pegylated-IFNs, 1-3 million units a day for 4-13 weeks) is effective at reducing the platelet count below 600 x 10⁹/L in approximately 90% of patients with an average dose of 3 million international units per day. It is not known to be teratogenic or leukemogenic, does not cross the placenta and is often the treatment of choice during pregnancy. However, the need for parenteral administration and its acute side effects, particularly flu-like symptoms, are significant problems and result in treatment withdrawal in a substantial proportion of patients; lower doses of ASA (ie, < 325 mg daily) might be associated with less hemorrhage and, perhaps, a beneficial effect on thrombosis^ref.
Management recommendations : the MRC PT1 trial demonstrates that hydroxyurea should be first-line therapy for most patients with high-risk disease. Interferon- and anagrelide are both reasonable second-line agents. A decision whether to use concomitant aspirin with anagrelide will require an assessment of the potential risks and benefits in each individual patient. The combination of hydroxyurea and anagrelide can be useful as a way of minimizing side effects associated with the 2 drugs. The optimal management of a patient with prior venous thrombosis will depend on individual circumstances, remembering that arterial thrombotic events during follow-up are > 3-fold more common than venous ones in ET.
• all patients : manage reversible cardiovascular risk factors aggressively (e.g., smoking, hypertension, hypercholesterolemia, obesity)
• high-risk patients (prior thrombosis or age > 60 years or platelets > 1500 x 10⁹/L): low-dose ASA + hydroxyurea (HU) (anagrelide or IFN-a_2b second line)
• intermediate-risk patients (age 40-60 years, no high-risk features): there are no good data to guide management and it is not clear whether cytoreduction is beneficial
• either enter into randomized trial (e.g., PT-1 intermediate risk arm : hydroxyurea (HU) + ASA versus aspirin alone)
• or low-dose aspirin (consider cytoreduction if other cardiovascular risk factors present)
• low-risk patients (age < 40 years and no high-risk features) : low-dose ASA alone (or equivalent)
There are few data to guide the management of ET in pregnancy^ref. It seems reasonable for patients to receive low-dose aspirin, but the decision whether to lower the platelet count is more contentious and there are conflicting reports as to whether the established factors for thrombosis in non-pregnant patients can predict poor pregnancy outcome. In the absence of clear data, it seems advisable to limit the use of platelet-lowering agents to patients thought to be at high risk of thrombosis and particularly to patients with a history of previous thrombosis or fetal loss. Anagrelide and hydroxyurea should be avoided because of the possibility of teratogenic effects, although there have been reports of normal pregnancies despite exposure to hydroxyurea. Interferon- is generally regarded as the treatment of choice and should be combined with heparin in patients at particularly high risk, with treatment continuing for several weeks postpartum.
Prognosis : risk score

	low	intermediate	high
age	< 40	40-60	> 60
platelet count	< 1,000	1,000-1,500	> 1,500
previous thrombosis/hemorrhage	no	no	yes

Protocols :
• fertile woman or programmed pregnancy : IFN-a_2b
• if PLT > 800,000/ml :
• low risk patients :
• if familial thrombophilia, splenomegaly, high LDH, progression, bone marrow fibrosis, disorders of microcirculation : cytoreductive therapy
• if no associated risk factors : wait-and-see
+ acetylsalicylic acid 100 mg/day on a patient basis
• intermediate risk :
• if familial thrombophilia, cardiovascular risk factors (tobacco smoking, systemic arterial hypertension, hypercholesterolemia, diabetes mellitus), splenomegaly, high LDH, progression, or bone marrow fibrosis : cytoreductive therapy
• if no associated risk factors : wait-and-see
• high-risk :
• age < 60 years : IFN-a_2b (1.5 MIU on Monday for 1 week => 3 MIU on Monday-Wednesday-Friday (PEG-IFN-a) adjusting the dose on response (up to 9 MIU))
• age > 60 years : hydroxyurea
• second-line therapy : anagrelide 0.5 mg/12 hr for 7 days, then increase by 0.5 mg/day each week up to 4 mg/day (monitor heart and blood pressure)
Follow-up : bone marrow trephine biopsy (BMTB) every 2 years
• chronic idiopathic myelofibrosis (IMF) (CIMF) (with extramedullary hematopoiesis) / aleukemic or nonleukemic myelosis / leukoerythroblastic anemia / myelofibrosis with myeloid metaplasia (MMM) is a clonal stem cell disease that is characterized by intense bone marrow stromal reaction including collagen fibrosis, osteosclerosis, and angiogenesis^ref.

Epidemiology : median age at diagnosis is 60 years, and > 90% of patients are diagnosed after age 40 years (0.5-1.5 per 100,000 a year) .
Aetiology :
• de novo (agnogenic myeloid metaplasia (AMM))
• develop in the setting of ... :
• polycythemia vera (post-polycythemic myeloid metaplasia)
• essential thrombocythemia (post-thrombocythemic myeloid metaplasia)
... at a rate of 10-20% after 15-20 years of follow-up.
Pathogenesis :
• the primary clonal process in myelofibrosis : it is generally agreed that MMM is a clonal disorder arising from an early stem cell, thereby affecting both the myeloid and lymphoid lineages^ref. However, the nature of the disease-causing genetic mutation remains elusive.
• the secondary bone marrow stromal reaction in myelofibrosis : fibrogenic and angiogenic cytokines derived from clonal megakaryocytes (PDGF, TGF-b₁, EGF, bFGF, calmodulin) and monocytes^ref => polyclonal fibroblast proliferation. However, the basis for megakaryocyte/monocyte proliferation and the specific cytokines responsible for the stromal reaction remain unknown.
Potential pathogenetic mechanisms in MMM :

In mice, either prolonged exposure to excess thrombopoietin (TPO)^ref or intrinsic underexpression of GATA-1 (a transcription factor that is important in the maturation of both megakaryocytes and erythrocytes)^refresults in megakaryocyte proliferation and an MMM phenotype (megakaryocytic hyperplasia, myelofibrosis, anemia, extramedullary hematopoiesis). In human MMM, however, there is no evidence of either over-expression of TPO^ref or underexpression of GATA-1^ref. Furthermore, neither TPO nor its receptor (TPO receptor (c-mpl)) is essential for endogenous megakaryocyte growth in human MMM^ref. However, MMM megakaryocytes from both the experimental and human disease models display decreased surface expression of Mpl^ref, and this might result in decreased clearance of TPO with subsequent contribution to cell proliferation and stimulation of other cytokine production. In TPO-induced murine MMM, both myelofibrosis (via stem cell-derived TGF-ß₁) and osteosclerosis (via stroma-derived osteoprotegerin (OPG)) have been directly linked to specific cytokines^{ref1, ref2}. Similarly TGF-ß₁, PDGF, bFGF, VEGF, and TIMPs have all been implicated in the bone marrow fibrosis and angiogenesis seen in human MMM^ref. Tissue abundance of these cytokines might result from pathologic interaction of megakaryocytes and neutrophils (emperipolesis) that is induced by increased expression of P-selectin by the former^ref. Similarly, neutrophil-derived elastase and MMP might contribute to the abnormal peripheral blood egress of myeloid progenitors in MMM^ref.
