B1 CELLS CHRONIC LYMPHOCYTIC LEUKEMIA (B-CLL) / SMALL LYMPHOCYTIC LYMPHOMA

Table of contents :

Epidemiology Aetiology Pathogenesis Symptoms & signs Laboratory examinations Differential diagnosis Therapy  Prognosis Richter's syndrome prolymphocytic leukemia (PLL) Web resources

Epidemiology : CLL is the most frequent type of leukemia in the Western world and affects mainly elderly individuals, but about a third of patients are < 60 years of age at diagnosis^ref. 95% of all CLLs. Prevalence : 15:100,000.
Aetiology : risk factors :

• male gender
• increasing age
• ethnicity (high in Caucasians, lowest in Asians) and family history : migration studies confirm that ethnic groups retain the risk associated with their origin rather than their new location, favoring a role for heredity. Kindreds with multiple cases of CLL have been well described in the literature and studies in large populations confirm that lymphoproliferative malignancies and especially CLL occur together at a rate that cannot be attributed to chance. Since environmental factors cannot readily explain the familial aggregations, a hereditary factor that affects susceptibility to CLL is likely. The identification of clones that are immunophenotypically identical to CLL in healthy individuals from CLL kindreds (14% to 18%) as well as in the general population (3.5% in age bracket >65 years) suggests a possible precursor condition, but longitudinal studies will be necessary to establish significance in the general population. Family (linkage) and population (candidate gene) studies to date have been too small to identify the specific genes that account for increased susceptibility; larger studies including planned consortia to identify additional high-risk kindreds for genetic studies, as well as the application of advanced technologies such as genomics, cytogenetic, expression, and proteomics, are widely expected to advance understanding over the next few years^ref. Some families show classic findings in support of an autosomal dominant mode of genetic transmission of CLL^ref
• farming and pesticide exposure
• ionizing radiation exposure^ref
• tobacco users and cigarette smokers (OR = 1.6)^ref, and environmental tobacco smoke (ETS) (OR = 2.3)^ref. Approximately 2% of patients with CLL develop lung carcinoma. In a study, 85% of the patients were smokers. These patients had a high risk of a third primary malignancy (including melanoma, basal cell carcinoma, laryngeal carcinoma, and colon carcinoma). Lung carcinoma was diagnosed a decade after CLL. Patients who develop both diseases die of lung carcinoma and not CLL or other solid tumors. CLL and poor performance status limit treatment, particularly for patients with unresectable lung carcinoma^ref
• familal B-CLL : genetic basis in about 10% of patients^ref. The lifetime risk of developing CLL in first-degree relatives of patients with the disease is increased by a factor of 8. The inherited predisposition to CLL may be mediated by a gene with pleiotropic effects as suggested by a two-fold increase, in relatives of CLL patients, of other B-cell disorders, mostly NHL and HL. In fact, in our database of over 300 families with CLL we have identified 12 with multiple myeloma in relatives^ref. By analogy with MGUS, when healthy relatives from CLL families were examined with sensitive flow cytometry methods, monoclonal B-cell lymphocytes are detected with high frequency^ref.
• HLA-B8 is a marker of mild disease while HLA-A2 and the closely associated antigens B12(44) and DR7 are markers for severe disease^ref
• HLA B12, alone or in combination with HLA A2, indicated better prognosis. Relatives HLA identical with the patients showed no evidence of white cell disorder when compared with haplo- or non-identical relatives, or controls. As a group, however, relatives of patients had fewer lymphocytes than relatives of controls^ref
• an increased incidence of lymphoid malignancy has been reported in western Ashkenazi Jews and in particular those of Russian descent.^ref

	IgVH unmutated B-CLL (U-CLL) (intraclonal variation < 95%) (50%)	IgVH mutated B-CLL (M-CLL) (50%)
intraclonal variation (ongoing SHM)	absent or very low	low
morphology	atypical according to the criteria of Matutes et alref as > 10% (but < 55%) prolymphocytes or > 15% cells with cleaved nuclei and/or lymphoplasmacytoid cells in the peripheral blood of patients whose predominant cell type was a small lymphocyte with coarsely clumped chromatin (dense cells (DC) / 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))	typical (Gumprecht shadow or basket cells (BC) are highly suggestiveref1, ref2)
stage at diagnosis	advanced (C)	early (A, B)
cell source	pregerminal center naive B-cells	germinal center exposed B-cells
CD38	high (> 30%)	low (< 30%)
ZAP70ref	high (> 20%)ref1, ref2	low (< 20%)
chromosomal aberrations	+12, del(11q), del(17p)	normal or del(13q14) (which contains 2 small miRNA genes that are turned off in about 60% of CLL cases)
miRNA signatureref	reduced expression of miR-16	reduced expression of miR-16
therapy	poor response	good response
survival	average life expectancy of about 8 years and is universally fatal	average survival of 25 years and most people with this form die of other causes rather than of CLL)

There is a bias in the use of certain V_H, D, and J_H genes among B-CLL cells. V_H genes of ~ 50% of the IgM⁺ B-CLL cells and ~ 75% of the non-IgM⁺ B-CLL cells can exhibit somatic mutations according to the V_H family expressed by the B-CLL cell (V_H3 expressers displaying more mutation than V_H1 and V_H4 expressers). In addition, the extent of mutation can be sizeable with ~ 32% of the IgM⁺ cases and ~ 68% of the non-IgM⁺ cases differing by > 5% from the most similar germline gene. Approximately 20% of the mutated V_H genes display replacement mutations in a pattern consistent with antigen selection. However, CDR3 characteristics (D and J_H gene use and association and HCDR3 length, composition, and charge) suggest that selection for distinct BCR occurs in many more B-CLL cells. Many B-CLL cells have been previously stimulated, placing them in the "experienced" or "memory" CD5⁺ B cell subset.

• a restricted set of variable (V) region genes, specifically V_H1-69 (also known as 51p1) and V_k3-A27 (also known as kv325), which bind to HCV E2 envelope protein, occurs in 10-20% of patients^{ref1, ref2}. Preferential use of the 51p1 gene has also been observed in CLL, with a prevalence of > 20% in particular geographic areas^ref. However, unlike the HCV-associated immunocytomas, the 51p1 V_H sequences in CLL are almost exclusively unmutated and usually have significantly longer CDR3 regions^{ref1, ref2}. V_H1-69 B-CLLs constitute a uniform group of patients that more often present at advanced clinical stages and require early treatment, but their survival does not differ significantly from patients with other unmutated V_H genes^ref.
• cutaneous manifestations of B-CLL comprise a wide spectrum of clinicopathologic presentations. In some cases, onset of skin lesions is triggered by antigenic stimulation, and specific skin infiltrates at sites of previous herpes simplex or herpes zoster infection have been well documented. Specific skin manifestations of B-CLL can also be observed at sites typical for lymphadenosis benigna cutis (nipple, scrotum, earlobe), a Borrelia burgdorferi-associated cutaneous B-cell pseudolymphoma. Histology of solitary erythematous plaques or nodules located on the nipple (4 cases) and scrotum (2 cases) from 6 patients with B-CLL (M : F = 4 : 2; mean age: 67.8) revealed in all cases a dense, monomorphous infiltrate of small lymphocytes showing an aberrant CD20⁺/CD43⁺ phenotype. In all cases monoclonality was demonstrated by PCR analysis of the J_H gene rearrangement. PCR analysis showed in four of the six cases the presence of DNA sequences specific for B.burgdorferi. Infection with B. burgdorferi can trigger the development of specific cutaneous infiltrates in patients with B-CLL^ref.

CLL cells do not undergo SHM in their V region genes

• lymphocytosis =>
• lymphadenopathy =>
• superficial
• deep : phlebothrombosis, lymphedema, obstructive jaundice, hydronephrosis
• hepatosplenomegaly =>
• bone marrow infiltration (anemia, neutropenia, thrombocytopenia) =>
• skin
• gastrointestinal involvement (gastric ulcers/hemorrhages, malabsorption enteritis, miliary pulmonary nodular infiltration, pleural effusion)
• pulmonary apparatus
• central nervous system =>
• autoimmune diseases :
• autoimmune hemolytic anemia (AIHA) occurs in approximately 10-25% of CLL patients at some time during the course of the disease^{ref1, ref2, ref3}. Although the pathogenic autoantibodies occasionally may be produced by the malignant B-cell clone^{ref1, ref2}. in most cases the autoantibodies appear to be made by remnant normal B lymphocyte^{ref1, ref2}. This view is supported by the observation that the antibodies eluted from the red blood cells (RBC) are typically “warm-reactive” polyclonal IgG antibodies that display activity against monomorphic antigens of the rhesus system. On the other hand, the antibodies produced by the B-CLL cells are usually IgM and are always monoclonal. Experiments with SCID mice reconstituted with peripheral blood lymphocytes from a B-CLL patient have also suggested that the anti-RBC antibodies are not related to the malignant clone^ref. A large proportion of these animals developed human IgG anti-RBC autoantibodies that were of polyclonal origin. Collectively, these data have suggested that the anti-erythrocyte autoantibodies in B-CLL occur as a consequence of progressive dilution or inhibition of nonneoplastic B or T lymphocytes whose role is to antagonize the autoreactive Bcell clones^{ref1, ref2}. This model, however, does not provide an explanation for the much higher frequency of AIHA in CLL versus other hematologic malignancies and the lack of correlation between the circulating levels of pathogenic anti-RBC autoantibodies and the duration and severity of the underlying lymphoproliferative disease : the expected frequency of 51p1 in CLL is approximately 15%, which is significantly lower than the frequency observed in the CLL-AIHA patients (47%). A significant correlation between V_H gene usage and AIHA was also observed for the DP-50 gene, which was not present among 75 surveyed sequences^ref, but accounted for 33% of the B-CLL-AIHA sequences^ref. Interestingly, this V_H gene has also been found in 5 of 14 investigated human monoclonal anti-Rh(D) antibodies, indicating that it is also overrepresented in the human anti-Rh(D) response^ref. The antibodies produced by the B-CLL cells could have a similar specificity and could promote the binding of RBCs, sensitized with polyclonal IgG antibodies, to the FcRs on macrophages. A subset of B-CLL cells produces secretory g-chain transcripts, which in 2 of the B-CLL-AIHA patients were predominantly of the g₂ and g₃ H-chain isotype. Antibodies of the IgG₃ subclass seem to play an important role in the initial adherence of sensitized erythrocytes to macrophages because of their greatest affinity for the FcgRIII^ref. Alternatively, the B-CLL antibodies might not bind directly to RBC antigens, but could represent second antibodies that bind to the Fc portion of the anti-RBC antibodies. Initial experiments with a recombinant Fv antibody that corresponds to the CLL Ig of patient HA2 show low-affinity reactivity against a number of self antigens including human IgG. Finally, the B-CLL antibodies could have antiidiotypic activity and could stimulate normal autoreactive B cells to produce anti-RBC antibodies. Antiidiotypic IgG antibodies that crossreact with some anti-Rh(D) antibodies have been detected in eluates from sensitized RBCs and have been implicated in the pathogenesis of AIHA^ref
• infections : reactivation of VZV

• polyadenopathic systemic form (mainly lymph node involvement)
• pure splenomegalic form (no lymphadenomegaly, favourable prognosis, first reported by Binet in 1977)
• dysimmunoglobulinic form
• erythrolytic variant : severe AIHA due to anti-D antibodies
• paraproteinemic or secretory form (5%) : monoclonal Ig in serum
• thrombocytopenic form (autoimmune thrombocytopenia)
• pure medullary form (very rare)
• erythrodermic form (Sezary-like syndrome)
• regional form (single organ or apparatus)
• insect bite-like reaction and true hypersensitivity to mosquito bites

• k (60%) > l (40%)
• the monoclonal B cells must represent the majority of leucocytes with an absolute lymphocyte count >5 · 10⁹ cells/l and < 55% prolymphocytes, which has persisted for at least 1 month^ref
• lymphocytosis > 30%
• hypogammaglobulinemia (80% at diagnosis)
• very high levels of sCD23
• cytomorphology : small mature lymphoid cells, with high nuclear-to-cytoplasm ratio, scant cytoplasm, and round nuclei with highly condensed chromatin and inconspicuous nucleolus. Admixed with these cells may be prolymphocytes and paraimmunoblasts, characterized by larger size and prominent nucleoli


Bone marrow infiltration pattern :
• nodular (10%)
• interstitial (40%)
• diffuse (25%)
• mixed (25%)
• never intrasinusoidal (differential diagnosis with SMZL)
• immunophenotype : pan-T cell marker CD5⁺ (differential diagnosis with MCL !)6⁺10 / CALLA^-11a⁺11c^-19⁺20^loFMC7^lo21⁺22^weak23⁺30^-38^+/-79b^lo sIg (IgM or IgD)^lo. CD5^- B-CLLs represent < 5% of B-CLLs. When compared with CD5⁺ B-CLLs, more cases have advanced disease, splenomegaly, and a cytologically mixed CLL pattern according to the French-American-British (FAB) classification. In addition, CD23 expression is frequently absent, whereas FMC7 positivity and expression of high amounts of SIg are more frequently observed than in CD5⁺ CLL^ref. Thus, CD5^- B-CLL seems to be intermediate between classical CLL and prolymphocytic leukemia^ref. Immunohistochemically, B-CLL differs from SMZL for a lower expression of CD20 and positivity for CD23
• atypical CLL :
• CD5^-ref
• CD8⁺ (< 0.5%)^ref
• CD11c⁺
• CD20^high
• FMC7^high
• CD22^high
• CD23^-
• CD56^+ref
• CD154^+ref
• IgL high
• cytogenetics :
• trisomy 12 (MDM2; 10-15% in conventional cytogenetics, 15% at FISH) : atypical/mixed-type CLL requiring therapy, reduced survival
• 13q14- (10% in conventional cytogenetics, 53% at FISH) : typical morphology, favorable prognosis
• 11q21- (8% at conventional cytogenetics, 19% at FISH) : typical morphology, massive lymphadenopathy, young age, reduced survival
• 6q- (6% at conventional cytogenetics, 9% at FISH) : artpical morphology, reduced survival
• 11q23- (20% : ATM) => increased TfR expression => more severe disease course, with earlier onset of symptoms, shortened lymphocyte doubling time, poor response to therapy, and shortened survival (may also partially explain why gallium, an atomically iron-like toxic metal that binds to transferrin and the TfR is incorporated into cells and was previously demonstrated to have anti-tumor activity in patients with lymphomas refractory to other chemotherapeutic treatments)^ref
• 17p- (4% at conventional cytogenetics, 8% at FISH : p53) : more common in atypical CLL and advanced stages of CLL, purine analog-refractary
• t(11;14)(q13;q32) (2-3%) : mixed type (CLL/PLL), frequent transformation into PLL, similar to leukemic MCL
• t(14;19)(q32;q13) : very rare, atypical morphology, young age, aggressive course
• molecular biology : a signature of microRNA (miRNA) gene expression profiling (based on the development of a microchip containing oligonucleotides corresponding to 245 miRNAs from human and mouse genomes) correlates with diagnosis and prognosis of B-CLL^ref. 2 signatures are found : one correlates with mutations in the variable region of the immunoglobulin gene—a good prognostic indicator, the other correlated with the deletion of chr13q14—a region containing 2 small miRNA genes that are downregulated in about 60% of CLL. There was only one common denominator between these 2 signatures, and that was the downregulation of miR-16, which is in fact the miR found deleted in CLL^ref. miR-15a and miR-16-1 are deleted or down-regulated in the majority of CLLs. miR-15a and miR-16-1 expression is inversely correlated to Bcl2 expression in CLL and both microRNAs negatively regulate Bcl2 at a posttranscriptional level. BCL2 repression by these microRNAs induces apoptopsis in a leukemic cell line model. Therefore, miR-15 and miR-16 are natural antisense Bcl2 interactors that could be used for therapy of Bcl2-overexpressing tumors^ref. A unique microRNA expression signature composed of 13 genes (of 190 analyzed) differentiates cases of B-CLL with low levels of ZAP-70 expression from those with high levels and cases with unmutated IgV_H from those with mutated IgV_H. The same microRNA signature is also associated with the presence or absence of disease progression. A germ-line mutation in the miR-16-1–miR-15a primary precursor causes low levels of microRNA expression in vitro and in vivo and is associated with deletion of the normal allele. Germ-line or somatic mutations are found in 5 of 42 sequenced microRNAs in 11 of 75 patients with CLL, but no such mutations are found in 160 subjects without cancer^ref
• abdominal and superficial lymph nodes echography
• chest X-rays
• CBC, SGOT, SGPT, total and direct bilirubin, alkaline phosphatase, gGT, uricemia, glycemia, azotemia, cretininemia, serum protein electrophoresis, b₂-microglobulin, LDH, ESR, PCR, hepatitis mosaic, direct and indirect Coombs tests, haptoglobin, IgG, IgM, IgA, ANA, ENA, ANCA, anti-endomysium, anti-dsDNA antibodies, RF

