Homo sapiens molecular nosology - T cell large granular lymphocyte leukemia (T-LGL)

T-CELL LARGE GRANULAR LYMPHOCYTE LEUKEMIA (T-LGL)

Table of contents :

Epidemiology

Pathogenesis

Symptoms & signs

Laboratory examinations

Differential diagnosis

Therapy

Prognosis

T-LGL is a clonal disorder of cytotoxic T lymphocytes (CTL)^ref1,
ref2. The establishment of T-LGL as a leukemia rather than a reactive disorder was based on nonrecurring chromosomal abnormalities observed in some patients with T-LGL in conjunction with the presence of tissue infiltration in the bone marrow, spleen, and liver^ref
Epidemiology : T-LGL represents only 4% of the chronic lymphoproliferative disorders^ref; however, it represents the most frequent T cell malignancy^ref. Patients tend to be older, with a median age of 55; there is an equal male/female distribution^ref. Pediatric cases have been recognized
Pathogenesis : central to the understanding of T-LGL leukemogenesis is the notion that T-LGL cells are antigen activated in vivo^ref. Multiple levels of evidence support this hypothesis, including immunophenotypic data demonstrating CD45RO, HLA-DR, perforin, and CD57 expression. Functionally, T-LGL display non-MHC–restricted cytotoxicity after anti-CD3 mAb stimulation. Molecular evidence of common usage of TCR-ß junctional motifs suggests antigenic pressure in the clonal evolution of the disease^ref1,
ref2. Finally, DNA microarray analysis has demonstrated upregulated proteases while protease inhibitors are downregulated. While the source of the antigenic activation is still uncertain, some patients with T-LGL show serum reactivity to gag p24 and env p21e of HTLV-I virus but do not have prototypical HTLV infection. These findings suggest a possible infection with a retrovirus with homology to HTLV-I as an underlying drive for the leukemic process. In contrast to normal CTL, T-LGL cells are resistant to Fas -mediated apoptosis. Unlike the mechanism of autoimmune lymphoproliferative disease, no mutation in the death domain of Fas in T-LGL has been observed. Alternatively, constitutive activation of STAT3 with upregulation of mcl-1 has been implicated in apoptotic resistance. Furthermore, novel splice variants of soluble Fas molecules resulting in "decoy receptors" allow T-LGL cells to circumvent immunosurveillance. A more detailed discussion of the leukemogenesis of T-LGL is provided in a recent review^ref. T-LGL is frequently associated with autoimmune disorders. Rheumatoid arthritis (RA) is the most common associated disorder, with T-LGL occurring in 25–33% of patients^ref. Similar to T-LGL, Felty's syndrome (FS) is likewise characterized by neutropenia with variable splenomegaly in conjunction with RA. Indeed it is our opinion that these disorders are part of a single disease process. The original diagnostic criteria for lymphoproliferative disorder of granular lymphocytes (including both T and NK forms of LGL) required an absolute large granular lymphocytosis in excess of 2.0 x 10⁹/L. Since that time, patients with lower numbers of LGLs have been shown to have similar clinical courses and response to therapy as those meeting the former diagnostic criteria^ref. Previously published historical studies had reported a low occurrence (8%) of LGL counts < 1.0 x 10⁹/L, while more recent studies have reported patients with T-LGL having LGL counts < 0.5 x 10⁹/L in 25%–30% of cases^ref1,
ref2. In such studies, the diagnosis is based on multicolor flow cytometric immunophenotyping and molecular analysis of the TCR genes. As would be expected from this shift, disorders originally diagnosed as FS in the past may well be classified as T-LGL in the present. Relative frequencies of Felty’s syndrome and T-LGL based on selected diagnostic criteria :