Symptoms & signs : hepatosplenic as well as non-hepatosplenic extramedullary (but uneffective) hematopoiesis (EMH) => enormous splenomegaly at diagnosis => dysphagia, malaise, night sweats, weight loss > 10 kg in the last 6 months; progressive anemia (66%; normocytic normochromic > microcytic hypochromic), thrombocytopenia (33%) or thrombocytosis > 600,000/ml (10-20%), leukopenia (15%)
Laboratory examinations :
• cytomorphology : leukoerythroblastic blood smear, anisopoikylocytosis and dacryocytes disappearing after splenectomy (quantification of CD34⁺ cells in peripheral blood); dysmegakaryocytopoiesis, dysgranulopoiesis, reticulin fibrosis of the bone marrow with resulting features of myelophthisis^ref
• stage I : trilineage hypercellularity with increase in megakaryocytes, focal increase in reticular fibres. In this early, prefibrotic stage of CIMF the classic findings are absent or minimal^{ref1, ref2} and shares a number of clinical, laboratory, and even morphologic features with the early stages of polycythemia vera (PV) and with essential thrombocythemia (ET). However, the prominent neutrophil proliferation, decreased numbers of erythroid precursors, and marked atypia of the megakaryocytic lineage often aid in distinguishing the prefibrotic stage of CIMF from the other CMPDs. Although these findings must always be correlated with other clinical, laboratory, and genetic studies, the worse survival of CIMF in comparison with ET or PV indicates that recognition of this phase of CIMF is important
• stage II : hypocellular bone marrow, disappearance of fatty component, thickening of reticulum
• stage III : hypocellular bone marrow, thickening of reticulum, appearance of collagen, bone neoformation
• stage IV : severe hypocellularity, diffuse fibrosis, increase of bone neoformation
• cytogenetics : recurrent abnormalities that are seen in approximately 50% of chemotherapy-naïve patients with MMM include del(20q11q13), del(13q12q22), trisomy 8, trisomy 9, t(1;7), del(12p11p13), monosomy or long arm deletions involving chromosome 7, and trisomy 1q^ref. However, the individual lesions occur in only the minority of patients, and none are specific to MMM. Furthermore, the application of molecular cytogenetic studies by FISH did not disclose additional, karyotypically occult, structural lesions^ref. Other genetic studies of pathogenesis have included mutation screening for type III receptor tyrosine kinases (c-kit, c-fms, flt-3)^ref and genome-wide scanning for LOH (Jones LC, Tefferi A, Idos GE, Hofmann WK, Kumagai T, Koeffler HP. RAR beta 2 is a candidate tumor suppressor gene in myelofibrosis with myeloid metaplasia. Blood. 2002;100:587a). The results from such studies have been largely unrevealing despite the identification of frequent (20-43%) LOH sites involving chromosomes 1p, 1q, 2p, 3q, and 3p (Jones LC, Tefferi A, Idos GE, Hofmann WK, Kumagai T, Koeffler HP. RAR beta 2 is a candidate tumor suppressor gene in myelofibrosis with myeloid metaplasia. Blood. 2002;100:587a). On the other hand, gene expression studies have suggested a more consistent alteration of gene function including both down-regulation (e.g., RARß2 located at 3p24, BCL1 located at 11q13, cdc2 located at 10q21) and up-regulation (e.g., HMGA2 located at 12q13-q15, FKBP51 located at 6p21, GATA2 located at 3q21, JUNB located at 19p13.2) in MMM-derived CD34 cells^{ref1, ref2} (Jones LC, Tefferi A, Idos GE, Hofmann WK, Kumagai T, Koeffler HP. RARb2 is a candidate tumor suppressor gene in myelofibrosis with myeloid metaplasia. Blood. 2002;100:587a; Jones LC, Tefferi A, Wachsman W, Hofmann WK, Koeffler HP. Detection of aberrant signaling pathways in CD34⁺ cells from patients with myelofibrosis with myeloid metaplasia using oligonulceotide microarrays. Blood. 2001;98:626a). Other studies have suggested that the JAK/STAT pathway is involved and STAT5 is constitutively activated in both CD34 cells and megakaryocytes from patients with MMM^ref. Whether or not the above set of observations will ultimately elucidate the underlying molecular mechanism in MMM remains unknown.
• increase in [uric acid, and vitamin B₁₂]_plasma
• high LDH₁ and LDH₂
• high LAP
• defective platelet aggregation
• low fibrinogen, factor V and VIII, high FDP
• ANA, RF, LAC, anti-RBC Abs, low complementemia, monoclonal gammopathies, circulating immune complexes
• CBC, creatinine, azotemia, SGOT, SGPT, gGT, alkaline phosphatase, ESR, PCR, serum protein electrophoresis, IgG, IgA, IgM, sideremia, ferritin, total cholesterol, urinalysis
• MRI to evaluate fibrosis and bone marrow cellularity
Differential diagnosis of myelofibrosis :
• nonneoplastic
• infections : tuberculosis, histoplasmosis
• renal osteodystrophy
• vitamin D deficiency
• hypoparathryoidism
• hyperparathyroidism
• gray platelet syndrome
• SLE
• scleroderma
• radiation exposure
• autoimmune myelofibrosis (AIMF)
• primary : absence of marked splenomegaly, peripheral blood cytopenias with mild teardrop poikilocytosis and leukoerythroblastosis, bone marrow lymphoid aggregates, and presence of autoantibodies. Primary AIMF appears to have an excellent prognosis, with all patients reported in this series responding to a short course of corticosteroid therapy^ref
• associated with SLE
• osteopetrosis
• Paget disease
• benzene exposure
• Gaucher disease
• neoplastic
• PV, CML (micromegakaryocytes without clusters, granulocyte hyperplasia), ET
• acute panmyelosis with myelofibrosis (APMF)
• AML M7
• ALL
• HCL
• HD
• MM
• MDS with myelofibrosis
• systemic mastocytosis
• NHL
• breast carcinoma, lung carcinoma, prostate carcinoma, gastric carcinoma
Therapy :
• conventional treatment^ref :
• first-line therapy for alleviation of anemia
• androgen preparations (e.g., oral fluoxymesterone 10 mg 2 times a day)
• corticosteroids (e.g., oral prednisone 30 mg/day)
• erythropoietin (e.g., 40,000 units subcutaneously once a week)
An approximate 30% response rate with a median remission duration of 1 year is expected from the use of one or more of these treatment modalities. In contrast, IFN-a therapy has limited value in alleviating anemia associated with MMM^ref
• symptomatic splenomegaly
• initially treated with hydrodyurea(e.g., starting dose of 500 mg 2 or 3 times a day)
• subsequently with splenectomy^ref. Splenectomy is indicated in the presence of drug-refractory splenic pain and/or discomfort, high RBC transfusion requirements, and symptomatic portal hypertension^ref. The procedure provides symptomatic relief for the majority of patients and a durable erythroid response in 25% of patients. Post-splenectomy occurrence of leukemic transformation in MMM represents the natural progression of the disease and is not the result of the procedure itself
• involved-field irradiation therapy works best for non-hepatosplenic EMH but has limited value in controlling symptomatic enlargement of the spleen and liver^ref
• other cytoreductive drugs that might be considered either prior to splenectomy or in those patients who are poor surgical candidates include
• intravenous cladribine (5 mg/m²/day in a 2-hour infusion for 5 consecutive days to be repeated for 4-6 monthly cycles)^ref
• oral melphalan (2.5 mg 3 times a week)^ref
• oral busulfan (2-6 mg/day for 10 days a month for 3 months, with close monitoring of blood counts)^ref (Shojania AM. Reversion of post polycythemia vera (PV) myelofibrosis (MF) to PV following busulfan therapy. Blood. 2002;100:343b)
• intravenous daunorubicin(50 mg/m² weekly for 4-8 weeks)
Associate with allopurinol 300 mg a day 3 times a week.
• investigational drug therapy : over the last decade, a group at the Mayo Clinic has explored experimental drug therapy intended to interfere with cytokines believed to mediate the bone marrow fibrosis and angiogenesis in MMM. In this regard, drugs that were shown to be clinically ineffective include :
• imatinib mesylate (inhibits PDGF receptor-associated tyrosine kinase activity)^ref
• IFN-a (non-specific myelosuppressive agent)^ref
• anagrelide (interferes with terminal differentiation of megakaryocytes and platelet production)^ref
• pirfenidone (impairs fibroblast proliferation and collagen synthesis)^ref
• suramin (inhibits TGF-ß binding on fibroblasts)^ref.
• calcitriol 0.5 mg daily^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9}, 2.5 mg daily for 6 months ^ref or 0.75 mg daily for 12 weeks^ref : 1.50-3.00 mg/day for at least 12 months is of doubtful benefit^ref
In contrast, the drugs that showed promise of activity include :
• thalidomide (has anti-angiogenic activity and also inhibits TNF-a production)^{ref1, ref2}. The demonstration of florid bone marrow angiogenesis in MMM^ref encouraged the development of several small pilot studies that evaluated the therapeutic value of thalidomide either
• alone^{ref1, ref2, ref3, ref4,ref5, ref6, ref7, ref8} (Grossi A, Gavazzi S, Biscardi M, et al. Thalidomide therapy effects on angiogenic growth factors (VEGF, TGF-b) and KDR expression in myeloid metaplasia with myelofibrosis. Blood. 2002;100:346b)
• in combination with other drugs^{ref1, ref2}
• the positive experience with the combination of low-dose thalidomide and prednisone therapy in MMM has encouraged an institution to evaluate the value of adding either etanercept (with the rational of optimizing anti-TNF-a activity) or cyclophosphamide (based on in vitro observation of drug synergism) to the combination in ongoing clinical trials. Thalidomide-associated drug side effects might be effectively addressed in the future by the development of thalidomide analogues that are more potent and yet less toxic than the parent drug. One of these agents, CC-5013, has shown significant activity in the treatment of both MM and MDS, and it is currently being tested in MMM
Studies that involved > 10 patients have demonstrated a response rate of 20-62% in anemia, 25-80% in thrombocytopenia, and 7-30% in splenomegaly^{ref1, ref2, ref3, ref4, ref5, ref6}. Information from these studies indicates that low-dose thalidomide (50 mg/day) is as effective as higher doses (> 200 mg/day) and that the addition of prednisone to the lower-dose schedule improves drug tolerance and may enhance the erythropoietic activity of the drug^{ref1, ref2}. Long-term analysis of thalidomide-based drug therapy suggests a durable response in 25% of the patients, including some in whom the drug was discontinued (Mesa RA, Elliott MA, Faoro L, Tefferi A. Durable and unmaintained remissions with thalidomide-based drug therapy in myelofibrosis with myeloid metaplasia: Long term outcome analysis of 2 prospective trials [abstract]. Blood. 2003;102:33a). Unusual thalidomide drug effects in MMM, all reversible, include extreme leukocytosis and thrombocytosis. Thalidomide responses in MMM have not been shown to affect either the degree of bone marrow fibrosis or angiogenesis.