• mantle cell lymphoma (MCL), which is also a tumor of CD5⁺ B cells and which has a much worse prognosis than CLL
• follicular lymphoma (FL) in leukemic phase; it is important to stress that t(14;18)(q32;q21) or t(18;22)(q21;q11) translocations have been previously reported in CLL and are not confined to FL^ref. B-CLL has not the intraclonal variation of immunoglobulin V_H genes seen in FL^ref , implying that cells showing somatic mutations are no longer under the influence of mutator enzymes and have therefore passed through the GC^ref
• splenic marginal zone lymphoma with villous lymphocytes, of which a small proportion are reportedly CD5^+ref
• nodal marginal zone lymphoma
• hairy cell leukemia
• prolymphocytic leukemia (B cell or T cell)
• lymphoplasmacytic lymphoma
• Sézary syndrome
• smoldering adult T cell leukemia/lymphoma

Binet's staging, 1981ref	criteria		Rai's staging, 1975ref	median survival, years
A (80% at diagnosis)	no lymphadenomegaly	lymphocytosis > 15,000/ml in peripheral blood or > 40% of bone marrow cells	0	>10
	1 or 2 lymphoid areas involved (cervical, axillary, inguinofemoral, or liver+spleen)	lymphocytosis + lymphadenomegaly	I	7
B	>= 3 lymphoid areas involved	lymphocytosis + hepatomegaly and/or splenomegaly	II	< 5
C (Fisher-Evans syndrome)	autoimmune hemolytic anemia (AIHA) < 10 g/dl in females, < 11 g/dl in males (from activation of non-neoplastic clones) independently from the number of lymphoid areas involved	lymphocytosis + autoimmune hemolytic anemia (AIHA) (HGB > 11 g/dl; from activation of non-neoplastic clones)	III	1.5-2
	autoimmune thrombocytopenia < 100,000/ml (from activation of non-neoplastic clones) independently from the number of lymphoid areas involved	lymphocytosis + autoimmune thrombocytopenia (PLT > 100,000/ml; due to activation of non-neoplastic clones)	IV	1.5-2

• clinical patient characteristics such as age, gender and performance status
• laboratory parameters reflecting the tumor burden or disease activity such as :
• absolute lymphocyte count
• LDH elevation
• bone marrow infiltration pattern
• lymphocyte doubling time (LDT)^{ref1, ref2, ref3}
• serum markers such as :
• soluble CD23
• ß₂-microglobulin (ß₂-MG)
• thymidine kinase (TK)^{ref1, ref2}
• cytogenetics :
• genomic aberrations^ref can be identified in about 80% of CLL cases by fluorescence in-situ hybridization (FISH) of interphase cell nuclei ("Interphase-Cytogenetics") with a disease-specific comprehensive probe set^ref :

	low-risk					high-risk
	13q-	13q-single	VH mutated	6q-	+12q	17p-	11q-	VH unmutated
single center cohort of CLL patients distributed over all stages	55%	36%	44%	7%	16%	7%	18%	56%
CLL1 trial of the GCLLSG for untreated Binet A patients with no classical indication for treatment	59%	40%	59%	2%	13%	4%	10%	41%
CLL4 trial (randomized F vs FC) of the GCLLSG for untreated Binet B/C patients up to 65 years of age with indication for treatment	53%	34%	31%	9%	11%	3%	21%	69%
CLL3 trial (early myeloablative radio-chemotherapy and autologous transplantation) of the GCLLSG for Binet B/C patients up to 60 years of age with maximum one line of prior therapy	52%	27%	32%	6%	12%	3%	22%	68%
CLL2H trial (subcutaneous alemtuzumab) of the GCLLSG for fludarabine-refractory patients with indication for treatment	48%	14%	19%	9%	18%	27%	32%	81%

Genomic aberrations provide insights into the pathogenesis of the disease since they point to loci of candidate genes (17p13: p53; 11q22-q23: ATM) and identify subgroups of patients with distinct clinical features. Specific genomic aberrations have been associated with disease characteristics such as marked lymphadenopathy (11q deletion) and resistance to treatment (17p deletion). Moreover, such aberrations define specific subgroups that differ in the rate of disease progression as determined by the time from diagnosis to first treatment and the overall survival time of CLL^ref.
Probabilities of disease progression as assessed by the treatment-free interval in the 5 dominant categories of genomic aberrations. The median treatment-free intervals for the 17p deletion (n = 23), 11q deletion (n = 56), 12q trisomy (n = 47), normal karyotype (n = 57), and 13q deletion (single abnormality; n = 117) groups were 9, 13, 33, 49, and 92 months, respectively :

Estimated survival probabilities from the date of diagnosis in 325 CLL patients divided into the 5 categories defined in a hierarchical model of genomic aberrations in CLL. The median survival times for the 17p deletion (n = 23), 11q deletion (n = 56), 12q trisomy (n = 47), normal karyotype (n = 57), and 13q deletion (as single abnormality; n = 117) groups were 32, 79, 114, 111, and 133 months, respectively.
Relation of V_H mutation status and genomic aberrations in 300 CLL cases^ref :

	VH mutated	VH unmutated	P-value at Fisher’s exact test
clonal aberrations	80%	84%	0.37
13q14 deletion appear to have a more favorable outcomeref	65%	48%	0.004
13q deletion single	50%	26%	< 0.001
trisomy 12	15%	19%	0.44
11q23 deletion have been associated with a shorter median survivalref1, ref2, ref3, ref4, ref5, ref6	4%	27%	< 0.001
17p deletion	3%	10%	0.03
17p or 11q deletion	7%	35%	< 0.001

V_H mutation status and genomic aberrations are 2 separate genetic parameters of prognostic relevance, but they appear to be correlated. Unfavorable aberrations (11q-, 17p-) occur more frequently in V_H unmutated tumors, and favorable aberrations (13q-, 13q- single) occur more frequently in the V_H mutated subgroup^{ref1, ref2, ref3}. This unbalanced distribution of genomic aberrations emphasizes the different biological background of the CLL subgroups with mutated or unmutated V_H and could in part explain their different clinical course. On the other hand, about 66% of the V_H-unmutated CLL cases show no unfavorable genomic aberrations, indicating a differential influence of these factors.
Incidence of genomic aberrations and V_H mutation status in one large single center fluorescence in situ hybridization (FISH) study compared with preliminary results from prospective multicenter trials of the German Chronic Lymphocytic Leukemia (CLL) Study Group (GCLLSG) for different clinical situations.
• gene abnormalities (p53 and ATM)
• CLL unmutated cases showed a strikingly higher level of CLLU1 expression (several hundred fold) compared with mutated ones, which in turn showed a median 5.7-fold up-regulation compared with normal B cells.
• the mutation status of the variable segments of immunoglobulin heavy chain genes (V_H), or surrogate markers for these factors (CD38, ZAP-70, LPL, etc.)^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9} (Orchard J, Davis Z, Ibbotson R, Richards S, Catovsky D, Oscier D. Comparison of ZAP-70, IgVH gene mutational status and CD38 in CLL patients requiring therapy: preliminary report from the UK CLL IV trial [abstract]. Blood. 2003;102:#105). Although early studies of the IgH gene in CLL failed to demonstrate somatic mutation^{ref1, ref2, ref3, ref4}, more recent analyses have shown that a substantial (20-50%) percentage of cases show somatic mutations, suggesting that CLL may arise from either pregerminal center naive B-cells, or germinal center exposed B-cells^{ref1, ref2, ref3, ref4, ref5}. These studies also suggest that the presence or frequency of somatic mutation correlates with immunophenotype^ref, the usage of variable region gene (V_H) families^ref and the presence of specific chromosomal abnormalities^ref. Fais et al^ref reported somatic mutation in 50% of the IgM⁺ expressing cases, with the highest incidence noted in the V_H3 family, compared with V_H1 and V_H4. Oscier et al^ref noted that cases with trisomy 12 lacked somatic mutation, whereas cases with 13q14 deletion showed significant levels of somatic mutation. The existence of CLL lymphocytes having undergone somatic mutation suggests that a subset of CLL is derived from memory B cells that have passed through the germinal center stage of B-cell differentiation. Taken together, the above observations indicate that CLL is more heterogeneous than previously thought and may develop either from ontogenically naive B lymphocytes or from a more mature antigen-exposed memory B cells. The mutation status of the V_H genes is one of the most important molecular genetic parameters defining pathogenic and prognostic subgroups of CLL^{ref1, ref2} :
• pregerminal center, unmutated (V_H homology > 98%) (50%) : unfavorable course with rapid progression (estimated median survival time = 79 months)
• germinal center, mutated (V_H homology < 98%) (50%) : slow progression and long survival (estimated median survival time = 152 months)
However, genome-wide gene expression profiling studies revealed a surprisingly homogeneous pattern of gene expression in both subtypes of CLL with only a limited set of genes being differentially expressed in the subgroups^{ref1, ref2}. Most importantly, it could be demonstrated that the V_H mutation status is clinically highly relevant^{ref1, ref2}. Survival curves for patients distributed over all stages (n = 300) and separately for patients diagnosed with Binet stage A disease (n = 189) from the largest published cohort^ref.
• independent of the mutation status, the usage of specific V_H genes such as V_H3-21 may be associated with an inferior outcome^ref.
To make the estimation of prognosis based on the V_H status accessible to the routine hematology laboratory, surrogate markers for the V_H status were identified :
• originally, a correlation was observed between the V_H mutation status and CD38 expression of the CLL cells pointing to CD38 expression as a prognostic marker^ref
• based on genome-wide gene expression studies other surrogate markers such as ZAP-70 expression were identified and validated^{ref1, ref2}. ZAP-70 expression appears to strongly correlate with V_H mutation status and was therefore a strong prognostic marker in a pivotal study^ref.
However, for both CD38 and ZAP-70, subsequent studies have yielded controversial results with regard to their validity as a surrogate marker for V_H and prognostic indicator. The facts that ... :
• divergent results have been obtained in different laboratories (CD38 and ZAP-70)
• the expression level may change over time (CD38)
• a careful separation of T cells is necessary (ZAP-70)
• different cut-off values to distinguish "positive" from "negative" cases were defined (CD38 and ZAP-70)
• approximately 10–30% of cases show discordant status for CD38 or ZAP-70 as compared to V_H in all series described
... indicate that these markers may not be as reliable as initially thought for routine diagnostics^{ref1, ref2, ref3, ref4} (Orchard J, Davis Z, Ibbotson R, Richards S, Catovsky D, Oscier D. Comparison of ZAP-70, IgV_H gene mutational status and CD38 in CLL patients requiring therapy: preliminary report from the UK CLL IV trial [abstract]. Blood. 2003;102:#105). Furthermore, the relationship of the V_H mutation status to other biological disease characteristics of potential pathogenic relevance such as ongoing V_H hypermutation as well as VDJ diversification, telomere length, ability for BCR signaling, expression of specific genes such as AID, and genomic aberrations are currently undergoing examination. Levels of F-actin-capping protein b subunit (CAPZB), 14-3-3b protein / YWHAB, and laminin-binding protein precursor / lectin, galactoside-binding, soluble, 3 (LGALS3) were significantly increased in mutated B-CLL relative to unmutated B-CLL. In addition, primary sequence data from tandem mass spectrometry showed that nucleophosmin was present as several protein spots in M-CLL but was not detected in unmutated B-CLL samples, suggesting that several post-translationally modified forms of nucleophosmin vary between these 2 sample groups. No specific differences were found between CD38⁺ and CD38^- patient samples using the same approach^ref.
IgG⁺ CLL is phenotypically similar to mutated IgM⁺IgD⁺ CLL (M-CLL) and variably expressed CD38 (4 of 14). ZAP-70, a tyrosine kinase preferentially expressed in unmutated CLL, was found in only 2 of 14 cases. The ability to signal via surface IgM (sIgM) varies between the main subsets of CLL and is associated with expression of ZAP-70. In IgG⁺ CLL, 9 of 14 responded to engagement of sIgG with no apparent requirement for expression of CD38 or ZAP-70. However, signal capacity correlated with intensity of sIgG expression. Most switched immunoglobulin variable region genes were somatically mutated without intraclonal variation, and no case expressed activation-induced cytidine deaminase. Derivation from a postgerminal center B cell is, therefore, likely, and a relationship with M-CLL is suggested. This is supported by a shared biased usage of the V4-34 gene. Similar bias in normal B cells developed with age, providing an expanded population for transforming events. However, conserved sequences detected in the CDR3 of V4-34-encoded gamma chains were not found M-CLL, indicating no direct path of isotype switch from M-CLL. IgG⁺ CLL is likely to arise from an age-related expanded pool of B cells, on a path parallel to M-CLL, and perhaps with a similar clinical course^ref
• longer survival in individuals heterozygous for 1513AC P2X₇ SNP, expecially in patients with mutated V_H genes^ref. P2X₇ receptor triggers IL-1 maturation and exteriorization^ref
• ongoing CSR occurs in only a fraction of the CLL clone, as only small proportions of CD5⁺CD19⁺ cells express surface IgG or IgA and lack IgM and IgD.

• A) among all 300 patients estimated median survival times were: 17p- 30 months, 11q- 70 months, V_H  98% 89 months. and V_H < 98% not reached (54% survival at 152 months)
• B) in Binet A patients only (n = 189) estimated median survival times were: 17p- 36 months, 11q- 68 months, V_H  98% 86 months months, and VH < 98% not reached (52% survival at 152 months).

• according to V_H mutation status :


• according to genomic aberrations :

• Richter's syndrome (RS)) (5-10%, high malignancy; Richter MN. Generalized reticular cell sarcoma of lymph nodes associated with lymphatic leukemia. Am.J.Pathol. 6:285-292 (1928)) :
• DLBCL (most) : despite DLBCL is generally thought to arise from the B-CLL clone, approximately 50% of the patients had genetically unrelated cancers^ref.
• Hodgkin’s disease variants (rare) arising probably only in V_H mutated CLL^ref
While overall response rates to various therapeutic strategies can vary between 5% and 43%, median survival for RS patients with cytotoxic therapy is 5 to 8 months. Approximately 50% have an age > 60 years, haemoglobin level < 11 g/dL, received > 2 prior therapies, LDH levels > 1.5 time the upper limit of normal, and tumour size >5 cm. Approximately 40% of patients with RS have platelet counts < 100,000, their time to transformation was > 5 years, and b₂-microglobulin levels were 3 times the upper limit of normal. 21% had a PST >1. Most subjects had prior treatment, including chemotherapy alone (61%), chemotherapy + rituximab (36%) and immunotherapy alone with rituximab and campath (3%). Some patients underwent subsequent allo-SCT in remission (7%), as salvage (6%) or as auto-SCT in remission (2%). There were no significant differences between the chemotherapy and chemotherapy/rituximab groups for either mean complete response (11% vs. 13%, respectively) or mean overall response (34% vs. 47%, respectively). The combination of these treated RS patients gave a complete response of 12% and an overall response of 39%, with a median failure-free survival of 7.1 months (95% confidence interval [CI], 5-10 months). Median overall survival was 9.1 months (95% CI, 8-11 months) for the full group of 143 RS patients. When divided according to subsequent transplantation, the patients that received allo-SCT in remission showed significantly (P =.004) greater survival than both the therapy responders without SCT and those that received SCT. In multivariate analysis, of the large range of significant factors for response and survival in the previously treated patients, the significant risk factors for poor survival were PST > 1 (P =.006), LDH level > 1.5 times the upper limit of normal (P =.003), platelet count below 100,000 (P =.012), tumour size > 5 cm (P =.022) and > 2 prior therapies (P =.024). However, when the SCT was included in this multivariate analysis, platelet count lost its significance, and the absence of allo-SCT in remission became a significant risk factor for poor survival (P =.002). As the use of chemotherapy with or without immunotherapy followed by allo-SCT resulted in improved survival when compared with the other therapies used in this trial, allogenic stem cell transplantation should be offered to all RS patients with available donors as post-remission therapy. At The University of Texas M.D. Anderson Cancer Center the incidence of RS is 3.9%. The large cells of RS may arise through transformation of the original CLL clone or represent a new neoplasm. RS may be triggered by viral infections, such as HHV-4 / EBV. Trisomy 12 and chromosome 11 abnormalities, as well as multiple genetic defects, have been described in patients with RS. These abnormalities may cause CLL cells to proliferate and, by facilitating the acquisition of new genetic abnormalities, to transform into RS cells. The therapeutic strategies for RS typically include therapies developed for NHL or acute lymphoblastic leukemia. The reported response rates with these therapies are 5% to 43%, and the median survival duration ranges from 5 to 8 months. The median overall survival duration at our institution of patients with RS is 9.1 months (95% confidence interval, 7.8 to 11 months), and the median FFS duration is 7.1 months (95% confidence interval, 5.1 to 10.4 months). Patients appear to benefit from cytoreductive therapy consisting of chemotherapy and immunotherapy, followed by allogeneic stem cell transplantation, as postremission therapy. As part of a program aiming to cure RS, we are currently conducting a clinical trial of oxaliplatin, fludarabine, and cytarabine in combination with rituximab and recommend postremission therapy, including allogeneic stem cell transplantation in patients with available donors^ref
• B-cell prolymphocytic leukemia (B-PLL) (5-10%; 60% of all PLLs)

Epidemiology : male-to-female ratio 4:1, age > 60 years; 20-fold lower incidence than B-CLL; a relatively rare form of chronic lymphoproliferative disease, first described by Galton in 1974^ref. Although PLL is considered to be distinct from CLL by many criteria, the delineation between the 2 is not clearcut because some cases of CLL can transform into PLL^ref and most PLL cases studied appeared to evolve from the B-CLL clone^ref. Furthermore, mixed forms of CLL-PL have been identified in which the number of prolymphocytes is 10-55%^ref
Symptoms & signs : massive splenomegaly and only rarely lymphadenopathy
Laboratory examinations :
• k (70%) > l (30%)
• according to French-American-British (FAB) diagnostic criteria, the hallmark of the disease is the presence of > 55% (or > 15,000/ml) circulating morphologically identified prolymphocytes^ref. It is a distinct clinicopathological entity characterized by massive splenomegaly without lymph node enlargement, frequent marked hyperleukocytosis (> 150,000/ml in 60%; the majority of which are prolymphocytes), and associated anemia and thrombocytopenia. Morphologically, prolymphocytes are large cells with condensed nuclear chromatin and a prominent central nucleolus. The vast majority of PLL are from the B-lineage origin and express high density monotypic sIg, usually IgM with or without IgD, and the CD19, CD20, and CD22 B cell marker (Catovsky D: Prolymphocytic leukemia, in Catovsky D, Foa R (eds): The Lymphoid Leukemia. London, UK, Butterworths, 1990, p 438).
• immunophenotype : CD5^-/+(30%)19⁺20⁺FMC7⁺21⁺22⁺23^-30^- sIg^hi
Prognosis is often poor (median survival = 24 months)
• acute leukemias (rare) :
• B cell acute lymphoblastic leukemia (B-ALL) (rare) : there is genetic relatedness to the B-CLL clone in some cases and unrelatedness in others^ref
• multiple myeloma : there is genetic relatedness to the B-CLL clone in some cases and unrelatedness in others^ref
• acute myeloid leukemia (due to chemotherapy)