• etanercept (a soluble TNF- receptor that produced a 60% response rate in constitutional symptoms)^ref
• allogeneic HSCT: treatment with AHSCT, either myeloablative^{ref1, ref2, ref3} or reduced-intensity^{ref1, ref2}, is directed at eradicating the mutant MMM clone. However, the particular treatment modality is associated with significant morbidity and mortality, and is not applicable to the majority of patients with MMM^ref. The 3 largest studies of myeloablative AHSCT (both related and matched unrelated) provide results in a combined total of 147 patients^{ref1, ref2, ref3}. Engraftment was not a problem, with > 80% of patients achieving neutrophil engraftment by day 30^ref. However, transplant-related morbidity and mortality were significant, resulting in a 5-year survival of only 14% for patients above age 44 years in one study^ref and a 2-year OS of 41% in another study^ref. In the most favorable of the 3 studies, 20 of the 56 patients had died within 3 years of the transplant, and the reported incidence of cGvHD was 59% at a median follow-up period of only 2.8 years^ref. On the positive side of the current experience with myeloablative AHSCT in MMM, younger patients did better in all 3 studies mentioned above, with projected 5-year survival rates of above 60%. Both clinical and histological remissions were documented in the surviving patients^{ref1, ref2} Furthermore, transplant outcome was significantly better in good-prognosis patients compared to the outcome in poor-prognosis patients. This latter observation suggests that a window of opportunity may be lost by delaying transplant until the disease progresses. However, it is important to realize that transplant-age patients with good-prognosis MMM can expect a median survival of 15 years^ref. This must be weighed against the risk of an upfront mortality rate of at least 20% and a high risk of CGVHD. Taking all these issues into consideration, an evolving guideline for transplant decision making in MMM can be formulated:


Low-risk disease is defined by the absence of hemoglobin < 10 g/dL, circulating blasts, and severe constitutional symptoms. The presence of one or more of these features define intermediate-risk and high-risk disease, respectively^ref
Reduced-intensity AHSCT has also been attempted in MMM. In a preliminary report from a multicenter study of 20 patients (median age 50 years) with MMM (including 5 who had undergone leukemic transformation), 1-year treatment-related mortality, relapse rate, and survival were 37%, 36%, and 54%, respectively. However, when reduced-intensity AHSCT was performed before transformation, 1-year survival increased to 77% (Hertenstein B, Guardiola P, Finke J, et al. Non-myeloablative (NMA) stem cell transplantation (SCT) for myeloid metaplasia with myelofibrosis (MMM): a survey from the chronic leukemia working party of the EBMT [abstract]. Blood. 2002;100:#253). A limited number of other studies, consisting of fewer than 6 study patients each, support the feasibility of reduced-intensity AHSCT in terms of engraftment, attainment of full donor chimerism, and ability to induce both clinical and pathological remissions^ref. However, a longer follow-up period as well as a larger sample size are needed to define the durability of full donor chimerism, long-term relapse rate, and quality of life. Such information is critical since the potential beneficiaries of reduced-intensity AHSCT are the subset of patients with good prognosis disease.
Prognosis : average life expectancy is estimated at 5-7 years but may approach 15 years in young patients with good prognostic factors. However, quality of life is often compromised by frequent RBC transfusions as well as a markedly enlarged spleen that is usually accompanied by both mechanical discomfort and profound constitutional symptoms. Cause of death in MMM includes :
• congestive heart failure (35%)
• transformation to acute myeloid leukemia (10-20% of patients in the first 10 years)
• hemorrhage (25%)
• infections (15%)
Dupriez risk score :
• low risk : HGB > 10 g/dl AND WBC = 4,000-30,000/ml
• intermediate risk : HGB > 10 g/dl OR (WBC <4,000/ml OR WBC >30,000/ml)
• high risk : HGB > 10 g/dl AND (WBC <4,000/ml OR WBC >30,000/ml)
Protocols : before starting therapy, collect 20 ml heparinated PB to assay CD34 colonies and call the lab before performing BMTB
• if asymptomatic, splenomegaly < 15 cm in largest diameter, no severe thrombocytosis and high LDH : wait-and-see
• otherwise : splenectomy if massive splenomegaly causing symptoms
• age < 60 years : hydroxyurea
• age < 45 with high-risk of progression : RIC allogeneic HSCT (TRM = 27%, 5-yr OS = 47%, DFS = 39%)
• age 45-60 years with high-risk, drug-refractary or transfusion-dependent : RIC allogeneic HSCT (5-yr OS : 14%)
• age > 60 years : busulfan (if refractory : hydroxyurea)
• age 60-70 years with high-risk, drug-refractary or transfusion-dependent : RIC allogeneic HSCT (5-yr OS : 14%)
• age > 70 years : evaluate PST before considering RIC allogeneic HSCT
• chronic myeloid leukemia (CML) [Ph chromosome, t(9;22)(q34;q11), BCR/ABL-positive] / chronic granulocytic leukemia / chronic myelocytic or myelogenous leukemia

Epidemiology : 15-20% of all leukemias; incidence = 1-1.5 cases per 100,000 per year; 10,000 new cases around the world a yearit is expected to strike about 4,500 people in 2006 and kill about 600. Occurring mainly between the age of 25 and 60 years. 40% of all leukemias in Hiroshima survivors and 12% in Nagasaki survivors.
Aetiology :
• t(9;22) (q34q11) (85-90%) => Philadelphia (Ph*) chromosome (city of Pennsylvania, USA, where Nowell^ref and Hungerford^ref identified it) with hybrid gene coding for BCR / c-ABL fusion protein : this fusion eliminates the first 2 or 3 exons of c-abl and results in unregulated tyrosine kinase activity. 3 breakpoint cluster regions have been characterized to date :
• p210 (major (M-bcr) breakpoint : the overwhelming majority), whose mRNA transcripts have a b3a2 and/or a b2a2 junction. There is apparently no significant difference between patients with a 5' or a 3' M-bcr breakpoint, except maybe for a slight predominance of b3a2-expressing cases among those with increased platelet counts (ET-like syndrome). p210 stimulates transcription of hnRNP2, which in turn prevents translation of C/EBPa mRNA. As the latter is the TF for G-CSFR, differentiation doesn't occur^ref.
• p190 (minor (m-bcr) breakpoint) is principally associated with Ph⁺ AML, Ph⁺ B-ALL (worse prognosis), and only rarely with CML (in these, the disease tends to have a prominent monocytic component, resembling CMML) : more acutely transforming in experimental settings
• p230 (micro (mu-bcr) breakpoint) is also rare, and has been associated with the CNL variant and/or with marked thrombocytosis



Exceptional CML cases have been described with :

• BCR breakpoints outside the 3 defined cluster regions
• unusual breakpoints in ABL resulting in BCR-ABL transcripts with b2a3 or b3a3 junctions
• aberrant fusion transcripts containing variable lengths of intronic sequence inserts
The reciprocal ABL-BCR gene found in the derivative 9q+ chromosome of the t(9;22) is transcriptionally active in nearly 66% of CML patients but has not been shown so far to have a functional role in CML. 'Ph-negative CML' comprises cases of typical CML in whom the BCR-ABL gene can be detected by molecular methods and others who are genuinely BCR-ABL negative and usually have an atypical CML^ref.