• chlorambucil 0.1 mg/kg/day PO daily is the oldest and best-known treatment for CLL. Emetogenic potential : level 1. 66 second malignancies in chlorambucil arm over 72 months. 48 second malignancies in observation arms over 67 months. It can induce partial remissions (PR) in 60–70% of previously untreated patients, but no significant complete remissions (CR)^ref
• chlorambucil + prednisone :
• chlorambucil 0.3 mg/kg/day PO days 1-5, prednisone 40 g/m2/day PO days 1-5. Repeat cycles every 28 days. Emetogenic potential days 1-5 level 1^ref
• chlorambucil 30 mg/m² PO day 1, prednisone 80 mg/day PO days 1-5. Repeat cycle every 14 days. Severe/life-threatening hematologic toxicity (25%), infection (7%), fever (2%), severe vomiting (3%). Emetogenic potential : day 1 level 1. Infectious mortality : 2%^ref
• alkylator/anthracycline combinations are frequently used with similar and perhaps better responses to those seen with chlorambucil, but no change in survival^ref
• CVP (cyclophosphamide, vincristine, prednisone)
• ChOP (cyclophosphamide, doxorubicin, vincristine, prednisone)
• purine analogues are nucleoside analogues that inhibit DNA polymerase and ribonucleotide reductase, promoting apoptosis^ref.
• fludarabine has been the most widely tested and used nucleoside analogue in CLL.
• the efficacy of fludarabine was studied in a large North American intergroup trial in which more than 500 patients were randomized to receive fludarabine, chlorambucil, and fludarabine and chlorambucil. Overall response (OR) favored the fludarabine group at 63% (20% CR + 43% PR) versus 37% (4% CR + 33% PR)^ref. The median duration of remission and the median progression-free survival (PFS) in the fludarabine group were 25 months and 20 months, respectively, whereas both values were 14 months in the chlorambucil group (P < 0.001 for both comparisons)
• Leporrier et al also reported their results in patients with previously untreated CLL treated with either fludarabine or an anthracycline-containing regimen (CAP [cyclophosphamide, doxorubicin, prednisone] or ChOP (cyclophosphamide, doxorubicin, vincristine, prednisone)). 938 patients (651 stage B and 287 stage C) were randomized in 73 centers. Compared to ChOP and fludarabine, CAP induced lower overall remission rates (58.2%; ChOP, 71.5%; fludarabine; 71.1%; P < .0001 for each), including lower clinical remission rates (CAP, 15.2%; ChOP, 29.6%; fludarabine, 40.1%; P = .003). By contrast, median survival time did not differ significantly according to randomization (67, 70, and 69 months in the ChOP, CAP, and fludarabine groups, respectively)^ref.
• fludarabine 25-30 mg/m²/day IV days 1-5 (phase 1/2 trial, 2 dose levels). Repeat cycle every 28 days. Grade 4 neutropenia (56%), FUO/minor infection (13%), pnemonia+/-septicemia (9%), thrombocytopenia (25%), stomatitis (2%), neuropathy (4%), nausea (3%), diarrhea (2%) => infectious mortality (3%). Emetogenic potential : days 1-5 level 1^ref
An important observation in all these trials is the higher percentage of CRs seen in the fludarabine treated groups. Fludarabine is well tolerated with major side effects being hematologic and immunologic toxicities. Also seen is autoimmune hemolytic anemia (AIHA) and, at very high doses, significant neurotoxicity. The issue of treating patients with fludarabine who have a history of AIHA or ITP, either primary or secondary to previous purine analogue therapy, is a topic of continued controversy (Montillo M, Tedeschi S, O’Brien SM, Lerner S, Morra E, Keating MJ. Autoimmune phenomena against hematopoietic cells and myelosuppression in CLL treated with fludarabine - including regimens as front-line therapy [abstract]. VIII International Workshop on CLL Programme and Abstract Book. 1999;54. Abstract P091; Hall R, Bolan S, Orchard J, Oscier D, Myint H, Hamblin TJ. Autoimmune phenomena following treating with fludarabine [abstract]. VIII International Workshop on CLL Programme and Abstract Book. 1999;58, Abstract P103). The decision to treat these patients should be done with extreme caution and close monitoring.
• cladribine (2-CdA) : there is evidence that produces similar responses as fludarabine in both previously treated and untreated populations. Juliusson and Liliemark reported a 31% CR and 27% PR in 52 patients with previously treated CLL, with a median PFS of 23 months for CR patients and 16 months for PR patients. Toxicities were similar to those observed with fludarabine^ref. Recently, Robak et al reported their experience with 2-CdA on 378 patients with CLL. 194 patients were previously untreated and 184 had relapsed or refractory disease. Results showed a CR of 45.4% and a PR of 82.5% in the previously untreated group with a median survival of 19.4 months, and a CR of 12.5% and PR of 48.4% in the previously treated group with a median survival of 16.3 months^ref. Ckadruvrube 0.12 mg/kg/day IV continuous infusion days 1-5. Repeat cycle every 28 days. Grade 3-4 neutropenia (9%), severe infections (13%), minor infections and FUO (34%), thrombocytopenia (23%) => infectious mortality (7%), hemorrhagic mortality (6%). Emetogenic potential days 1-5 level 1^ref
• cladribine (2-CdA) + prednisone : cladribrine 0.12 mg/kg/day IV over 2 hours days 1-5, prednisone 30 mg/m2/day PO days 1-5, repeat cycle every 28 days for 3 courses, grade 3-4 neutropenia (9%), anemia grade 1-2 (6%), grade 3-4 (2%), FUO/infection (56%), thrombocytopenia (9%), eosinophilia (9%), autoimmune hemolytic anemia (AIHA) (6%: related-deaths in 3% of patients), hepatic (6%), skin reaction (6%), CNS (2%), diarrhea (2%), nausea/vomiting (2%), neuropathy (2%) => herpes zoster reactivation in 21% of patients, herpes simplex infections in 11% of patients; URI or pneumonia in 30% of patients. Emetogenic potential : days 1-5 level 1^ref
The accumulating 2-CdA data is provocative but not much different from the fludarabine data. Unfortunately, crossresistance is seen among all the nucleoside analogues, so a sequential treatment algorithm is unlikely. Reports of synergism between alkylators and nucleoside analogues by alkylators inducing DNA damage and nucleoside analogues inhibiting DNA repair prompted trials combining these 2 modalities^ref (Koel U, Li L, Nowak B. Fludarabine and cyclophosphamide: synergistic cytotoxicity associated with inhibition of interstrand cross-link removal [abstract]. Proc Am Ass Cancer Res. 1997;38:2). Early reports of the combination of fludarabine + cyclophosphamide showed higher OR rates than fludarabine alone in previously treated patients with alkylators or fludarabine^{ref1, ref2, ref3} (O’Brien SM, Kantarjian H, Beran M. Fludarabine and cyclophosphamide therapy in chronic lymphocytic leukemia [abstract]. Blood. 1996;88:480).
• cyclophosphamide 300 mg/m²/day IV over 1 hour days 1-3, fludarabine 30 mg/m²/day IV over 30 minutes days 1-3. Repeat cycle every 28-42 days. Neutropenia grade 3 (75%), grade 4 (48%), fever/infection (40%), thrombocytopenia (19%), nausea/vomiting (6%), fatigue/aches (2%), rash (2%) => documented sepsis or pneumonia (25%), FUO or neutropenic fever (25%), 6 atypical infections were reported, herpes zoster infection (5$), reactivation of herpes simplex (8%), 29% of patients at the 300 mg/m2 dose level required a dose reduction of cyclophosphamide^ref
• fludarabine + cyclophosphamide + filgrastim :  in a multicenter Phase II study conducted by ECOG, 36 patients received cyclophosphamide 600 mg/m² intravenous (iv) day 1 and fludarabine 20 mg/m² IV days 1 through 5, followed by filgrastim 5 mg/kg subcutaneous starting approximately day 8. 15 of 36 (41.7%) evaluable patients achieved complete response and 8 (22.2%) achieved a partial response, giving an OR rate of 63.9% (90% CI: 48.4%, 77.2%) (Flinn IW, Jemiai Y, Bennett JM, et al. Fludarabine and cyclophosphamide achieves high complete response rate in patients with previously untreated chronic lymphocytic leukemia: E1997 [abstract]. Blood. 2001;98:633a)
• Preliminary results from a North American Intergroup trial comparing this combination regimen to single-agent fludarabine revealed the combination produced a superior CR rate compared to fludarabine alone, 23% versus 6%, respectively
• Alternative regimens using 3-day schedules of fludarabine and cyclophosphamide have produced encouraging results. The German CLL Study Group noted a higher CR rate with fludarabine and cyclophosphamide than with fludarabine alone (20.2% vs 8.6%) (Eichhorst B, Busch R, Hopfinger G, et al. Fludarabine + cyclophosphamide induces higher remission rates and longer progression free survival than fludarabine alone in first line therapy of advanced chronic lymphocytic leukemia: results of a Phase III study (CLL4 Protocol) [abstract]. Blood. 2003;102:72a). While these CR rates were disappointingly low in both arms, the difference in response translated into a longer PFS in the combination arm. In a single center Phase II arm study, CR rates were not found to be substantially increased. However, the quality of these CRs was improved, since only 8% of CR patients demonstrated residual CD5⁺ cells in the marrow compared with 33% after fludarabine and prednisone^ref.
Taken together these studies suggest that the combination of fludarabine and cyclophosphamide improves CR in patients with previously untreated CLL. However, it is not clear as to what regimen might serve as the best backbone by which to add new agents such as rituximab. Numerous other fludarabine combination regimens have been tested with promising results in Phase II trials^{ref1, ref2, ref3}.
• single-agent antibodies :
• anti-CD20 mAbs : rituximab has afforded excellent responses in patients with B-cell lymphomas. Early disappointing results in small lymphocytic leukemia (SLL)/CLL from the pivotal trial led to skepticism about its ultimate utility in CLL. However, subsequent studies examining alternative schedules, doses, and patient populations have clearly demonstrated rituximab’s efficacy in CLL. One explanation for the meager response rates in patients with SLL is the low serum trough levels seen in these patients, which are known to be inversely related to response rates. To overcome the apparent altered pharmacokinetics, the antibody has been given thrice weekly for 4 weeks at standard doses (375 mg/m²) as well as very large single doses weekly for 4 doses. The thrice weekly regimen has resulted in 45% OR rate in patients with previously treated CLL^ref. 5 of 6 previously untreated patients responded. A dose escalation study has also been completed in which cohorts of patients with CLL and other mature B cell malignancies received increasing doses of rituximab weekly for 4 doses. Response correlated with dose and 75% of patients at the highest dose (2250 mg/m²) responded^ref. 44 previously untreated patients with CLL/SLL received rituximab 375 mg/m² weekly for 4 consecutive weeks. All patients were required to have one or more indications for treatment^ref. Patients with objective response or stable disease continued to receive identical 4-week rituximab courses at 6-month intervals, for a total of 4 courses. The response rate after the first course of rituximab was 51% (4% CR). While many patients treated with rituximab will respond, these are predominantly partial responses, with the bone marrow being the most difficult compartment to treat adequately. Grade 3 anemia (3%), grade 3-4 infection (17%), grade 3 flu-like symptoms (14%), grade 3 abnormal liver function (7%), grade 3 hypotension (7%), grade 3 pulmonary (7%), grade 3 bone pain (3%), skin grade 3 (3%) and 4 (zoster : 3%). Due to a fatal treatment-related complication, the 375 mg/m2 dose in week 1 was fractionated. Patients received 50 mg on day 1, 150 mg on day 2, and the remainder of the dose on day 3. 1 patient died of late pulmonary aspergillosis. Emetogenic potential level 1^ref
• anti-CD52 mAbs : alemtuzumab
• 30 mg IV over 2 hours 3 times a week (initiate dosing at 3 mg IV daily; when tolerated (infusion-related toxicities are < grade 2) increase to 10 mg). When the 10 mg dose is tolerated, the maintenance dose of alemtuzumab 30 mg can be initiated. Maintain at 30 mg 3 times a week for up to 12 weeks. Grade 3-4 neutropenia (64%), grade 1-2 anemia (42%), grade 3-4 anemia (38%), grade 3-4 sepsis (10%), fever (19%), bronchitis/pneumonitis (13%), pneumonia (10%), thrombocytopenia (50%), rigors (16%), dyspnea (9%), fatigue (5%), hypotension (5%), urticaria (5%). Gradual escalation to the recommended maintenance dose is required at the initiation of therapy and after interruption of therapy for 7 days or longer. Hematological toxicity is common. In patients with heavily pretreated B-CLL, a response rate of 30–40% has been reported with a median response duration of 12 months. The toxicity related to the infusion of the antibody has hampered its early use in patients.
• 30 mg subcutaneously 3 times a week up to a maximum of 18 weeks; dosing was initiated at 3 mg on day 1, 10 mg on day 2, and then to full dose of 30 mg on day 5 if tolerated. In a Phase II study of previously untreated patients given alemtuzumab for 18 weeks, an OR of 87% (95% CI, 76%–98%; CR, 19%; PR, 68%) was achieved in 38 evaluable patients (81% of intent-to-treat population). CLL cells were cleared from blood in 95% patients in a median time of 21 days. CR or nodular PR in the bone marrow was achieved in 66% of the patients and most patients achieved this after 18 weeks of treatment. An 87% OR (29% CR) was achieved in the lymph nodes. Transient injection site skin reactions were seen in 90% of patients (grade 3 in 2%). Rigor, rash, nausea, dyspnea, and hypotension were rare or absent.  Grade 2-3 neutropenia (53%), grade 4 neutropenia (21%), anemia grade 0-1 (61%) or 2-3 (39%), grade 3 fever (2%), thrombocytopenia (grade 2-3 : 11%; grade 4 : 5%), grade 3 fatigue (2%), grade 3 rigor (2% : much lower than in IV administration !), the incidence of hypotension is significantly reduced. Upward titration to ful dose was achieved by 31% of patients during week 1. 88% of patients had grade 1-2 local injection site reaction. Therapy was temporarily stopped in 7 patients due to neutropenia. Reactivation of CMV in 4 patients^ref.
Emetogenic potential : level 1. Appropriate premedication. Alemtuzumab induces a profound lymphopenia, which may have contributed to the high incidence of infectious complications seen in the early studies. This risk has been substantially reduced by the use of prophylactic trimethoprim/sulfamethoxazole, acyclovir, and fluconazole. Prophylaxis against PCP and herpes virus infections is strongly recommended, and should continue for 2 months after completion of alemtuzumab therapy, or until the CD4⁺ is >= 200 cells/ml, whichever is later. In this study of previously untreated patients infections were rare, but 10% of patients developed cytomegalovirus (CMV) reactivation. In contrast to rituximab, alemtuzumab has its most pronounced effects in blood and bone marrow, with minimal effect on bulky disease. Subcutaneous administration of MabCampath shows similar efficacy as the intravenous application. Most importantly alemtuzumab is active in high risk CLL as defined by the presence of 17p- deletion.' (Stilgenbauer). The median survival by FISH category after a median follow-up of 70 months is: for ‘abnormality 17p-‘ median survival 2.5 years; for ‘no abnormality' median survival 9 years; and for ‘abnormality 13q-‘ median survival 11 years. Also, the median survival for patients with early stage disease with non-mutated IgV_H gene - detected by FACS of peripheral blood - have been reported to be about 8 years, whereas patients with early-stage disease with mutated-type clones have a median survival > 24 years^ref. (See Shanafelt TA, Call TG. Mayo Clin Proc. 2004;79:388-398)
Campath-1 has shown significant and durable effects in the treatment in patients with previously treated, recurrent, indolent CLL. A 42% overall response rate was achieved among 29 patients in one phase III study^ref. Other encouraging results have demonstrated the efficacy of campath-1 for patients suffering from fludarabine refractory disease^ref, and a 73% response rate observed among a limited number of patients with prolymphocytic leukemia^ref
The increased specificity of mAbs for CLL compared to chemotherapy with relative sparing of normal tissues has raised the question as to whether MABs could be used as adjuvants to chemotherapy to target MRD or in a maintenance approach to maintain remissions.
• rituximab has been used in other low-grade lymphoid malignancies for maintenance and it has prevented relapse but the duration of rituximab benefit was not superior to using the antibody when patients relapsed^ref. In patients with CLL, PFS (18.6 months) was relatively poor when rituximab was used as front-line therapy and then administered at 6 month intervals as maintenance^ref. Further investigation is needed to learn whether rituximab might be useful in this setting if used at different doses or schedules.
• the efficacy and safety of alemtuzumab in patients with CLL who had residual disease after chemotherapy have also been investigated. Alemtuzumab was administered to 41 patients 3 times weekly for 4 weeks in a Phase I/II study following maximum response to induction chemotherapy. The OR was 46%. The major reason for failure to respond was the presence of adenopathy. Residual bone marrow disease cleared in most patients, and 11 of 29 patients (38%) achieved a molecular disease remission. Infections were reported to occur in 15 patients (37%), and 9 of these infections were reactivation of CMV. 3 patients developed EBV⁺, large cell lymphoma^ref. Given the significant infectious toxicity, larger studies are needed before this approach can be used on a routine basis.
• combination chemo-immunotherapy : the encouraging high rate of durable responses seen with fludarabine-based regimens may be further improved by the addition of other novel agents.
• fludarabine + cyclophosphamide + rituximab (FCR) is currently under investigation as a first-line therapy for patients with CLL. A single center study has shown good preliminary results including a 71% CR rate (Keating MJ, O’Brien S, Lerner S, Wierda W, Kantarjian H. Chemoimmunotherapy with fludarabine (F), cyclophosphamide (C), and rituximab (R) improves complete response (CR), remission duration and survival as initial therapy of chronic lymphocytic leukemia (CLL) [abstract]. J Clin Oncol. 2004;23:571), with 57% of complete responders tested (35/61 patients) showing molecular remission (Keating M, Manshouri T, O’Brien S, et al. A high proportion of molecular remission can be obtained with a fludarabine, cyclophosphamide, rituximab combination in chronic lymphocytic leukemia [abstract]. Blood. 2002;100:205a.)
• pentostatin + cyclophosphamide + rituximab : cyclophosphamide 600 mg/m² IV day 1 followed by pentostatin 4 mg/m² IV day 1, rituximab 375 mg/m² IV day 1. Repeat cycles every 21 days. Appropriate premedications. For cycle 1, patients did not receive rituximab. Prophylaxis with trimethoprim/sulfamethoxazole and acyclovir were administered to all recipients. Prophylactic filgrastim was administered to all patients. Grade 3-4 neutropenia (45%), infection (15%), thrombocytopenia (5%). Emetogenic potential day 1 level 5 (Weiss MA et al, Blood 2003, 102, 673a, Abstract 2494)
• fludarabine + rituximab (FR) in combination have also been reported to result in very high response rates in previously untreated patients with CLL, with CRs seen in 33% of patients when FR was administered concurrently. The CR rate improved to 47% when responding patients were consolidated with rituximab^ref.
• cycle 1 : fludarabine 25 mg/m²/day IV over 20-30 minutes days 1-5
• rituximab 50 mg/m² IV day 1 infused over 4 hours with no rate escalation
• rituximab 325 mg/m² IV day 3 infused at an initial rate of 50 mg/hour and increaased in 50 mg/hour increments every 30 minutes to a maximum rate of 400 mg/hour as tolerated.
• rituximab 375 mg/m² IV day 5 infused at a rate of 100 mg/hour for the first 15 minutes and then rate increased to infuse remainder over the next 45 minutes
Neutropenia grade 3 (33%) or 4 (43%), anemia grade 1 (47%), 2 (18%), or 3 (4%), grade 3 infection (20%), thrombocytopenia (20%), dyspnea (14%), grade 3 hypotension (6%)
• cycles 2-6 : fludarabine 25 mg/m²/day IV over 20-30 minutes days 1-5, rituximab 375 mg/m² IV day 1 infused at a rate of 100 mg/hour for the first 15 minutes and then rate increased to infuse remainder over the next 45 minutes. Repeat cycle every 28 days. Appropriate premedications. Neutropenia grade 3 (11%) or 4 (8%). anemia grade 1 (14%), 2 (5%) or 4 (3%), grade 3-4 infection (6%), grade 3 dyspnea (6%)
8 opportunistic infections noted. 3 cases of grade 3-4 pulmonary toxicity. Emetogenic potential days 1-5 level 1. This group retrospectively compared the treatment outcome of the patients on this trial (n = 104) to patients with similar clinical characteristics enrolled on a US Intergroup who received fludarabine alone (n = 178). In multivariate analyses controlling for pretreatment characteristics, the patients receiving fludarabine and rituximab had a significantly better PFS (P < 0.0001) and overall survival (OS) (P = 0.0006) than patients receiving fludarabine therapy. 2-year PFS probabilities were 0.67 versus 0.45, and 2-year OS probabilities were 0.93 versus 0.81. Infectious toxicity was similar between the 2 treatment approaches^ref.
• fludarabine + alemtuzumab in patients with previously untreated CLL. Treatment included fludarabine 25 mg/m²/day for 5 days IV, monthly x 4, followed by observation for 2 months when an evaluation of initial response was performed. Patients were evaluated 2 months after completing fludarabine and those who achieved stable disease (SD) or better were treated with a 6-week course of alemtuzumab. Alemtuzumab was given at 30 mg IV 3 times a week after a stepped up dosing during the first week (3 mg as the first dose and increasing, as tolerated, to 10 mg, and to 30 mg). Prophylaxis with trimethoprim-sulfamethoxazole and acyclovir was required during and for 6 months after alemtuzumab therapy. Among 36 patients who entered the alemtuzumab phase of therapy, there were 15 CRs (42%) and 18 PRs (50%) with an OR rate of 92%^ref. Out of 11 patients with SD after fludarabine, 3 achieved CR and 5 had PR following alemtuzumab. Among all 56 patients (intention-to-treat population) the CR and PR incidence was 27% and 43%, respectively. Grade 3 or worse major infections were recorded in 12 patients during or following alemtuzumab therapy. Infections with CMV occurred in 8 patients during or within 4 months following alemtuzumab. The outcome of CMV was fatal in 1 case, complete or partial resolution occurred in 6, and persistent CMV in 1 (Rai K, Byrd JC, Petersen BL, Larson RA. A phase II trial of fludarabine followed by alemtuzumab (campath-1H) in previously untreated chronic lymphocytic leukemia (CLL) patients with active disease: Cancer and Leukemia Group B (CALGB) study 19901 [abstract]. Blood. 2002;100:205–206a)
• HSCT : the use of BMT in CLL was slow to develop given the perceived "good prognosis" of these patients, their higher median age, the unproven benefit of BMT and the potential toxicity of the treatment. Nonetheless, as our understanding of the pathogenesis of this disease improves and our ability to identify patients with aggressive disease and poor prognosis improves, the need for aggressive, potentially curative therapy becomes paramount. Also, as the mortality with transplantation decreases, as well as recent innovations like nonmyeloablative allogeneic transplants (Khouri J, Keating MJ, Przepiorka D. Engraftment and induction of GVL with fludarabine (FAMP)-based non-ablative preparative regimen in patients with chronic lymphocytic leukemia and lymphoma [abstract]. Blood. 1996;88:301a) emerge, this modality will likely become an important facet in the treatment of some CLL patients. The first report of BMT was by Michallet et al in 1988^ref. Since then, multiple other investigators have shown the ability of patients with CLL to tolerate and respond to both allogeneic and autologous bone marrow transplants^ref.
• in autologous HSCT, the transplant mortality is < 10% with the status of disease and the number of previous lines of treatments being the most important prognostic factors. Unfortunately, the survival plots do not show a plateau, with about 50% of patients relapsing at 4 years. However, durable remissions have been achieved in patients who received autologous grafts purged of leukemia^ref and a survival advantage has been noted even in patients with poor prognostic factors^ref.
• allogeneic HSCTs, on the other hand, result in survival curves plateaus of about 40%. In a recent update, the European Bone Marrow Transplant Registry reported that OS and event-free survival at 10 years was 41% and 36.6% in a series of 54 patients who underwent allogeneic BMT from a matched sibling^ref. Unfortunately, 48% of patients died from TRM, which is not surprising in light of the fact that 85% of the patients were refractory to chemotherapy at the time of transplant. The use of BMT early in the course of the disease dramatically reduces TRM^ref and should be considered for patients with poor-risk features. However, most patients with CLL are over the age of 55 and thus usually not considered for allogeneic BMT with a standard preparative regimen. The realization that much of the efficacy of allogeneic transplantation came from the GvL effect and the subsequent development of reduced intensity conditioning (RIC) regimens is particularly important to patients with CLL. Although the use of RIC regimens is still in relative infancy compared to standard regimens, early studies suggest that the efficacy is as good with RIC while the risks are significantly less. For example, in a study of 448 patients with CLL, 228 who received an RIC and 222 who received a standard myeloablative regimen, Dreger et al found that the hazard ratio for treatment-related mortality and OS were 0.5 ([95% CI 0.3–0.83]; P = .007) and 0.56 ([95% CI 0.37–0.86]; P = 0.007) respectively favoring RIC. Furthermore, no increased risk of relapse was seen.39 RIC regimens significantly broaden the utility of allogeneic BMT to patients well into their 70s and many institutions now favor RIC over standard regimens for nearly all of their patients. Durable remissions have been achieved with BMT. Only time will tell whether these remissions will develop into survival advantages and possibly cures. The use of peripheral stem cells, nonmyeloablative (mini BMT) approaches and graft purging methods such as chemotherapy, B cell specific mAbs, positive cell selection and others continue to improve upon the results and tolerability of transplantation and will likely make these procedures more widely available to CLL patients.
Risk versus benefit evaluation of autologous and allogeneic HSCT in CLL : they are increasingly considered in the management of medically fit patients with active CLL. These procedures may confer therapeutic benefit but are also associated with considerable toxicity and cost. The efficacy of autologous HSCT relies solely on the cytotoxic therapy administered. Allogeneic SCT offers the potential additional benefit of the immune-mediated GvL effect but also harbors the danger of graft-versus-host disease. Hence, there is a need to identify prognostic factors that may help to determine whether a patient is a candidate for HSCT and if an allogeneic HSCT or autologous HSCT should be considered. Emerging data from prospective autologous HSCT trials are demonstrating safety, improved remission after transplant and long survival times. In the Medical Research Council (MRC) series the early transplant-related mortality (TRM) was 1.5% and the 5-year overall and disease-free survival following transplantation were 77.5% and 51.5%, respectively^ref. In the multicenter prospective autologous HSCT study of the GCLLSG (CLL3) the TRM was 5% with a 2-year overall survival rate of 88% among 105 patients^ref. This result appears promising considering the high-risk features present in the majority of patients (see also Table 1 for the CLL3 trial: 68% unmutated V_H, 25% 11q- or 17p-). However, the continuing clinical and molecular relapses observed in all series of autologous HSCT in CLL are evidence against the curative potential of the procedure in the majority of patients. Furthermore, genetic risk factors appear to retain their adverse impact after autologous SCT. In the CLL3 trial the median time to recurrence of a clonal CDR3 PCR analysis as molecular evidence of disease was 17 months in the group with 11q- versus 37 months in the group without 11p- (P = .0019). Similarly, the time to clinical relapse and the time to disease recurrence as assessed by consensus primer CDR3 PCR was significantly shorter among patients with unmutated VH genes^ref. Nevertheless, the median treatment-free interval of 49 months in the V_H unmutated cohort suggested a beneficial effect of autologous HSCT for this high-risk population. This was confirmed in a matched pair analysis of autologous GSCT versus conventional treatment including the V_H mutation status as matching variable^ref. In this study auto-GSCT resulted in significantly longer overall survival as compared to conventional chemotherapy (autologous HSCT: last death at 139 months, corresponding survival rate of 0.57, conventional: median survival 119 months) and this effect was primarily based on the benefit of the subgroup of cases with unmutated V_H Matched pair analysis based on the matching variables age, Binet stage, V_H mutation status, and lymphocyte count between patients receiving autologous stem cell transplantation (autologous HSCT) and conventional treatment (conventional)^ref

Survival from the time of diagnosis (n = 88) of all patients treated with autologous HSCT (broken line) and conventional chemotherapy (solid line), respectively.
As compared to autologous HSCT the primary therapeutic advantage of allogeneic HSCT after dose-reduced conditioning is the graft-versus-leukemia effect, which may offer long-term disease control and eventual cure. Indeed, a recent comparative study of minimal residual disease (MRD) as detected by consensus primer CDR3 PCR provided evidence that the graft-versus-leukemia effect is operational in CLL with unmutated V_H^ref. In this study only a modest decrease in MRD levels was observed immediately after allogeneic HSCT, but MRD became undetectable in 7 of 9 (78%) CLL patients with unmutated V_H after tapering immunosuppression, chronic graft-versus-host disease or donor lymphocyte infusions. After a median of 25 (14–37) months, these 7 patients remain in clinical and molecular remission. In contrast, PCR negativity was achieved in only 6 of 26 (23%) control CLL cases with unmutated V_H after autologous SCT after dose-reduced conditioning and was not durable. MRD levels (number of CLL specific DNA copies per total DNA copies in the sample compared to the reference [pre-therapeutic] sample) of patients after myeloablative conditioning and auto-HSCT and after non myeloablative allogeneic HSCT. Black-filled symbols (circles and triangles) denote MRD-positive samples whereas blank symbols denote the sensitivity of PCR^- samples (calculated minimum MRD level, which would have been detected in this particular sample).

Therefore, allogeneic HSCT appears to combine the favorable features of low TRM with the activity of the graft-versus-leukemia effect, making this procedure a valid option when aiming at cure for high risk CLL.
Complications of therapy and treatment choices : an important consideration in deciding on an initial therapy is the potential morbidity and mortality of the therapy in both the short-term and long-term treatment plans for a patient. Infections and hemolytic anemia are the most common initial complications of the therapy. The nucleoside analogues and alemtuzumab can have prolonged effects on cellular immunity. Combination regimens are particularly problematic. However, the risk of infection can usually be managed. Many clinical trials of combination regimens with fludarabine have successfully utilized prophylactic antibiotics to prevent Pneumocystis carinii pneumonia^{ref1, ref2}. The depression in CD4 counts persists for at least 6 months after completing chemotherapy and thus so should prophylaxis. Similarly, in high-risk populations such as patients treated with the combination of fludarabine and cyclophosphamide, acyclovir and its analogues have been used to prevent herpes zoster. Re-activation of CMV is a prominent complication of alemtuzumab, especially when used in combination with fludarabine or for MRD. Prophylaxis is more cumbersome and less well established. However, surveillance for CMV using a PCR-based approach has been used successfully (Rai K, Byrd JC, Petersen BL, Larson RA. A phase II trial of fludarabine followed by alemtuzumab (campath-1H) in previously untreated chronic lymphocytic leukemia (CLL) patients with active disease: Cancer and Leukemia Group B (CALGB) study 19901 [abstract]. Blood. 2002;100:205–206a). The treatment of CLL in patients with a history of AIHA is highly controversial. The incidence of AIHA in patients treated with fludarabine or other nucleoside analogues may be increased^ref. In randomized trials there do not appear to be huge differences in the incidence of this important complication^ref. However, in patients that are direct antibody positive or have a history of AIHA, the risk appears high enough that these patients have been excluded from some clinical trials^ref, a paradigm useful in clinical practice. The late consequences of initial treatment, most notably damage to normal bone marrow, are harder to incorporate in the initial management decisions of patients with CLL given the paucity of information. It is, however, clear that aggressive therapies that produce higher CRs also have deleterious side effects to bone marrow reserve. This side effect is probably most easily quantifiable in terms of the ability to collect autologous stem cells for transplantation. While autologous stem cell transplantation applies to a relatively small number of patients with CLL, the ability to collect stem cells reflects hematopoietic reserve. Although stem cells can still be collected when fludarabine is used judiciously^ref, numerous groups have now reported that fludarabine-containing regimens impair the ability to collect autologous stem cells and perhaps even the ability of these stem cells to engraft^{ref1, ref2}. In addition, a concern about damage to normal hematopoietic precursors that results in an increased incidence of myelodysplasia has been raised with the combination of alkylating agents with fludarabine^{ref1, ref2}.
• low-risk : minimally toxic therapy (rituximab, chlorambucil, fludarabine)
• intermediate risk : nucleoside analog combination regimens (fludarabine and cyclophosphamide, fludarabine and rituximab, fludarabine, cyclophosphamide, and rituximab)
• high risk : clinical trial or BMT (myeloablative or nonmyeloablative)