=> abnormal, excessive, unrestrained overgrowth of granulocytes in the bone marrow
• other (5-10%) : in virtually all cases thus far reported these patients are found to be positive for BCR-ABL on molecular analysis^ref
• other translocations, most commonly complex variants that involve one or more chromosomal regions in addition to bands 9q34 and 22q11, but also simple variants that typically involve 22q11 and a chromosome other than 9
• t(8;22)(p11;q11) : BCR gene translocates into a breakpoint within fibroblast growth factor receptor 1 (FGFR1), the receptor tyrosine kinase that is known to be rearranged and constitutively activated in patients with a distinct, aggressive biphenotypic hematologic malignancy that has been termed the 8p11 myeloproliferative syndrome (EMS) / stem cell leukemia-lymphoma syndrome (SCLL)^{ref1, ref2}. Although it is difficult to generalize from just 2 cases, patients with BCR-FGFR1 appear to resemble classical CML more closely than individuals with other FGFR1 fusion proteins. One possible explanation for this is that the BCR moiety contributes to specific features of the patient phenotype. This could be by several mechanisms, for example, by providing a tyrosine residue that serves as a Grb2 binding site when phosphorylated, a function that is believed to be important for the action of BCR-ABL^ref, by its intrinsic Ser/Thr kinase activity^ref, by driving a particular level of transcription of the fusion gene, or as a consequence of a reduced level of normal BCR expression. Simple variants of the t(9;22) are seen in a small minority of patients with CML. For example, the Medical Research Council CML III trial included 9 such individuals, corresponding to 1.5% of all patients or 18% of those with variant translocations. Of those cases that have been reported, virtually all have been BCR-ABL positive by RT-PCR or FISH analysis, or have had a rearrangement within the M-Bcr as determined by Southern blotting^{ref1, ref2}. Thus, in common with individuals who are Ph positive, patients who present with apparently typical CML and a variant translocation are not always evaluated for BCR-ABL by molecular tests : occasional patients may therefore be misdiagnosed. This is not simply an academic point: BCR-ABL positive cases are amenable to treatment with STI571^ref, but data from a cell line model indicate that FGFR1 fusion proteins are resistant to this compound. However, cells transformed with BCR-FGFR1 or ZNF198-FGFR1 are sensitive to SU5402, a compound known to have inhibitory activity against FGFR1^ref. RT-PCR confirmed the presence in both cases of an in-frame messenger RNA fusion between BCR exon 4 and FGFR1 exon 9 (not significantly inhibited by treatment with STI571, but was inhibited by SU5402, a compound with inhibitory activity against FGFR1, with IC₅₀ = 5 µM). Clinical and morphologic picture is essentially indistinguishable from typical, BCR-ABL⁺ CML^ref
• the status of the remaining 5% of patients, many of whom have distinctive clinical features, has been controversial. Until recently these patients were typically classified as either Ph^-, BCR-ABL^- CML, or as atypical CML, and many cases were considered eligible for entry into CML clinical trials^{ref1, ref2, ref3}. More recently BCR-ABL^- cases have been classified into a spectrum of myeloproliferative disorders/myelodysplastic syndromes (MPDs/MDSs); the term "CML" is reserved for Ph⁺ and/or BCR-ABL⁺ cases only^ref
Pathogenesis : an apparently normal circulating DC compartment carrying the Ph+ chromosome can be identified in CML patients; however, these cells appear unable to trigger a protective anti-leukemic immune response in autologous T cells^ref
Symptoms & signs^{ref1, ref2} (Brunning RD, McKenna RW. Tumors of the Bone Marrow. Washington, DC: Armed Forces Institute of Pathology; 1994) : mild hepatomegaly (15%) and splenomegaly (80%) with common splenic infarctions, dysphagia ("small stomach syndrome") => cachexia, scotomas, acuphems, vertigo due to occlusion of retinal vessels, priapism, malaise and fatigue, thromboses
Staging :
• CML, chronic phase (CP)^ref (95% at diagnosis) : myelocytes, promyelocytes and monoblasts in peripheral blood smear. Diagnose if all of the following are present :
• blasts in PB and BM < 15%
• basophils in PB < 20%
• PLT > 100,000/ml (thrombocytopenia (10%), normal (60%) or thrombocytosis (30%))
• [leukocytosis (WBC > 100,000 / mL)
• leukopenia
• HGB < 10 g/dl]
‡
• CML, accelerated phase (AP) : diagnose if one or more of the following is present^{ref1, ref2} :
• blasts 15-30% of peripheral blood white cells or bone marrow cells
• blasts + promyelocytes in PB or BM > 30% with blasts < 30%
• peripheral blood basophils > 20%
• persistent thrombocytopenia (< 100 × 10⁹/L) unrelated to therapy, or persistent thrombocytosis(> 1,000 × 10⁹/L) unresponsive to therapy
• [increasing spleen size and increasing WBC count unresponsive to therapy
• cytogenetic evidence of clonal evolution (ie, the appearance of an additional genetic abnormality that was not present in the initial specimen at the time of diagnosis of chronic phase CML)
• megakaryocytic proliferation in sizable sheets and clusters, associated with marked reticulin or collagen fibrosis, and/or severe granulocytic dysplasia, should be considered as suggestive of CML-AP. These findings have not yet been analyzed in large clinical studies, however, so it is not clear if they are independent criteria for accelerated phase. They often occur simultaneously with one or more of the other features listed]
‡ 70%
• CML, blast phase (BP) / blast crisis (BC) : a sudden, severe transformation of CML to a more aggressive course; the proportion of blast cells in bone marrow increases rapidly to > 30%, and the clinical picture resembles that in acute myelogenous leukemia. The granulocyte-macrophage progenitor (CFU-GM) pool from patients with CML in blast crisis and imatinib-resistant CML is expanded, expresses BCR-ABL, and has elevated levels of nuclear b-catenin as compared with the levels in progenitors from normal marrow. Unlike normal granulocyte-macrophage progenitors, CML granulocyte-macrophage progenitors form self-renewing, replatable myeloid colonies, and in vitro self-renewal capacity was reduced by enforced expression of axin. Activation of b-catenin in CML granulocyte-macrophage progenitors appears to enhance the self-renewal activity and leukemic potential of these cells^ref. Conversion to chronic phase with chemotherapy in 10-30% : otherwise median survival is 6 months
• myeloblastic BC (60%)
• megakaryoblastic or erythroid (10%)
• lymphoblastic BC (15-20%) : 40-70% can be reconverted to chronic phase with chemotherapy
• undifferentiated (10%)
Diagnose if one or more of following is present:
• blasts > 30% of peripheral blood white cells or bone marrow cells
• extramedullary blast proliferation other than hepatosplenomegaly
• [large foci or clusters of blasts in bone marrow biopsy]
Laboratory examinations : although in most cases the diagnosis is easily made from morphologic evaluation of the blood smear (including alkaline phosphatase), confirmation by genetic studies is essential, particularly in view of the advent of therapy that targets the BCR/ABL fusion protein^ref
• CBC (anemia, leukocytosis)
• creatinine, azotemia, SGOT, SGPT, gGT, ESR, PCR, serum protein electrophoresis, IgG, IgA, IgM quantification, sideremia, ferritin, total cholesterol, urinalysis
• increase in [LDH₁, hyperuricemia, transcobalamin and vitamin B₁₂]_plasma
• absent or reduced LAP (except during blast phase)
• BMAB : hypercellularity of granulocytic (and sometimes megakaryocytic but never erythrocytic) lineage

Co-diagnosis : CP-CML and HCL^{ref1, ref2}
Evolution : CML => HCL^ref (atypical in a few ways, with an initial high white blood cell count, the absence of monocytopenia, and atypical morphology. Immunophenotyping also showed that hairy cells were CD25 negative)
Therapy :
• BCR-c-Abl inhibitors :
• chronic phase : 400 mg PO daily. the dose was increased to 400 mg PO twice daily in those in whom a complete hematologic response was not achieved after 3 months of treatment; in those whose disease relapsed within 3 months after the achievement of a complete hematologic response; and in those in whom a major cytogenetic response had not been achieved after 12 months of therapy. Neutropenia grade 3 (27%) or 4 (8%), leukopenia grade 3 (22%) or 4 (2%), anemia grade 3 (6%) or 4 (1%), grade 3-4 thrombocytopenia (20%), weight gain (4%), rash and related events (3%), nausea (2%), superficial edema (1%) => median time to first grade 3 or 4 episode of neutropenia was 62 days. Drug-related adverse events led to treatment discontinuation in 11 patients. Emetogenic potential daily level 3^ref
• blast crisis : 400 mg or 600 mg PO daily (administered once a day with a meal). Starting dose increased to 600 mg PO daily after first 37 patients; dose was permitted to be increased to a maximum of 400 mg PO twice daily in patients who relapsed, and in patients who did not achieve a hematological response after at least 1 month of treatment. Neutropenia grade 3 (14% at 400 mg, 16% at 600 mg) or 4 (54% at 400 mg, 47% at 600 mg), anemia grade 3 (43% at 400 mg, 40% at 600 mg) or 4 (16% at 400 mg, 10% at 600 mg), grade 3-4 thrombocytopenia (62%), dermatitis/rash (4%), grade 3 hyperbilirubinemia (4%), superficial edema (4%), other fluid-retention events (3%), hemorrhage (2%), nausea (2%), grade 3 transaminases (2%), diarrhea (1%), vomiting (1%) => hematologic side effects managed with a dose reduction or interruption (see prescribing information for recommendations). Adverse events led to a reduction in the initial dose of imatinib on 1 or more occasions in 46% taking 400 mg daily and in 47% taking 600 mg daily^ref
• accelerated phase : 400 mg or 600 mg PO daily (administered once a day with a meal). Starting dose increased after first 77 patients; dose was able to be escalated to 400 mg PO twice daily in patients who had a relapse, or in those patients who did not achieve a hematologic response after at least 1 month of treatment. Neutropenia grade 3 (21% at 400 mg, 25% at 600 mg) or 4 (35% at 400 mg, 35% at 600 mg), leukopenia grade 3 (27% at 400 mg, 35% at 600 mg) or 4 (18% at 400 mg, 13% at 600 mg). anemia grade 3 (35% at 400 mg, 32% at 600 mg) or 4 (9% at 400 mg, 15% at 600 mg), grade 3-4 thrombocytopenia (43%), arthralgia (3%), edema (3%), fatigue (3%), nausea (3%), grade 3 transaminases (3%), hemorrhage (2%), hyperbilirubinemia (2%), dermatitis (1%), fluid retention (1%), vomiting (1%). Hemtologic side effects managed with a dose reduction or interruption (see prescribing information for recommendations). Adverse events led to dose reduction in 49% of patients on 400 mg and 52% on 600 mg dose. 1 death duspected to be related to liver failure^ref.