• management of patients with fludarabine-refractory CLL : for some patients CLL is an indolent disease that never progresses to require therapy. However, when patients become symptomatic with their CLL, the OS with older therapy is quite short, ranging from a mean of 1.5 to 6 years^{ref1, ref2}. Applying traditional therapies utilized in CLL such as chlorambucilor fludarabine results in palliative responses and a PFS = 20 months in one study^ref. Combination strategies with fludarabine + cyclophosphamide or fludarabine-based combinations with rituximab may extend PFS, but no plateau is appreciated with any of these studies. At the time of relapse from initial response to fludarabine, 40% can be retreated and will respond again to the same regimen^ref. Data with respect to retreatment with fludarabine and cyclophosphamide or fludarabine-based combinations with rituximab are currently not available. Ultimately, virtually all CLL patients who are treated become fludarabine-refractory. Therapy for CLL for many years consisted only of alkylator-based therapy with or without corticosteroids. Until the approval of fludarabine for use in alkylator-resistant CLL in 1991, no salvage therapies existed for the treatment of CLL. This un-met medical need served as the basis for fludarabine’s FDA approval for use to treat alkylator-resistant CLL. Soon after the approval of fludarabine, a new class of CLL patients emerged, those resistant to fludarabine. The definition for fludarabine-refractory disease generally utilized in publications and accepted by the FDA and other regulatory agencies is no response to therapy or initial response to fludarabine but subsequent progression within a 6-month period from completion of therapy^ref. From 1991 to 2001, new therapies for fludarabine-refractory CLL were investigated in clinical trials, and several have emerged that can be effectively used for this patient population. Despite the > 13-year time period since fludarabine was approved, a paucity of data are available describing clinical features associated with this subset of CLL. One series published by the MD Anderson Cancer Center reported on 147 such patients followed over 13 years who either did not respond (138, 94%) or relapsed within 6 months (9, 6%) following completion of therapy with fludarabine^ref. Valuable demographics were provided by the study. These patients had a median age of 60, a median of 3 prior therapies, and a majority had Rai high-risk disease. With initiation of different investigational or standard therapies, early death within 3 months was noted in 13% of patients and life-threatening infections in 58 (40%). Response to therapy was only 12% in those patients who had primary resistance to fludarabine and 22% in those who had previously had short responses. Responses were predominately noted with fludarabine-based combinations such as fludarabine and cyclophosphamide or cladribine and cyclophosphamide. Of interest, infection risk was lowest in patients who responded to therapy. For the entire group of patients, the median survival was 10 months. A second retrospective study by our group examined demographics and infectious morbidity of fludarabine-refractory CLL and SLL patients who received alternative forms of commercially available chemotherapy or rituximab after they were classified as fludarabine-refractory^ref. This study included 27 patients with a median age of 67 and a median of 3 prior therapies. Overall, 24 of 27 patients (89%) developed serious infections requiring intravenous antibiotics with a 17% frequency of hospital admission per month. Overall an 11.4% response to therapy was noted, predominantly in patients receiving rituximab. Median survival of this cohort was 13 months. Overall, both of these studies uniformly demonstrate fludarabine-refractory CLL patients have a high frequency of infections even in the absence of any treatment. Additionally, survival for this patient group is quite short, ranging from 9–13 months. Understanding the molecular features present in fludarabine-refractory CLL that mediate resistance to therapy remains a high priority. P53 mutations and deletions are one such feature, being present in only 5–10% of patients at diagnosis^ref but in 40–50% at the time fludarabine-refractory disease is present^{ref1, ref2}. Identifying the presence or absence of p53 mutations remains quite important to picking therapy even in the absence of fludarabine-refractory status. Indeed, p53 mutations and deletions predict for lack of response to monotherapy with fludarabine, chlorambucil, and rituximab^{ref1, ref2, ref3}. Furthermore, p53 mutations and deletions also predict for markedly inferior survival as compared to those patients who have wild type p53^ref. The biologic significance of other molecular markers and events associated with progression to refractory CLL has not been adequately explored. Such studies will be important if new, effective therapies are to be developed.
• alternative nucleoside analogues : during the 1990s significant debate existed over the crossresistance between fludarabine, cladribine and pentostatin. A preliminary report that noted responses to cladribine (2-CdA) in patients with fludarabine-refractory CLL prompted great interest^ref; however, subsequent experience from several other groups failed to confirm these results and demonstrated considerable toxicity from cladribine due to myelosuppression and infections^{ref1, ref2}. The majority of patients treated on these trials had significant baseline cytopenias and advanced disease. To address this question, the Cancer and Leukemia Group B (CALGB) performed a Phase II study in fludarabine-refractory CLL^ref. 28 patients were enrolled on this study with 13 having intermediate (Rai stage I or II) and 15 high (Rai stage III and IV) risk stages. No patient had a complete remission, but 9 (32%; 95% confidence interval, 15%–49%) attained a partial remission using the NCI criteria (1996)^ref. Responses were noted predominately in patients without anemia or thrombocytopenia (7 of 13, 54% with PR) versus only 2 of 15, 13% with PR, in patients with modest thrombocytopenia or anemia. The median time to relapse for responders was 12 months, while median PFS for the entire group was 9 months. Of interest, the median OS for this highly selected group of patients was 2.2 years. Further supporting the selective nature of the eligibility is that a large cooperative group required 4 years to enroll the 28 patients. Despite the modest benefit in terms of response, this regimen was quite toxic with grade 3–5 neutropenia (75%), thrombocytopenia (68%), and infections (43%) noted. Trials with clofarabine using the 5 consecutive day schedule of administration have also demonstrated minimal activity in this patient population^ref. Overall, the use of an alternative nucleoside analogue such as cladribine or pentostatin in the setting of fludarabine-refractory CLL is not recommended.
• rituximab : the activity of rituximab in previously treated CLL is highly dependent upon schedule/dose of administration and has been reviewed previously^ref. Dosed at the standard 375 mg/m² weekly for 4–8 weeks as would be done in lymphoma, very modest activity is observed. Based upon the pharmacokinetic considerations of rapid antibody clearance in SLL, investigators have taken 2 different strategies to improve efficacy. In one single institution study, 50 patients with previously treated CLL (n = 40) or other B cell leukemias (n = 10) received weekly rituximab dose-escalated from 500 mg/m² to 2250 mg/m^2ref. Although no CLL patient achieved complete response, the overall response rate was 40%, and a statistically significant dose-response relationship was observed; 22% of patients treated with 500–850 mg/m2 responded, compared to 75% of patients treated with 2250 mg/m². The overall response rate was 36% for CLL and 60% for other B cell leukemias; median response duration was 8 months. Eight of 12 patients (67%) at 2250 mg/m² developed grade 2 toxicity, primarily fatigue, but no grade 3 or 4 toxicity was observed. However, for the patients who were fludarabine-refractory in this trial, the response rate was 20%. A group treated 33 patients with relapsed or refractory SLL/CLL with thrice weekly rituximab (375 mg/m²) for 4 weeks^ref. Patients received 100 mg over 4 hours on the first day of therapy and 375 mg/m² thereafter. This "stepped up" dosing schedule was designed to minimize infusion-related toxicity. The overall response rate was 45% (complete response 3%), and median response duration was 10 months. While response rate was 42% in the fludarabine-refractory patients, the response duration was only 6 months. Examination of molecular features in this trial demonstrated that the CLL patients with del(17p13.1) did not respond to therapy^ref. An ongoing follow-up study by our group demonstrated absence of response in a second series of 8 del(17p13.1) patients (personal communication, JC Byrd). In general, it is the author’s approach not to consider rituximab monotherapy for fludarabine-refractory CLL unless the patient lacks a del(17p13.1) on interphase cytogenetics and is not a candidate for more aggressive therapy.
• fludarabine + cyclophosphamide : a number of trials throughout the 1990s studied the combination of fludarabine and alkylating agents. Preclinical data^ref suggest synergistic interaction between DNA damaging agents and the purine analogues. Such synergy likely occurs as a consequence of alkylator-induced DNA damage and subsequent inhibition of DNA repair by the nucleoside analogues. Combination studies with alkylating agents and each of the purine analogues have been performed. Early clinical studies demonstrated that myelosupression was more problematic and compromised the total administered dose of each agent. Recent Phase II reports on fludarabine and cyclophosphamide with or without filgrastim support have been published. The MD Anderson experience included the largest series of fludarabine-refractory patients^ref. O’Brien et al describe a total of 128 patients with CLL who were treated with fludarabine 30 mg/m² intravenously daily for 3 days and cyclophosphamide at either 500 mg/m² daily for 3 days (n = 11), 350 mg/m²/d for 3 days (n = 26), or 300 mg/m² daily for 3 days (n = 91). The cyclophosphamide dose was decreased because of myelosuppression in the early part of the study. Patients were divided into 4 groups based on the expectation for response to single-agent fludarabine, including previously untreated patients (n = 34), patients previously treated with alkylating agents (n = 20), patients successfully treated with alkylating agents and fludarabine but relapsing (n = 46) and patients refractory to fludarabine with or without alkylating agents (n = 28). Fludarabine and cyclophosphamide produced 80% response rates in all patients not refractory to fludarabine at the start of therapy; however, it had a 38% response rate in patients who were refractory to fludarabine. 28 patients who were refractory to an alkylating agent + fludarabine had an overall response rate of 39%, with 3% complete response, 13% nodular partial response, and 26% partial response. The median TTP in patients who had previously received fludarabine and alkylating agents was 20 months. However, the estimated median survival was 12 months for those patients who were fludarabine-refractory. Infections or FUO occurred in almost half of all patients. Documented sepsis or pneumonia was noted in 25% of patients at some time during the therapy. FUO, frequently associated with neutropenia and requiring hospitalization, was observed in another 25% of patients. Significant infections were seen in 48% of fludarabine-refractory patients compared with 18% of patients not refractory to fludarabine with or without alkylating agents. Thus, while the combination of fludarabine and cyclophosphamide does produce palliation in a subset of patients with refractory CLL, the toxicities are significant. Because of this, pursuit of the potentially less myelosuppressive pentostatin + cyclophosphamide has occurred, with preliminary data being quite positive in one single center Phase II study :
• cyclophosphamide 600 mg/m² IV day 1 followed by pentostatin 4 mg/m² IV day 1, filgrastim 300 mg/day (for <= 70 kg) or 480 mg/day (> 70 kg) subcutaneously from day 2 until ANC > 5000 or > 1500 for 2 consecutive days. Repeat cycles every 21 days. Prophylaxis with trimethoprim sulfamethoxazole (1 DS BID, 3 times a week) and acyclovir (800 mg PO BID) were administered to all recipients. Neutropenia grade 3 (12%) or 4 (29%), anemia grade 1 (47%) or 2 (29%), grade 3-4 infection (12%), thrombocytopenia (30%) => neutropenia of brief duration (median 1 day; range 1 to 16 days), overall 65% of patients developed infections, 1 patient died of pneumonia. Emetogenic potential day 1 level 5^ref
Further pursuit of this strategy in larger Phase II trials is warranted to confirm these findings.
• fludarabine + cyclophosphamide + rituximab : rituximab has several biologic properties in vivo in primary CLL cells including downmodulation of anti-apoptotic proteins that mediate resistance to fludarabine and alkylator-based therapy. In symptomatic, but untreated CLL, the addition of rituximab to fludarabine or fludarabine and cyclophosphamide improves CR rate, PFS and OS as compared to historical control studies that did not use rituximab. In relapsed CLL, a large 177 patient study from MD Anderson Cancer Center examining fludarabine, cyclophosphamide, and rituximab was reported^ref. This study has demonstrated that 73% of patients responded with 25% attaining a CR. For the 37 patients who had fludarabine-refractory disease, a 5% complete response rate and 59% overall response was observed. Response duration for this group was not reported. Toxicity including grade 3 and 4 neutropenia was noted in 81% of patients while infections requiring hospitalization and antibiotic therapy occurred with 5% of the cumulative treatment courses. AML or MDS following therapy with this regimen was noted in 3% patients. Given the success of this combination, it is reasonable to consider this in fludarabine-refractory CLL patients who have bulky lymphadenopathy who are not good candidates for ...
• alemtuzumab was approved for marketing in fludarabine-refractory CLL based upon treatment efficacy in 93 heavily pretreated, fludarabine-refractory CLL patients^ref. This trial demonstrated an ITT response rate of 33%, although only 2% of patients achieved complete response. Median TTP for responders was 9.5 months, with a median OS of 16 months for all patients and 32 months for responders. While the median peripheral blood CLL count decreased by > 99.9%, alemtuzumab was less effective against nodal disease with only 12% of patients responding who had lymph nodes > 5 cm. Additionally, patients with an ECoG performance status of 2 did markedly worse than patients with no or minimal symptoms from their disease. As a result of this pivotal CAM211 study, alemtuzumab was approved for the treatment of fludarabine-refractory CLL in the USA. The activity of alemtuzumab in CLL was confirmed by a multi-institutional study in 136 patients with fludarabine-refractory B-CLL who received 30 mg thrice weekly for up to 12 weeks on a compassionate basis (Coutre S, Rai KR, Keating MJ, et al on Behalf of the Campath Study Group. Efficacy and safety of alemtuzumab (Campath-1H) in refractory B-CLL patients treated on a compassionate basis [abstract]. Blood. 2001:98(11):365a). The overall response rate was 40% (CR rate 7%), and the median PFS and OS of responders were 7.3 and 13.4 months, respectively. Currently several groups are combining alemtuzumab with fludarabine^ref or rituximab^ref as part of clinical trials. One intriguing finding with alemtuzumab is its ability to eliminate CLL disease with p53 mutations or deletions. This was first reported by Stilgenbauer and Dohner, who demonstrated a complete response in a single patient with del(17)(p13.1) receiving alemtuzumab^ref. Based upon this intriguing observation, a group sought to determine if alemtuzumab was an effective therapy in this patient group^ref. 36 patients with fludarabine-refractory CLL were treated with alemtuzumab, 15 of whom (42%) had p53 mutations or deletions. Clinical responses in patients with p53 mutations and/or deletions were noted in 6 of 15 (40%) versus 4 of 21 (19%) of patients without p53 abnormalities. The median response duration for this subset of patients was 8 months (range 3–17 months). These data confirm the results of Stilgenbauer and Dohner and suggest that alemtuzumab may be an effective therapy for CLL patients with p53 mutations or deletions. This also serves to contrast the divergent response patterns observed with mAbs, as CLL with p53 deletions is not responsive to rituximab therapy^ref. The major complications of alemtuzumab therapy include infections, infusion toxicity, and myelosuppression, which have limited its implementation into wide practice^{ref1, ref2}. With respect to infusion-related toxicity, subcutaneous administration has demonstrated promise in Phase II trials of previously untreated patients, but may have to be administered for a more prolonged period and have different pharmacokinetics. This option should therefore not be considered outside of a clinical trial until more data are available. Patients receiving alemtuzumab should receive prophylaxis for Pneumocystis carinii and VZV. In addition, patients should also be monitored carefully for CMV reactivation during and immediately after therapy (for a minimum of 2 months after therapy). Some consider this therapy for patients with a PST of 0–1 who have lymph nodes < 5 cm. In general alemtuzumab should be avoided in patients with active infections or who have a contraindication to prolonged immunosuppression.
• cyclin-dependent kinase inhibitor flavopiridol using a modified schedule of administration where promising early results have been observed
• monoclonal antibodies including engineered anti-CD20 antibodies, anti-HLA-DR antibodies, anti-CD40 antibodies, TRAIL receptor DR4 and DR5 directed antibodies, and antibody-like molecules targeting CD37 are also entering early Phase I trials. Finally a recent trial with denileukin diftitox was reported in 18 fludarabine-refractory patients where 2 of 18 (11%) patients had partial responses^ref. Significant constitutional symptoms were noted in a large proportion of these patients. Based upon these results a confirmatory study is ongoing at this time with this agent. Given the cost, toxicity, and marginal efficacy of this therapy, use of Ontak should be confined to ongoing Phase II trials. Indeed, it is through performance of clinical trials of promising new therapies in this patient population, with subsequent confirmation in less treated patients, that therapeutic options of CLL will ultimately improve.
• preclinical studies suggest Xcellerated T Cells have the potential to produce a potent anti-tumor effect, restore broad immune function and reduce the risk of infectious complications in patients with CLL. Unlike other cancer settings, T cells constitute only a small fraction of CLL patients' PBMC. To generate large numbers of Xcellerated T Cells of high purity from CLL patients' PBMC, a reproducible, streamlined and cost-effective good manufacturing process (GMP) is required. The 10-L volume Wave Bioreactor-based Xcellerate III Process using Xcyte Dynabeads in a single custom 20-L Cellbag container was adapted, qualified and implemented for GMP operations. For n=17 CLL patients, starting with approximately 1.34 x 10⁹ CD3⁺ T cells at 6.8+/-7.5% purity in the PBMC leukapheresis products, using the 10-L volume Wave Bioreactor-based Xcellerate III Process, it was feasible to manufacture 137.0+/-34.3 x 10⁹ Xcellerated T Cells at 98.5+/-1.0% CD3⁺ T-cell purity. An average 400-fold clearance of malignant B cells was documented during the manufacturing process. The Xcellerated T Cells produced from the Xcellerate III Process exhibited high in vitro biologic activity and have their T-cell receptor repertoire restored to a normal diversity. In-process T-cell activation was reproducibly robust, as measured by increase in cell size, up-regulation of CD25 and CD154 expression and the secretion of IL-2, IFN-g and TNF-a. A low-volume, high-yield bioreactor-based process has been developed, qualified and implemented for the reproducible, GMP manufacture of high purity, biologically active Xcellerated T Cells for the treatment of CLL patients in clinical trials^ref
• CpG ODN alters B-CLL cell phenotype and increases their immunogenicity. In contrast to the classic understanding of CpG ODN as inhibitors of B cell apoptosis, IS ODN including CpG ODN induce apoptosis in B-CLL cells. It is important that these changes are seen not only with CpG ODN but with ODN that lack the classical CpG motif. B-CLL cells from 20 subjects were treated in vitro with IS ODN for up to 7 days. IS ODN treatment resulted in increased numbers of apoptotic cells in 13 out of 20 B-CLL samples. IS ODN enhanced apoptosis in samples with 13q deletion as a single aberration and had a heterogeneous effect on apoptosis in samples with other aberrations including 17p deletion, 11q deletion, or trisomy 12. Induction of apoptosis did not correlate with expression of the CpG ODN receptor TLR9. Apoptosis was dependent on the activation of caspases and was accompanied by up-regulation of CD95/Fas and its ligand. IS ODN including CpG ODN can induce apoptosis of most B-CLL samples. The ability of IS ODN to induce apoptosis differs based on cytogenetic status. Up-regulation of CD95/Fas may play a role in IS ODN-induced apoptosis of B-CLL^ref.
• PNP inhibitors : forodesine was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T-lymphocytes. A phase I clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T-cells may be dependant on the levels of deoxynucleoside kinases. Because B-CLL cells have high activity of deoxycytidine kinase (dCK) these lymphocytes respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization, phosphorylation of p53 at ser15, and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation, changes in mitochondrial membrane potential, and PARP cleavage. Based on these data a phase II clinical trial of forodesine has been initiated for CLL patients^ref.
• low-dose thalidomide + oral fludarabine + cyclophosphamide is ineffective in heavily pre-treated patients with chronic lymphocytic leukemia. Elevated levels of TNF-a have been associated with progressive disease in patients with CLL. Thalidomide has been shown to inhibit production of TNF-a. The effects of thalidomide on clinical outcome and TNF-a serum levels were investigated in 5 pre-treated CLL patients. The schedule consisted on daily thalidomide (Thal), oral fludarabine (Flu) and oral cyclophosphamide (CTX). Median duration of treatment was 60 days; four patients stopped treatment for disease progression and one patient for neurological toxicity. Serum TNF-a levels did not show any decrease during treatment^ref
• IVIg^ref

• 1967 (published again in 1973 as the report of the Chronic Leukemia-Myeloma Task Force [1] and 1978 of Cancer and Leukemia Group B (CALGB) [2])
• 1988 : National Cancer Institute (NCI)-sponsored Chronic Lymphocytic Leukemia (CLL) Working Group was convened to develop a set of standardized eligibility, response, and toxicity criteria for clinical trials^ref
• 1989 : International Workshop on Chronic Lymphocytic Leukemia: Chronic lymphocytic leukemia: Recommendations for diagnosis, staging, and response criteria^ref
• 1996 : Cheson^ref : for diagnosis, the NCI-WC requires a lymphocyte count of 5 x 10⁹/ml, which is lower than the 10 x 10⁹/mL required by the IWCLL, unless the lymphocytes are B cells and the bone marrow is involved. To be considered a complete remission (CR), the NCI-WC criteria specify that < 30% lymphocytes must be present in the bone marrow, with a recommendation that the clinical significance of lymphoid nodules be assessed prospectively; the IWCLL allows focal infiltrates or nodules in the bone marrow aspirate and biopsy for CR. The IWCLL uses a shift in clinical stage as the sole index of partial remission (PR), whereas the NCI-WC provides more specific criteria and recommends validation of the relevance of stage shift. The major differences were the well-defined criteria in the NCI guidelines regarding when to initiate therapy, hematologic toxicity, and other important components for clinical trials design. The purpose of this report is to present those revisions as considered necessary in view of advances in the past 8 years. Many of these revisions evolved as the guidelines were used in a systematic fashion in large clinical trials and, also, with the experience following the use of newer, more effective

agents, such as fludarabine^{ref1, ref2, ref3, ref4, ref5, ref6} (Sorensen JM, Vena D, Fallavollita A, Cheson BD: Treatment of refractory chronic lymphocytic leukemia (CLL) with fludarabine monophosphate (FAMP). Proc Am Soc Clin Oncol 11:264, 1992 (abstr 867)). Although this report will focus on those changes recommended by the NCI-sponsored CLL Working Group, it will include sufficient details from the original guidelines so that the reader would find it a complete document by itself without having to refer to the older version. The initial NCI-WC Guidelines were primarily designed as recommendations for the conduct of clinical trials. An important addition is that these revisions will distinguish practice guidelines from research issues in the diagnosis, decision to treat, and monitoring response in patients with CLL. It is increasingly clear that a more biologically relevant staging and response assessment of patients is needed if we are to continue to make progress in defining clinically disparate patient subsets and generate more innovative and effective treatment options.
Recommendations regarding evaluation and monitoring of CLL patients :

recommendation		general clinical (the use of accepted treatment options for a CLL patient not enrolled on a clinical trial)	clinical trial
pretreatment evaluation	history and physical	always	always
	examination of PBS	always	always
	immunophenotyping of PBLs	always	always
	bone marrow at diagnosis	desirable	always
	BM prior to therapy	always if a study not performed recently, eg, at diagnosis	always
	cytogeneticimolecular studies	not generally indicated	if a research question
	CT scans, MRI, lymphangiogram, gallium scan	not generally indicated	not generally indicated
indications for treatment	treat with stage 0-1	not generally indicated	if a research question
	treat for active/progressive disease (newly dx)	always	always
	treat without active/progressive disease (newly dx)	not generally indicated	if a research question
	treat without active/progressive disease (relapsed/refractory)	not generally indicated	if a research question
	treat beyond maximum response	not generally indicated	if a research question
response assessment	CBC, differential	always	always
	bone marrow	desirable	always
	phenotype	not generally indicated	desirable
	cytogenetics/FISH	not generally indicated	if a research question

Comparison of NCI-Working Group and IWCLL guidelines for CLL :

			NCI	IWCLL
diagnosis	lymphocytes (x 109/L)		> 5; >= 1 B-cell marker (CD19, CD20, CD23) + CD5	>= 10 + B-phenotype or bone marrow involved OR < 10 + both of above
	atypical cells (%) (e.g. prolymphocytes)		< 55%	not stated
	duration of lymphocytosis		none required	not stated
	bone marrow lymphocytes (%)		>= 30%	> 30%
staging			modified Rai, correlate with Binet	IWCLL
eligibility for trials			active disease (details in document)	A : lymphs > 50 x 109/L; doubling time < 12 mo; diffuse marrow B, C : all patients
response criteria	CR	physical exam	normal (no lymphadenopathy, splenomegaly, hepatomegaly or B symptoms)	normal
		symptoms	none	none
		lymphocytes (x 109/L)	<= 4	< 4
		neutrophils (x 109/L)	>= 1.5	> 1.5
		platelets (x 109/L)	> 100	> 100
		Hb (g/dl)	> 11 (untransfused)	not stated
		bone marrow lymphs (%)	< 30 (no nodules)	normal, allowing nodules or focal infiltrates
	PR	physical exam (nodes, and/or liver, spleen)	>= 50% decrease	downshift in stage
		neutrophils (x 109/L)	>= 1.5
		platelets (x 109/L)	> 100
		hemoglobin (g/dl)	> 11 or 50% improvement
	duration of CR or PR		>= 2 mo	not stated
	progressive disease	physical exam	>= 50% increase or new	upshift in stage
		circulating lymphocytes	>= 50% increase
		other	Richter's syndrome
	stable disease		all others	no change in stage

• diagnosis of B-CLL
• peripheral blood : the clinical diagnosis of CLL requires an absolute lymphocytosis with a lower threshold of > 5,000 mature-appearing lymphocytes/mL in the peripheral blood, in part to separate CLL from small lymphocytic non-Hodgkin's lymphoma. Morphologically, the lymphocytes must appear mature. Nevertheless, it is common to find admixtures of larger or atypical cells, cells that are cleaved, as well as those consistent with prolymphocytes; however, the percentage that should be used to distinguish CLL from prolymphocytic leukemia (PLL) is controversial. A value of up to 55% is still consistent with the diagnosis of CLL^ref. The presence of > 55% prolymphocytes and/or > 15,000/mL of prolymphocytes establishes a diagnosis of primary PLL or progression to prolymphocytoid leukemia. However, the markers on these cells should be different (eg, PLL cells are negative for CD5 in half of the cases). Prospective assessment of the significance of the proportion of peripheral blood prolymphocytes, their phenotypic characteristics, and the patterns of clonal evolution remain important research questions. The peripheral blood should also be carefully examined to rule out a leukemic phase of mantle-cell lymphoma, another CD5⁺ lymphoid malignancy (Catovsky D: Diagnosis and treatment of CLL variants, in Cheson BD (ed): Chronic Lymphocytic Leukemia: Scientific Advances and Clinical Developments. New York, NY, Dekker, 1993, p 369)^ref. In the original guidelines^ref, a duration of lymphocytosis of > 4 weeks was required to substantiate the diagnosis. Since the clinical features, histology, and phenotypic characteristics are sufficient to permit an accurate diagnosis of CLL in the majority of patients, only in rare patients with questionable or indolent/smoldering CLL is a reassessment of the lymphocyte count needed after >= 4 weeks^{ref1, ref2}. The routine availability of peripheral blood lymphocyte immunophenotyping has facilitated the diagnosis of CLL in patients with a monoclonal lymphocytosis^{ref1, ref2, ref3}. 3 main phenotypic features define B-CLL: the predominant population shares B-cell markers (CD19, CD20, and CD23) with the CD5 antigen, in the absence of other pan-T-cell markers; the B cell is monoclonal with regard to expression of either k or l; and surface immunoglobulin (sIg) is of low density. Not only are these characteristics generally adequate for a precise diagnosis, but, importantly, they distinguish CLL from uncommon disorders such as PLL, hairy-cell leukemia, mantle-cell lymphoma, and other lymphomas^{ref1, ref2}. These guidelines have been proposed for B-CLL. Therefore, the following lymphoid malignancies are specifically excluded from protocol studies directed at patients with BCLL: T-CLL, prolymphocytic leukemia (B and T cell), hairy-cell leukemia and variant forms, splenic lymphoma with villous lymphocytes, large granular lymphocytosis, Sézary-cell leukemia, adult T-cell leukemid lymphoma, and leukemic manifestations of non-Hodgkin's lymphomas, including follicular center-cell and mantle-cell lymphoma types^{ref1, ref2, ref3}
• bone marrow examination : a bone marrow aspirate and biopsy are generally not required to make the diagnosis of CLL. Nevertheless, CLL is a disease of the bone marrow, and it is appropriate to evaluate a major site of involvement. The aspirate smear must show >= 30% of all nucleated cells to be lymphoid. A bone marrow examination also provides useful prognostic information by determining whether there is diffuse or nondiffuse involvement^ref, and permits an assessment of the erythroid precursors and megakaryocytes.
• immunophenotype : as noted earlier, a thorough immunophenotypic profile of the malignant lymphocytes from the peripheral blood is necessary for the initial diagnosis of the patient with CLL.
• molecular biology/cytogenetics : not only do cytogenetic analyses provide useful prognostic information, but they help identify potentially important nonrandom genetic alterations and oncogenes (Catovsky D: Diagnosis and treatment of CLL variants, in Cheson BD (ed): Chronic Lymphocytic Leukemia: Scientific Advances and Clinical Developments. New York, NY, Dekker, 1993, p 369)^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}. Sequential analysis of established genetic alterations may also be helpful in evaluating the evolution of the disease process. However, given the expense and limited availability of these studies, they should be restricted to a research setting in which to evaluate their potential prognostic and biologic importance.
• clinical staging : we recognize that there are two somewhat different major staging methods that are currently in use throughout the world: the Rai system^ref and the Binet system^ref. In 1981, the IWCLL recommended that the two systems be integrated so that each of the Binet stages be subclassified with the Rai stage. However, the IWCLL-integrated system has not received widespread usage, and physicians continue to use either the Rai or Binet method in both patient care and in clinical trials. For clinicians using the Rai classification, we recommend the use of the modified version, which reduces the number of prognostic groups from 5 to 3 (Rai KR: A critical analysis of staging in CLL, in Gale RP, Rai KR (eds): Chronic Lymphocytic Leukemia. Recent Progress and Future Direction. New York, NY, Liss, 1987, p 253). These 2 systems are outlined following.
• Rai system : in the 3-stage Rai system low risk encompasses Rai stage 0, with the clinical features of lymphocytosis in blood and bone marrow only. Intermediate risk encompasses stage I, with lymphocytosis and enlarged nodes, and stage II, with lymphocytosis plus splenomegaly andor hepatomegaly (nodes positive or negative). High risk encompasses stage III, with lymphocytosis plus anemia, and stage IV, with lymphocytosis and thrombocytopenia.
• Binet staging system : staging is based on the number of involved areas, and the level of hemoglobin (Hb) and platelet count. Whether significant adenopathy (> 1 cm in diameter) is bilateral or unilateral is recorded. Area of involvement considered for staging
• head and neck, including the Waldeyer ring (this counts as one area even if more than one group of nodes are enlarged).
• axillae (involvement of both axillae counts as one area).
• groins, including superficial femorals (involvement of both groins counts as one area).
• palpable spleen.
• palpable liver (clinically enlarged).
Stage A. Hb >= 10 g/dL and platelets >= 100 X 10⁹/L and up to two of the above involved.
Stage B. Hb >= 10 g/dL and platelets >= 100 X 10⁹/L and organomegaly greater than that defined for stage A, ie, 3 or more areas of nodal or organ enlargement.
Stage C. All patients, irrespective of organomegaly in whom Hb less than 10 g/dL and/or platelets < 100 x 10⁹/L.
• eligibility criteria for clinical trials :
• clinical stage : the stage of CLL eligible for a clinical trial should reflect the therapeutic objectives, anticipated toxicities, and desired end results for each study. For example, a phase I study should involve only patients in advanced stages (Rai high risk, poor prognosis), while phase II and, particularly, phase III studies may also include patients in the intermediate-risk group. Decisions will be based on pilot data from phase I and early phase II trials with the particular agent or regimen. Patients with Rai stage 0 disease should generally not be entered into clinical trials. Other requirements for eligibility for clinical trials with respect to age, clinical stage, performance status, organ function, and status of disease activity should be defined for each study.
• performance status  : for phase I clinical trials, only patients with an Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0 to 2 should be eligible. For phase II and III clinical trials, patients with PS 0 to 3 may be eligible; however, these limits may be individualized on the basis of the drugs or therapies being tested. For trials in which it appears reasonable to include patients with PS 3, yet where there is a concern over the potential toxicities of an agent or therapy, it may be advisable to initially start those patients at a lower dose of the treatment (eg, SO% reduction) and to gradually increase the dose over subsequent courses to the standard dose. if the treatment is well tolerated and toxicity is within an acceptable range. This approach should be individualized for each relevant protocol and may not be appropriate for some therapies (eg, high-dose therapy with stem-cell support).
• organ function eligibility for clinical trials : most chemotherapy agents possess the potential for toxicity to the liver, kidneys, heart, lungs, central or peripheral nervous system, or other organ systems. Therefore, organ function requirements must be guided by the known toxicities of each drug based on observations from animal studies and previous therapeutic trials. Normal function for organs for which there is a well-recognized, specific toxicity must be required. Otherwise, as a general principle:
• baseline liver enzymes (ie, transaminase levels) should be no worse than 1.5 times the upper range of normal values. Serum bilirubin concentration should be >= 2.0 mg/dL, unless resulting from documented hemolysis.
• baseline renal function (ie, blood urea nitrogen [BUN], creatinine) should be no worse than 1.5 times the upper range of normal values.
• baseline requirements for other studies (eg. systolic ejection fraction, pulmonary function tests) should be decided individually for each study.
• infection status
• patients with active infections requiring systemic antibiotics should be excluded from B-CLL clinical trials until resolution of infection.
• patients who are HIV⁺ should be excluded because of their poor tolerance to chemotherapy and the potential risks from the immunosuppressive effects of new agents such as fludarabine^ref
• second malignancies : patients with a second malignancy, other than non-basalcell carcinoma of the skin or in situ carcinoma of the cervix, should not be entered onto a CLL clinical trial unless the tumor was treated with curative intent at least 2 years previously.
• required pretreatment evaluation : as already noted, the parameters that are considered necessary for a complete pretreatment evaluation differ whether the patient is being treated in a general practice setting or on a clinical research protocol. In general, and where feasible, these studies should be quantified within 48 hours of placing a patient on a treatment protocol (except for bone marrow aspirate and biopsy), and CT scans (see later). They should also be repeated at appropriate intervals to assess the maximum response to therapy.
• complete blood cell count (CBC; white blood cell count, hemoglobin and hematocrit, platelet count) and differential, including both percent and absolute number of lymphocytes and prolymphocytes, and reticulocyte count.
• unilateral bone marrow aspirate and biopsy should be performed within 2 weeks prior to entering the study, unless a previous diagnostic specimen was diffusely involved and there has been no intervening systemic therapy. It is preferable to evaluate the bone marrow at diagnosis for prognostic purposes; however, it is mandatory in clinical trials, and highly desirable in clinical practice, to perform a unilateral bone marrow aspirate and biopsy prior to treatment to provide a baseline for further response assessment. If a repeat bone marrow is obtained, it should be reviewed along with the original diagnostic sample.
• lymph node evaluation
• physical examination should record the diameter, in 2 planes, of the largest palpable nodes in each of the following sites: cervical, axillary, supraclavicular, inguinal, and femoral.
• chest radiograph.
• CT scans are generally not necessary in the initial evaluation of patients with CLL, but should only be performed if clinically indicated. A chest CT may be useful if the chest radiograph shows hilar adenopathy. These should be obtained within 2 weeks prior to entering the protocol.
• lymph node biopsy is generally not indicated, unless such tissue is necessary for companion scientific studies.
• liver and spleen size should be assessed by physical examination. CT scans should only be performed if clinically indicated or if part of a research question (see section 3.4.3.3).
• serum chemistries (eg, creatinine, bilirubin).
• assessment of PS (ECOG).
• baseline immunobiologic, cytogenetic, and molecular assessment for CLL trials. Those that should be performed on all patients include serum immunoglobulin determination, including quantitative immunoglobulins and immunoelectrophoresis, direct and indirect antiglobulin (Coombs’ test), and immunophenotypic evaluation of the B-cell clone
• Indications for treatment
• primary treatment decisions : once the diagnosis of CLL has been made, the treating physician is faced with the decision of not only how to treat the patient, but when to initiate therapy. Criteria for initiating treatment may be quite different between clinical practice and clinical trial conduct. A subset of patients are considered as having smoldering CLL; they include those with Rai stage 0 (Binet A), with a nondiffuse pattern of bone marrow involvement, a serum Hb concentration >=13.0 g/dL, peripheral blood lymphocytes < 30 X 10⁹/L, and a lymphocyte doubling time > 12 month^{ref1, ref2}. Therapy should not be offered to these patients until they exhibit clear evidence of disease progression. Other newly diagnosed patients with early stage disease (Rai 0 to I, Binet A), should be monitored without therapy until evidence of disease progression. Studies from both the French Cooperative Group on CLL and the Cancer and Leukemia Group B (CALGB) in patients with early-stage disease confirm that early therapy of patients with early-stage disease does not prolong survival^{ref1, ref2}, but may be associated with an increased frequency of fatal epithelial cancers^ref. However, these studies were conducted with alkylator-based regimens, and the potential benefit of earlier therapy using nucleoside analog therapy is an important research question. Whereas most patients with Rai stages III and IV require treatment at presentation, many can still be monitored without therapy until they exhibit evidence of progressive or symptomatic disease. Active disease should be clearly documented for protocol therapy. The following criteria must be met:
• a minimum of any one of the following disease-related symptoms must be present:
• (a) Weight loss 210% within the previous 6 months.
• (b) Extreme fatigue (ie, ECOG PS 2 or worse; cannot work or unable to perform usual activities).
• (c) Fevers > 100.5°F for >= 2 weeks without evidence of infection.
• (d) Night sweats without evidence of infection.
• evidence of progressive marrow failure as manifested by the development of, or worsening of, anemia and/or thrombocytopenia
• autoimmune anemia and/or thrombocytopenia poorly responsive to corticosteroid therapy
• massive (ie, >6 cm below the left costal margin) or progressive splenomegaly
• progressive lymphocytosis with an increase of >50% over a 2-month period, or an anticipated doubling time < 6 months
• marked hypogammaglobulinemia or the development of a monoclonal protein in the absence of any of the above criteria for active disease is not sufficient for protocol therapy
Patients with CLL may present with a markedly elevated leukocyte count; however, the symptoms referable to leukocyte aggregates that develop in patients with acute leukemia rarely occur in patients with CLL. Therefore, the absolute lymphocyte count should not be used as the sole indicator for treatment, but should be included as a part of the total clinical picture, which includes the lymphocyte doubling time (see earlier).
• second-line treatment decisions : treatment of CLL is generally palliative in intent; therefore, patients who have relapsed may be followed without therapy until they experience disease-related symptoms or progressive disease, with deterioration of blood counts, discomfort from lymphadenopathy or hepatosplenomegaly, recurrent infections, or associated autoimmune disorders. A possible exception is allogeneic bone marrow transplantation. Recent data suggest that, in selected patients, allogeneic bone marrow transplantation or high-dose chemotherapy with autologous stem-cell support may be reasonable treatment options, particularly in the context of a clinical research protocol^{ref1, ref2, ref3}. The acceptable extent of prior therapy for protocol entry must be decided separately for each study.
• for all phase III therapeutic trials, it is recommended that only those patients who have not received previous cytotoxic or biological therapy be eligible. It is appropriate to include patients who have received previous corticosteroids if this is compatible with the therapeutic objectives of the trial. However, it may be necessary to analyze these previously treated patients as a separate group.
• for phase I and II studies, we recommend that < 2 types of prior therapy (eg, fludarabine, chlorambucil with or without prednisone) be allowed for entering patients. Certain trials may require previously untreated patients; this will be determined separately depending on the objectives of the study.
• definition of response : assessment of response should include a careful physical examination and evaluation of the peripheral blood and bone marrow. The response criteria in the original NCI-WG guidelines have been retained.
• complete remission requires all of the following for a period of at least 2 months:
• absence of lymphadenopathy by physical examination and appropriate radiographic techniques.
• no hepatomegaly or splenomegaly by physical examination, or appropriate radiographic techniques if in a clinical trial.
• absence of constitutional symptoms.
• normal CBC as exhibited by :
• PMN leukocytes >= 1,500/mL.
• platelets > 100,000/mL.
• hemoglobin > 11.0 g/dL (untransfused).
• bone marrow aspirate and biopsy should be performed 2 months after clinical and laboratory results demonstrate that all of the requirements listed in 5.1.1 to 5.1.4 have been met to demonstrate that a CR has been achieved. The marrow sample must be at least normocellular for age, with < 30% of nucleated cells being lymphocytes. Lymphoid nodules should be absent. If the bone marrow is hypocellular, a repeat determination should be made in 4 weeks. Samples should be re-reviewed in conjunction with the prior pathology.
Grading scale for hematological toxicity in CLL studies :