• should all newly diagnosed patients be treated with imatinib? This question is frequently asked and opinions still vary. However, there is an inexorable shift toward a trial period of imatinib therapy for most if not all newly diagnosed patients because of the efficacy of imatinib and its minimal morbidity and mortality compared to allografting. As described below, it is now possible to formulate evidence-based criteria that allow the definition of "suboptimal response" to the drug and it is patients with such suboptimal response who might reasonably be offered an allograft. Such an approach can be fairly easily justified for a newly diagnosed 40 year old for whom only a matched unrelated donor is available; however, the decision is much more difficult for a 20 year old with a sibling donor. Although the ongoing response rates with imatinib monotherapy continue to be impressive^ref, < 10% of patients appear to be "disease free" based on PCR analysis^ref, and there are small numbers of concerning reports of patients, occasionally children^ref, developing blast crisis from complete cytogenetic response. Allogeneic transplant should therefore be considered for younger patients especially if a sibling donor is available. An update (3.5 years' follow-up on all patients) of the IRIS (0106) study^ref was presented at the ASH 2004 meeting and the maturing data from this study continue to be the most valuable source of information on the longer-term use of imatinib in newly diagnosed patients. In the original report, after a median follow-up of 19 months, imatinib showed significantly superior tolerability and hematologic and cytogenetic responses and also reduced time to progression when compared with IFN-a combined with cytosine arabinoside (Ara-C)^ref. In 553 patients major and complete cytogenetic responses at 18 months were 87.1% and 76.2%, respectively, in the imatinib arm and 34.7% and 14.5% in the IFN and Ara-C arm. The rate of freedom from progression was 96.7% in the imatinib-treated group and significantly less at 91.5% in the combination treatment group. As well as being clinically superior, a comprehensive quality-of-life study has also demonstrated the superiority of imatinib over IFN^ref. A review of the 2.5-year follow-up data at the 2003 ASH meeting confirmed that there had been a slight increase in imatinib response rates with further follow-up: 90% major cytogenetic response rate and 82% complete cytogenetic response rate. It is also clear that diagnostic Sokal and/or Hasford scores have an impact on response rates, , and on progression-free survival. After 2.5 years of follow-up, the overall survival in the study was 95%. Whether these excellent results will be maintained requires longer follow-up, but so far imatinib resistance does not seem to be a major problem in patients treated in chronic phase. Some groups have reported improved survival in single cohorts of patients^ref, but only longer observation will allow a robust analysis of the impact of imatinib on the long-term survival of CML patients. Imatinib offers clear quality of life (QoL) advantages over IFN as first-line treatment of chronic-phase CML. In addition, patients who crossed over to imatinib reported higher QoL than those who remained on IFN^ref
• 95% of patients are in chronic phase at diagnosis; in these patients who are treated with imatinib, blood counts will return to normal in 98 percent, and in 86% the Philadelphia chromosome will disappear — in other words, they will have a complete cytogenetic response. With a median follow-up of 54 months, the overall relapse rate for patients with newly diagnosed chronic-phase CML who were treated with imatinib was 16%, but for patients with a complete cytogenetic response, the relapse rate was 7% (Simonsson B. Beneficial effects of cytogenetic and molecular response on long-term outcome in patients with newly diagnosed chronic myeloid leukemia in chronic phase (CML-CP) treated with imatinib (IM): update from the IRIS study. Blood 2005;106:52a-52a) Among patients in whom BCR-ABL messenager RNA (mRNA) levels were reduced by at least 3 log, the relapse rate at 54 months was only 3%, and none of these patients had progression to the accelerated phase or blast crisis^ref.
• Studies of patients in relapse have shown that mutations in the kinase domain of BCR-ABL are the predominant mechanism underlying acquired drug resistance to imatinib; another mechanism in some patients is amplification of the BCR-ABL fusion gene^ref. Mutations have been observed to affect at least 25 amino acids scattered throughout the ABL kinase domain, and each of them reduces the sensitivity of the kinase to imatinib. Structural biology has made major contributions to the understanding of imatinib resistance and the design of new inhibitors. The crystal structure of ABL in a complex with imatinib shows that imatinib binds to a unique, inactive conformation of the ABL kinase domain in which the activation loop is in a closed position^ref. Mutations that confer resistance to imatinib affect residues that are directly involved in drug binding, that impair the ability of the kinase domain to undergo the extensive conformational changes required for imatinib binding, or that favor the active conformation of the kinase to which imatinib is unable to bind. Information from the crystal structure suggested two approaches for the circumventing of resistance. One avenue was the modification of imatinib in such a way that the drug binds more tightly to the ABL kinase. This modification was accomplished by chemists at Novartis in the design of nilotinib^ref. A second approach was the development of a compound with an entirely different chemical scaffold. This tack, taken by scientists at Bristol-Myers Squibb, was based on the finding that SRC inhibitors also inhibit the wild-type ABL kinase and most imatinib-resistant mutations^ref. (SRC is a protein tyrosine kinase that acts as a signal transducer in several molecular pathways within the cell.) Thus, dasatinib, originally synthesized as a SRC-family inhibitor, was tested for its ability to inhibit ABL and imatinib-resistant mutations^ref. Both nilotinib and dasatinib are more potent ABL inhibitors than is imatinib and inhibit all tested imatinib-resistant mutations except T315I. Consistent with the preclinical profiles of nilotinib and dasatinib, responses in the phase 1 trials of these drugs were seen in patients with all imatinib-resistant mutations except T315I^{ref1, ref2}. Responses were also seen in patients with resistance that was not caused by mutations, a finding that may relate to the high potency of these drugs. Both drugs have unique side effects, including pleural effusions with dasatinib and elevated liver enzyme levels and a prolongation of corrected QT intervals with nilotinib. The question as to whether additional side effects will be observed over time will require larger studies and longer follow-up. Response rates in patients with chronic-phase CML were impressive and have thus far been durable, but responses in accelerated-phase and blast-phase disease were lower, and relapses have been common. For this reason, other strategies are required for the advanced phases of CML and for patients with the T315I ABL mutation. Nilotinib and dasatinib have immediate applicability for patients with imatinib resistance, but the drugs may also be useful at earlier stages of CML. As noted, in most patients treated with imatinib, CML is well controlled but not eradicated. The molecular basis of persistent disease is poorly understood, but BCR-ABL kinase mutations appear not to be a major factor. Given that the greatest protection from relapse occurs in patients with reductions of 3 log₁₀ or greater in BCR-ABL transcript levels, it may be possible to achieve this goal in more patients with more potent inhibitors. Another strategy for reaching the 3-log₁₀ end point or preventing resistance is to combine inhibitors; preclinical data support this approach^ref. The value of combination treatment will depend on any increase in adverse events and the development of resistance. For example, if T315I mutant clones are prevalent, it would be necessary to add a T315I inhibitor to combination therapy. Regardless, the good news for patients with CML is that the long-term prospects for control of the disease are excellent. These drugs were developed rapidly. A driving factor was the high quality of the basic science associated with clinical trials in CML. Mechanisms of relapse were rapidly identified, and the solution to the crystal structure of ABL in complex with imatinib prompted rapid modification of the structure of the drug. Only a continued investment in a basic understanding of disease mechanisms will make possible similar advances. It would be logical to ask why two large pharmaceutical companies would have an interest in a disease that affects fewer than 5000 patients per year in the United States — and one in which drug resistance is relatively uncommon. One reason is that imatinib had gross sales of $2.1 billion in 2004. Although this volume of sales is not entirely attributable to its use in CML, it does mean that CML therapy is an attractive market. An encouraging thought is that some large pharmaceutical companies recognize the power of genomic medicine. They have an interest in testing a drug in a small, molecularly defined population, such as patients with imatinib-resistant CML. By limiting testing to a population with a high probability of having a response to therapy, the company reduces the risk of drug failure, and the number of patients who are required to demonstrate clinical activity of the drug can be relatively small. These 2 factors minimize the costs of drug development, could lead to rapid approval by the Food and Drug Administration, and might result in decreased drug costs^ref.
• how should patients on drug therapy be monitored? There are essentially 4 laboratory tests that can be used to monitor drug therapy:
• peripheral blood counts
• cytogenetic analysis
• real-time quantitative PCR (RQ-PCR) for BCR-ABL mRNA
• assessment of ABL kinase domain mutations^{ref1, ref2, ref3, ref4}.
The aim of ongoing assessment is to identify those patients who are not responding optimally to standard-dose imatinib monotherapy so that an alternative treatment strategy can be considered. As described below, there is now good evidence to suggest that achievement of complete cytogenetic response should be the major therapeutic target and it has also been demonstrated that the achievement of a 3-log reduction in BCR-ABL/ABL ratio confers superior PFS^ref, suggesting that a good "molecular" response is also important. ABL kinase domain mutations that cause most cases of imatinib resistance are well-defined and easily detectable, and provide a powerful tool for predicting which patients may develop drug resistance^ref. Realistically most hematologists do not have the facilities and resources to perform all of these studies on patients at every visit. So a practical approach to molecular monitoring is necessary. An excellent recent study by the Australian group has suggested an appropriate approach to "molecular"monitoring of patients on imatinib therapy^ref. In 214 imatinib-treated patients with chronic-phase disease Branford et al determined whether there was a difference in the incidence of mutations between the patients with a greater than 2-fold rise in BCR-ABL and those with stable or decreasing levels. Of 56 patients, 34 (61%) with a greater than 2-fold rise had detectable mutations. Only 1 of 158 patients (0.6%) with stable BCR-ABL levels had a detectable mutation. This approach (selecting for mutation analysis only those patients with a 2-fold increase in BCR-ABL levels) therefore detected 34 of 35 (97%) patients who were found to harbor a mutation. This PCR-based selection of patients for mutation analysis is a very useful practical method that is time, labor and cost-effective and paves the way for a more rational, and indeed very powerful, molecular approach to identifying patients in whom imatinib might not be the best treatment in the longer term. Furthermore, recent technological advances in the analysis of heteroduplex DNA by denaturing high-performance liquid chromatography (dHPLC-"Wave" technology) have made the routine detection of mutations more straightforward^{ref1, ref2, ref3}. One could make a case for molecular monitoring alone: no more marrows, forget morphology, dispense with laborious cytogenetic testing. However, until PCR and mutation analysis are more broadly and reliably quality assured, such a move may be a little premature and some would argue strongly that conventional cytogenetics should not be dropped at this stage^ref.
• how should a suboptimal response to imatinib be defined? This is becoming perhaps the key question in imatinib therapy at present as it is likely to become the major determinant of which patients proceed to allografting, a much riskier therapy than taking imatinib but one with the potential to cure. An analysis of data from the IRIS0106 study presented at the ASH 2003 meeting has proven very useful in attempting to predict, based on early cytogenetic response, which patients are likely to achieve a complete cytogenetic response (CCR) over time. These data indicate that :
• the achievement of CCR is important in avoiding disease progression
• achievement of CCR is beneficial no matter how long it takes to get there
• a patient is unlikely to achieve CCR if they have not achieved any significant cytogenetic response by 6 months
• a patient is unlikely to achieve CCR if they have not achieved a major cytogenetic response by 12 months.
Other studies have also shown that failure to achieve a significant cytogenetic response on imatinib therapy is an adverse prognostic feature (O'Dwyer ME, Mauro MJ, Blasdel C, et al. Lack of cytogenetic response is an adverse prognostic factor for relapse of chronic phase CML patients treated with imatinib mesylate (STI571). Blood. 2001;98:137a). There is also an increasing body of evidence that the development of cytopenias while on imatinib therapy is associated with a significantly reduced chance of obtaining a complete cytogenetic response^ref but this is somewhat difficult to quantify. Based on these data, and incorporating what we know from the RQ-PCR and mutation data described above, one can formulate a set of criteria to define sub-optimal response. These criteria would suggest that during the first year from diagnosis, as well as routine blood counts and chemistry analyses at clinic visits, patients should ideally have 20 ml blood taken (in 4 EDTA tubes) for PCR at 3-month intervals and have their bone marrow cytogenetic response analyzed at 6 month intervals. Definition of "suboptimal" response. These criteria are based on imatinib monotherapy, 400 mg daily for 6 to 12 months
• broadly applicable:
• 1. failure to achieve a complete hematological response after 3 months
• 2. failure to achieve any significant cytogenetic response after 6 months
• 3. failure to achieve a major cytogenetic response after 1 year
• applicable with access to reliable specialized facilities :
• 4. failure to achieve a 3-log reduction in BCR-ABL/ABL ratios compared to pre-treatment levels
• 5. detection of ABL kinase domain mutations, particularly those in the P loop. Mutation analysis triggered by 2-fold rise in BCR-ABL/ABL ratio by real-time quantitative polymerase chain reaction (RQ-PCR)
• how should patients with suboptimal response be managed? Having established that we now have reasonably reliable methods for identifying patients who may not do well on imatinib, the obvious question is how such patients should be treated. The key issue for suboptimal responders is whether, if a suitable donor is available, the patient should be transplanted and this possibility should be actively explored. Some, but certainly not all, patients with suboptimal response have mutations in the ABL kinase domain^ref, and various strategies have been suggested to bypass the effects of such mutations^{ref1, ref2}. None are particularly satisfactory. Increasing the dose of imatinib seems, in some patients, to overcome resistance or improve suboptimal response, but in most cases this response is short lived^ref. The best hope for the future is to switch to, or add in, one or more of the new drugs described below.
• can response to therapy be predicted at diagnosis? As noted above, the Sokal (and Euro/Hasford) scores seem to still be useful in selecting good and poor prognosis patients although these scores have not been specifically formulated for imatinib-treated patients. Other laboratory approaches such as assessment of derivative 9q deletion status^ref, and assessment of telomere shortening^ref appear useful. Gene expression profiling as applied to CML is still in its infancy but the technology seems to show promise^{ref1, ref2, ref3,ref4, ref5} in identifying patients who respond well (or don't respond well) to imatinib and changes in gene expression profiles during therapy may also be informative^ref. MDR1 expression would play an important role in Imatinib resistance when the disease is not fully controlled (e.g., progressive disease or during the first months of treatment)^ref.
• can imatinib be safely stopped in patients who have achieved a good response? This is another rather imponderable question to which there is no good answer at present. There are anecdotal reports of patients who have achieved and maintained a complete cytogenetic response and the outcome has varied^ref. In contrast there are reports of patients developing blast crisis shortly after stopping imatinib^ref, so it remains uncertain how safe it is to stop the drug. Investigators are beginning to consider for the first time whether it might be reasonable with very close monitoring to stop therapy in patients who have maintained, for example, a complete molecular response for 2 years, but at the moment there are very few data to inform this particular debate.