decrease in platelets (if at any level of decrease the platelet count is <20,000/mL, this will be considered grade 4 toxicity, unless a severe or life-threatening decrease in the initial platelet count (eg, ~20,000/mL) was present pretreatment, in which case the patient is inevaluable for toxicity referable to platelet counts) or Hbt (nadir; baseline and subsequent Hb determinations must be performed before any given transfusions) from pretreatment value (%)	grade (death occurring as a result of toxicity at any level of decrease from pretreatment will be recorded as grade V)	ANC/ml (nadir) (if the absolute neutrophil count (ANC) reaches < 1,000/mL, it should be judged to be grade 3 toxicity. Other decreases in the white blood cell count, or in circulating granulocytes, are not to be considered, since a decrease in the white blood cell count is a desired therapeutic end point. A gradual decrease in granulocytes is not a reliable index in CLL for stepwise grading of toxicity. If the ANC was < 1,000/mL prior to therapy, the patient is inevaluable for toxicity referable to the ANC)
0-10%	0	>= 2,000
11-24%	1 / mild	1,500-2,000
25-49%	2 / moderate	1,000-1,500
50-74%	3 / severe	500-1,000
>= 75%	4 / life-threatening	< 500

• for patients who fulfill all of the previous criteria for a CR, an abdominal CT scan may be performed to confirm this clinical and hematologic impression if clinically indicated or if required testing for a clinical research study.
• PR is considered in a broad sense to enable the detection of agents with biological effect. To be considered a PR, the patient must exhibit 250% decrease in peripheral blood lymphocyte count from the pretreatment baseline value and 250% reduction in lymphadenopathy and/or size of the liver and/or spleen (if abnormal prior to therapy), as well as one or more of the remaining features for at least 2 months. In addition, the presence or absence of constitutional symptoms will also be recorded.
• 250% decrease in peripheral blood lymphocyte count from the pretreatment baseline value.
• 250% reduction in lymphadenopathy.
• 250% reduction in the size of the liver and/or spleen.
• PMN leukocytes >= 1,500/mL or 50% improvement over baseline.
• platelets > 100,000/mL or 50% improvement over baseline.
• hemoglobin > 11.0 g/dL or 50% improvement over baseline without transfusions.
• in a subset of patients who are otherwise in a complete remission, bone marrow nodules can be identified histologically. It is, unfortunately, difficult with currently available techniques to determine the clonality of these nodules. The original NCI-WG guidelines suggested that patients with a CR and persistent nodules should be analyzed carefully to compare their outcome relative to others who are more conventionally classified as a CR or PR^ref. Robertson et al^ref have since demonstrated that patients with a nodular CR had a shorter time to disease progression compared with patients with a CR. Therefore, nodular CRs should be reported separately from CRs, and should not be used to inflate the percentage of CRs. We recommend that they be referred to as nodular PRs (nPR) and included with the PRs.
• a controversial issue is how best to categorize the response of patients who fulfill all the criteria for a CR, but who have a persistent anemia or thrombocytopenia apparently unrelated to disease activity and more likely the consequence of persistent drug toxicity. The long-term outcome of these patients may differ from the more routine complete responders. Therefore, these patients should not be considered CRs or a separate response category, but should be considered PRs. However, they should be monitored prospectively to better characterize their outcome, and may be described within the context of results of clinical trials.
• progressive disease (PD) will be characterized by at least one of the following:
• 250% increase in the sum of the products of at least two lymph nodes on two consecutive determinations 2 weeks apart (at least one node must be >= 2 cm); appearance of new palpable lymph nodes.
• 250% increase in the size of the liver and/or spleen as determined by measurement below the respective costal margin; appearance of palpable hepatomegaly or splenomegaly, which was not previously present.
• 250% increase in the absolute number of circulating lymphocytes to at least 5,000/mL.
• transformation to a more aggressive histology (eg, Richter’s syndrome or PLL with >55% prolymphocytes).
• in the absence of progression, as defined earlier, the presence of a >= 2 g/dL decrease in Hb, or 250% decrease in platelet count, and/or absolute granulocyte count will not exclude a patient from continuing the study. Each protocol will define the amount of drug(s) to be administered with such hematological parameters. Bone marrow aspirate and biopsy are strongly encouraged to better define the cause of the suppressed counts.
• patients who have not achieved a CR or a PR, or who have not exhibited PD, will be considered to have stable disease.
• responses that should be considered clinically beneficial include CR, nPR and PR; all others, eg, stable disease, nonresponse, progressive disease, and death from any cause, should be rated as a treatment failure.
• because current criteria for response are arbitrary and often not validated by prospective studies, alternative criteria may also be evaluated; however, to ensure comparability  with other studies, these should be studied within the framework of the current schema and be well defined, with adequate rationale. Should such a schema be determined to have important clinical relevance following prospective evaluation, it will be considered for incorporation into criteria for future studies.
• duration of response should be measured from the time the patient has exhibited the features of maximum response until evidence of progressive disease. Survival duration should be measured from the time of entry onto the clinical trial.
• prognostic factors requiring stratification
• previous treatment versus no previous treatment in studies for which prior therapy is allowed.
• if > 1 clinical stage is allowed, patients should be stratified for stage (eg, if intermediate and poor risk are eligible, intermediate v poor), depending on the nature of the study and the available patient resources.
• application of new prognostic factors : in the interval since the initial publication of the guidelines, several modifications have been recommended.
• decrease in lymphocyte count: In several recent studies, a decrease in the peripheral blood lymphocyte count has been used as the primary index of response? Although this parameter may identify a therapy that has lymphocytotoxic activity, there is no evidence that it has long-term clinical implications. It has, therefore, not been incorporated into the current response criteria.
• immunobiological assessment
• quantification of the serum immunoglobulin concentration in responders is recommended at the time of maximal clinical response, but it is not an established indicator of response^ref.
• repeat immunophenotyping at the time of a response is not part of standard practice. Moreover, progression of disease after a CR should not be based purely on the basis of a small number of clonal cells identified using flow cytometric determinations.
• in the clinical trials setting, not only should the peripheral blood smear and bone marrow aspirate and biopsy be carefully examined, but immunophenotype, cytogenetics, (including fluorescent in situ hybridization [FISH]), and molecular biologic studies provide important data and should be performed at diagnosis, at the time of maximal response, and at recurrence if part of a research question.
• serum b₂-microglobulin is recommended as an inexpensive prognostic marker (Keating MJ, Lerner S , Kantarjian H, Freireich W, O’Brien S: The serum beta2-microglobulin (beta2M) level is more powerful than stage in predicting response and survival in chronic lymphocytic leukemia (CLL). Blood 86:606a, 1995 (abstr, suppl 1))
• other optional studies that may be of interest include markers of B-cell proliferation such as Ki-67, which might identify alterations in the malignant cell population, soluble CD23, adhesion molecules, or molecular analysis of specific genes (eg, oncogenes, tumor-suppressor genes). These scientific parameters that assess the biology of the malignant clone may help us to identify new therapeutic strategies.
• MRD : the optimal approach to the patient with minimal residual disease remains another important research issue. Careful assessment for minimal residual disease as determined by flow cytometry, cytogenetics, or similar studies is not indicated outside of a research study at the time of CR and at recurrence. Additional treatment decisions on the basis of minimal residual disease remains an issue for clinical investigation.
• assessment of toxicity : an evaluation of potential treatment-induced toxicity in patients with advanced malignancies may be quite difficult, requiring careful consideration of both the manifestations of the underlying disease, as well as adverse reactions to the therapies under study. Moreover, some of the conventional criteria for toxicity are not applicable to studies involving patients with hematological malignancies in general, or CLL in particular. An example is hematological toxicity; patients with advanced CLL may exhibit a deterioration in blood counts, which may represent either treatment-related toxicity or progressive bone marrow failure from the disease itself. This discrimination may become increasingly difficult as new agents are tested earlier in their development at a point where the complete spectrum of their toxicities has not yet been elaborated. A few guidelines are presented recognizing that evaluation methods will be determined to a large extent within the therapy involved.
• hematological toxicity : as is the case with virtually all of the hematological malignancies, an evaluation of hematological toxicity in patients with CLL must consider the high frequency of hematological compromise at the initiation of therapy. Therefore, the standard criteria used for solid tumors cannot be applied directly; many patients would be considered to have grade II to IV hematological toxicity at presentation. Also, in the past, the peripheral blood neutrophil level has rarely been used as a criterion for dose modification since these values were felt to be unreliable in CLL. However, the increasing use of more effective therapeutic agents, particularly those with neutropenia as a dose-limiting toxicity (eg, nucleoside analogs), has resulted in clinically significant myelosuppression. Therefore, we have proposed a new dosemodification scheme for quantifying hematological deterioration in patients with CLL, which includes alterations in the dose of myelosuppressive agents based on the absolute neutrophil count
• infectious complications : in CLL, as with many other hematological malignancies, it may be difficult to distinguish between the occurrence of infections related to the disease itself or to the consequences of therapy. However, such an analysis is of value when comparing the results of various treatments, particularly with immunosuppressive agents such as the nucleoside analogs^ref. The etiology of the infection should be reported and categorized as bacterial, viral, or fungal, and proven or probable. The severity of infections should be quantified as minor (requiring either oral antimicrobial therapy or symptomatic care alone), major (requiring hospitalization and systemic antimicrobial therapy), or fatal (death as a result of the infection).
• nonhematological toxicities : other nonhematological toxicities should be graded according to the NCI Common Toxicity Criteria (Wittes RE (ed): Manual of Oncologic Therapeutics: 1991/1992. Philadelphia, PA, Lippincott, 1991, p 446)
• reporting of clinical response data : clear and careful reporting of data is an essential part of any clinical trial. In clinical studies involving previously treated patients, patients who are relapsed or refractory should be clearly distinguished. Relapse is defined as a patient who has previously achieved the clinicopathologic criteria to be classified as a CR or PR, but, after a period of >= 6 months, demonstrated evidence of disease progression. For those patients who have relapsed, it is also useful to describe the quality and duration of their prior response. Refractory disease refers to the clinical situation in which a patient fails to achieve at least a PR or progresses while on therapy.

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