• what are the questions being asked in current large-scale clinical trials? A number of investigator-led Phase III prospective randomized trials are currently underway that seek to address similar questions. Most of these Phase III trials are evaluating the role of higher doses of imatinib^ref or imatinib in combination with interferon or Ara-C^ref and have both survival and molecular endpoints^ref. Higher doses of imatinib (800 mg daily) have been evaluated, particularly by the MD Anderson Cancer Center group, in a nonrandomized context, and very encouraging data have emerged. However, the utility of higher doses of imatinib needs to be evaluated in a Phase III setting, and it will be some years before robust answers emerge from these current studies.
• Bcr-Abl-related :
• Bcr-Abl downregulation^{ref1, ref2}
• leptomycin (nuclear entrapment of Bcr-Abl)^ref
• imatinib mesylate + anagrelide is safe and is associated with an 89% complete hematologic response (CHR) rate and 50% cytogenetic response rate in patients with CML in chronic phase and persistent thrombocythemia. Anemia is assumed to be a rather rare and late (early occurence in young children) adverse effect.
• IFN-a_2b can modulate both the tumour cell and the immune system to achieve clinical benefits^ref. Interferon-consensus-sequence-binding protein (ICSBP) is a transcription factor that helps to regulate the expression of many interferon-stimulated genes^ref. Icsbp-knockout mice develop a CML-like syndrome^ref, and the forced expression of Bcr-Abl in mouse cells leads to a CML-like disease that is associated with downregulated Icsbp expression^ref. In addition, Icsbp-knockout mice show marked decreases in plasmacytoid DCs, a principal in vivo source of IFN-a^ref. Together, these findings raise the possibility that IFN-a deficiency can contribute to CML pathogenesis. Consistent with this idea, most CML samples from untreated patients have impaired ICSBP levels, whereas IFN-a therapy substantially increased ICSBP expression^ref. IFN-a treatment might further sensitize leukaemia cells to p53-mediated apoptosis^ref. Patients who responded to IFN-a therapy also generated high-affinity CD8⁺ T cells that specifically recognize CML cells^{ref1, ref2}. 5 MIU/m² subcutaneously daily. Appropriate premedication. Grade 3 hematologic toxicity (3%), fever, flu-like syndrome (2%), thrombocytopenia (2%), mucositis (1%), other toxicity (9%), depression (6%), weight loss/asthenia (6%), other psychologic (5%), nausea/vomiting/diarrhea (4%), skin rash (2%), central neurologic symptoms (1%), cytolytic hepatitis (1%), other GI (1%), peripheral neuropathy (1%). Emetogenic potential level 1^ref
• IFN-a_2b + cytarabine : IFN-a_2b 2 MIU/m²subcutaneously daily, cytarabine 20 mg/m²/day subcutaneously days 1-10 every month. Appropriate premedications. If hematologic control is not achieved, then increase cytarabine to 40 mg/m²/day subcutaneously days 1-15. Grade 3-4 hematoogic toxicity (9%), fever, flu-like syndrome (3%), thrombocytopenia (6%), mucositis (6%), other toxicity (9%), depression (4%), weight loss/asthenia (13%), other psychologic (4%), nausea/vomiting/diarrhea (13%), skin rash (5%), central neurologic symptoms (1%), cytolytic hepatitis (3%), other GI (1%), peripheral neuropathy (1%). Emetogenic potential days 1-10 level 2^ref
• IFN-a_2b + imatinib mesylate
• hydroxyurea (HU) 40 mg/kg/day PO. Grade 3-4 cytopenias, mild nausea, dermatologic and drug fever. Treatment discontinued due to toxicity in < 1% of patients. Emetogenic potential daily level 1^ref
• 17-AAG (hsp90 chaperone function inhibition) (Topaly J, Schad M, Zeller WJ, Ho AD, Fruehauf S. Strong synergism of different signal transduction inhibitors in chronic myelogenous leukemia. Blood. 2001;98:617a)
• tyrosine kinase inhibitor
• adaphostin^ref
• AG490^ref
• Abl & src kinase inhibitor
• AP23464^ref
• BMS354825* (Abl & src kinase inhibitor)^ref
• combined abl and src tyrosine kinase inhibitor, "825," has only recently gone into Phase I clinical studies. Promising initial results have been obtained with an encouraging toxicity profile. The agent can bypass in vitro the effects of ABL kinase domain mutations that cause imatinib resistance but, for reasons that are not clear as yet, 825 is not effective against the T315I mutation^{ref1, ref2}
• As₂O₃ (induction of mitochondrial damage)
• bestatin, actinonin (aminopeptidase inhibition) (Sawafuji K, Miyakawa Y, Weisberg E, Griffin JD, Ikeda Y, Kizaki M. Effects of aminopeptidase inhibitors on STI571 resistant cell lines. Blood. 2001;98:309a)
• decitabine (DNA hypomethylation : La Rosée P, Johnson K, Moseson EM, O'Dwyer ME, Druker BJ. Preclinical evaluation of the efficacy of STI571 in combination with a variety of novel anticancer agents. Blood. 2001;98:839a)
• homoharringtonine (inhibition of protein synthesis)^ref
• PD184352 (MAPK inhibition)^ref
• wortmannin, LY294002 (PI3K inhibition)^{ref1, ref2}
• PS341 (proteasome inhibition) (Gatto SR, Scappini B, Verstovsek S, et al. vitro effects of PS-341 alone and in combination with STI571 in Bcr-Abl positive cell lines both sensitive and resistant to STI571. Blood. 2001;98:101a)
• SCH66336, L-744,832 (farnesyl transferase inhibitors (FTI))^ref1,ref2 (Topaly J, Schad M, Zeller WJ, Ho AD, Fruehauf S. Strong synergism of different signal transduction inhibitors in chronic myelogenous leukemia. Blood. 2001;98:617a)
• TRAIL (induction of apoptosis)^ref
• trichostatin A (histone deacetylase inhibition) (La Rosée P, Johnson K, Moseson EM, O'Dwyer ME, Druker BJ. Preclinical evaluation of the efficacy of STI571 in combination with a variety of novel anticancer agents. Blood. 2001;98:839a)
• anti-BCR-c-Abl vaccines. Bcr-abl⁺ monocytes provide a unique opportunity to generate dendritic cells (DC) expressing a broad spectrum of leukemic antigens, and bcr-abl⁺ DC vaccines may allow immunological eradication of leukemic cells persisting under treatment with the tyrosine kinase inhibitor imatinib. However, the efficiency of bcr-abl⁺ DC vaccines will critically depend on the absence of deleterious effects of bcr-abl and of imatinib on DC functions. Bcr-abl⁺ monocytes, devoid of contamination of CD14^low granulocytic precursors, differentiate into DC with typical immunophenotypical and functional features, and bcr-abl transcription decreases simultaneously. During differentiation, imatinib induces a slight increase of DC apoptosis and prevents CD1a up-regulation in a dose-dependent manner in bcr-abl⁺ and normal monocyte-derived DC, but at most, 25% of DC fail to acquire CD1a. When DC maturation is induced in the presence of imatinib, bcr-abl⁺ and normal monocyte-derived DC up-regulate major histocompatibility complex and costimulatory molecules, CCR7 and CD83. However, secretion of IL-12p70 is decreased in a dose-dependent manner. Imatinib exposure of bcr-abl⁺ and normal monocyte-derived DC during differentiation and maturation is not detrimental to T cell immunostimulatory functions of DC. In sharp contrast, imatinib, when added to DC-T cell cultures, profoundly suppresses DC-mediated T cell proliferation, despite reciprocal DC-T cell activation attested by up-regulation of CD25 on T cells and of CD86 on DC. T cells, not normal or bcr-abl⁺ monocyte-derived DC, are major targets for imatinib immunomodulatory effects. It can be envisioned already that imatinib-free windows will be required to enable vaccination-induced, leukemia-specific T cell expansion^ref.
• all-trans retinoic acid (ATRA)
• allogeneic HSCT was first used to treat patients with CML in chronic phase (CP) in the early 1980s^{ref1, ref2, ref3, ref4}; with clinical experience over 25 years, it is clear that some patients have survived for long periods after transplantation without evidence of relapse and may be regarded as "cured"^ref, although the mechanism underlying such cures is not well defined. Moreover, occasional patients who were treated by allogeneic stem cell transplantation in chronic phase have relapsed > 10 years after an otherwise "successful" transplantation^{ref1, ref2}, and data collated by the Center for International Blood and Marrow Transplant Research show that the cumulative incidence of relapse at 15 years for patients in remission at 5 years after transplantations involving a matched sibling donor for CML in chronic phase was 17% (Goldman JM, Sobocinski KA, Zhang M-J et al. Long-term outcome after allogeneic hematopoietic transplantation (HCT) for CML [abstract]. Biol Blood Marrow Transplant. 2006;12(suppl 1): 17. Abstract no. 40). Of 8738 CML patients who had undergone allogeneic HSCT between 1978 and 1998, nearly 3000 patients were identified as living > 5 years, and of those long-term survivors, approximately 10% experienced late relapse of their CML, even as late as 15 years after the transplantation (Goldman JM, Sobocinski KA, Zhang M-J, et al. Long-term outcome after allogeneic hematopoieitic cell transplantation (HCT) for CML. Biol Blood Marrow Transplant. 2006;12(suppl 1): 17). In another study, of 243 patients with CML who underwent allogeneic HSCT,  53% of patients relapsed as determined by molecular criteria alone (BCR-ABL/ABL ratio > 0.02% on 3 occasions or > 0.05% on 2 occasions) or by progression to abnormal cytogenetic or hematologic states. In addition, in the remaining 47%, the majority had multiple episodes when low positive BCR-ABL PCR results were observed after transplantation, from which the probability of future relapse could be defined. Thus, in the entire cohort, only approximately 15% were classified as persistently negative, defined as always PCR negative (6.6%; 16 patients) for BCR-ABL or with a single, detectable low-level positive result (8.2%; 20 patients). Patients were found with relapse or with BCR-ABL PCR signals whether or not T-cell depletion was used, thus raising the question, to these authors, whether some or potentially even all patients after allogeneic HSCT continue to harbor small quantities of residual leukemic cells^ref. These data clearly indicate a critical need for ongoing serial monitoring of all CML patients who have undergone and will undergo allogeneic HSCT^ref. The importance of this monitoring is highlighted by the desire to identify CML patients early, prior to hematologic relapse, so that they can be assessed for treatment options, such as donor leukocyte infusions, imatinib, or perhaps other second-generation kinase inhibitors^ref. Aat least one group has reported lower PCR BCR-ABL positivity and subsequent relapse rates in similar CML cohorts, although with less lengthy follow-up and in the absence of T-cell depletion^ref
Assessing response to therapy :
• hematological response (HR) :
• complete HR (CHR) :
• 0110 study : WBC < 10,000/ml, PLT < 450,000/ml, myelocytes+metamyelocytes < 5% in PB, no blasts and promyelocytes in PB, basophils < 20%, no extramedullary involvement
• 0102 and 0109 studies : ANC >= 1,500/ml, PLT >= 100,000/ml, no blasts in PB, BM blasts < 5% and no extramedullary disease
• no evidence of leukemia (NEL) : same as for CHR but ANC >= 1,000/ml and PLT >= 20,000/ml (only 0102 and 0109)
• return to chronic phase (RCP) : < 15% blasts in BM and PB, < 30% blasts+promyelocytes in BM and PB, < 20% basophils in PB, no extramedullary disease other than hepatosplenomegaly (only for 0102 and 0109 studies)
• cytogenetic response :
• no cytogenetic response (96-100%)
• minimal cytogenetic response (66-95%)
• minor cytogenetic response (36-65%)
• partial cytogenetic response (1-35%)
• complete cytogenetic response (CCR) (0%)
• MCR : PR (1-35% of metaphases Ph⁺) + CCR (0%)
Protocols : imatinib 400 mg/day =>
• age < 35 years :
• MUD or sibling allogeneic HSCT with busulfan conditioning
• age > 36 years :
• complete molecular response for 6 months (2 measurements) :
• age 36-70 : continue imatinib 400 mg/day => HSC apheresis after G-CSF mobilization
• age > 70 : continue imatinib 400 mg/day
• incomplete molecular response (positive quantitative PCR) for 6 months (2 measurements)
• if complete hematological response (WBC_PB < 10,000/ml for >= 4 weeks, no blasts in PB, myelocytes+metamyelocytes < 5%, no extramedullary disease), major cytogenetic response (Ph+ metaphases < 35% (out of >= 20) in BM blood), and stable (<= 2 log₁₀ variation) molecular response at real-time RT-PCR in the last >= 2 measurements (separated by >= 3 months) :
• age 18-70 : imatinib + IFN-a
• inclusion criteria : serum creatinine < 2.5 mg/dl, negative pregnancy test, PST <= 3, serum bilirubine, ALT, AST, alkaline phosphatase < 3-folds the highest value, no uncontrolled diabetes mellitus, thyroid diseases, infections, cardiopathies, neuropsychiatric disorders
• if increase > 2 log₁₀ in 2 measurements (even separated by < 6 months) : imatinib 800 mg/day or AMN107
• if still increasing : allogeneic HSCT
• if age 36-55 years :
• BMT risk score 0-2 : BM HSCT with busulfan conditioning
• BMT risk score 3 : myeloablative OR RIC-allogeneic HSCT
• BMT risk score 4-7 : RIC PB HSCT
• if age 56-70 years : RIC PB HSCT
Prognosis : average survival = 4-5 years, depending on
• Sokal score, 1984^ref is calculated for patients aged 5-84 years by hazard ratio = exp (0.0116 (age [yrs] - 4.34) + 0 .0345 (spleen [cm] - 7.51 cm) + 0.188 [(platelets [ x10⁹/l]/700)² - 0.563] + 0.0887 (myeloblasts in peripheral blood [%] - 2.1) :
• < 0.8 : low risk/good prognosis
• 0.8-1.2 : intermediate risk/moderate prognosis
• > 1.2 : high risk/poor prognosis
• Hasford score / Euro score, 1998^{ref1, ref2} : 0.6666 x age (0 when < 50 years, 1 otherwise) + 0.042 x spleen size (cm below costal margin) + 1.0956 x platelet count (0 when < 1500, 1 otherwise) + 0.0584 x blasts (%) + 0.0413 x eosinophils (%) + 0.2039 x basophils (0 when < 3%, 1 otherwise) x 1000
• < 780 : low risk group
• 780-1479 : intermediate risk group
• >= 1480 high risk group
• Gratwohl score, 1998^ref
• BMT risk score :
• donor :
• HLA-identical sibling : 0
• HLA-matched unrelated donor : 1
• disease stage :
• CP : 0
• AP : 1
• BC : 2
• age :
• < 20 years : 0
• 20-40 years : 1
• > 40 years : 2
• donor/recipient sex match :
• female donor/male recipient : 1
• other marches : 0
• time lapse between diagnosis and HSCT :
• < 1 year : 0
• > 1 year : 1
5-yr OS :
• 0-1 : 73%
• 2 : 62%
• 3 : 50%
• 4 : 40%
• 5-7 : 15%
• other mutations (monosomy 1, ..)
Experimental animal models : murine bcr-abl⁺ leukemia, 12B1
Web resources : The UnOfficial Gleevec (STI571) Site
• chronic eosinophilic leukemia (CEL) (and the hypereosinophilic syndrome (HES))
• chronic neutrophilic leukemia (CNL) : the major question regarding CNL is whether it is a due to abnormal cytokine production by an associated tumor or abnormal inflammatory response

Epidemiology : < 150 cases have been reported in the literature, mostly in elderlies, and in a number of these cases CNL was found in association with another neoplasm, particularly multiple myeloma^{ref1, ref2, ref3}
Symptoms & signs : gastrointestinal hemorrhages and pruritus (25-30%)
Laboratory examinations :
• neutrophilia > 25,000/ml (promyelocytes + myelocytes + metamyelocytes = 5-10%); increased LAP
• no dysplasia or reticulin fibrosis
• cytogenetics : normal (90%), +8, +9, del(20q), del(11q)
• in a few of the latter cases, molecular studies showed that the neutrophils were not clonal^ref
• normal cytogenetic studies and the "toxic" neutrophil morphology in most reported cases
However, there are well-characterized cases that do meet the criteria for CNL for which cytogenetic or molecular genetic studies have confirmed clonality of the neutrophil lineage^{ref1, ref2, ref3}. In view of these latter reports, the WHO included CNL in the CMPDs, with the recommendation that the possibility of an underlying disease be carefully excluded. If another neoplasm, such as MM, is present, the diagnosis of CNL should be made only if there is genetic evidence of a myeloid neoplasm.
Differential diagnosis : neutrophilia
Therapy : hydroxyurea (HU)
Prognosis : survival ranges from 6 months to 20 years
• chronic myeloproliferative disease, unclassifiable

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