The number of CD3⁺, CD8⁺, CD57⁺ cells is measured by flow cytometry. TCR clonal rearrangement is assessed by PCR.
Given the subtleties in diagnostic distinction, it is not surprising that these disorders share a common immunogenetic link. Patients with FS and T-LGL with RA have a similar frequency of the HLA-DR4 allele (80–90%)^ref. However, T-LGL patients without RA lack the elevated allele frequency and are similar to normal racially matched controls (33%)^ref. More recently, the immunohistochemical evaluation of the T cell infiltrate within the bone marrow of patients with T-LGL have shown the quantity and pattern of infiltration to be specific to the diagnosis^ref1,
ref2; however, similar patterns of T cell infiltration have been observed in patients with FS as those of T-LGL. What can safely be said about FS and T-LGL with RA is that they appear to be related disorders whose distinction is somewhat arbitrary. These disorders can be separated by the presence of clonal TCR gene rearrangements in T-LGL, but not in FS, recognizing that clonally expanded CD3⁺, CD57⁺ lymphocytes (LGL) may be detected using sensitive techniques in nonneutropenic patients with RA as well as some normal elderly individuals. What is not understood yet is why patients with RA are especially prone to develop clonal expansions of T-LGL. Nevertheless, it is likely that the pathogenesis of neutropenia in these disorders overlaps. Early studies into the mechanisms of neutropenia in FS revealed 2 distinct groups of patients :

one group exhibited evidence of humoral mediated destruction (60%)^ref. These patients had either monomeric (non-complexed) immunoglobulin, presumed to be neutrophil autoantibodies and/or immune complexes of varying sizes^ref
alternatively, about 40% of patients lacked neutrophil-bound immunoglobulin and were reported to have depressed granulocyte colony growth in bone marrow co-culture experiments using patient lymphocytes^ref.

While neutrophil autoantibody-mediated autoimmune neutropenia is perhaps the easiest mechanism to conceptualize, it is the most difficult to evaluate in the laboratory. Unlike red cells in autoimmune hemolytic anemia

, granulocytes are more difficult to isolate and therefore a direct "Coombs test" for neutrophils is not practical. As such, indirect assays using patient serum and banked normal neutrophils, namely the granulocyte immunofluorescent test (GIFT) and granulocyte agglutination test (GAT), represent the screening methods of choice. To add further complexity, neutrophils, unlike red cells, express HLA-antigens as well as Fc-receptors for immunoglobulin, which limits the ability of the test to distinguish between neutrophil autoantibodies, HLA alloantibodies, and immune complexes. The antibody-dependent lymphocyte-mediated granulocytotoxicity test (ADLG) can detect neutrophil-bound monomeric immunoglobulin and, with prior absorption using random pooled platelets, can distinguish between confounding HLA alloantibodies and neutrophil autoantibodies. When a battery of such tests is performed with FS sera, 77% have bound immunoglobulin via either the GIFT or GAT tests. Based on the results of the ADLG test, showing positivity on only 14%, the bound antibody appears to be made of predominantly immune complexes. Of those with positive ADLG test, all were inhibited by platelet absorption indicative of HLA alloantibodies, which implies that true neutrophil autoantibodies are not present^ref. A similar analysis of 5 patients with T-LGL without RA revealed low levels of immune complexes in only 1 patient and no definitive neutrophil autoantibodies in the remaining 4 patients^ref. Testing for antineutrophil antibodies is not recommended for routine clinical practice. Because of the complexity of this approach, newer techniques have been employed to assess neutrophil-specific autoantibodies. Using an antibody phage display library made from the RNA of FS bone marrow lymphocytes, a putative neutrophil autoantibody was discovered. Panning against a myeloid antigen expression library with this antibody revealed a protein identified as eukaryotic elongation factor-1A-1 (eEF1A-1). An ELISA test, using this factor to screen sera of patients with FS, revealed positivity in 66% of 62 patients. The antibody does not appear to be specific to FS, as positive results were also seen in RA without neutropenia (23%) and SLE (9%), but not normal controls^ref. Similarly, adults with atopic dermatitis

without neutropenia also express antibody to eEFA1-1^ref. The frequency of eEFA1-1 autoantibodies has yet to be tested in patients with T-LGL. Immune complexes represent another humoral mechanism by which neutropenia may occur. Using column separation, ultracentrifugation, and C1q binding assays, patients with FA have increased numbers of immune complexes as compared to normal controls and patients with RA without neutropenia. Furthermore, the neutrophil-bound immune complexes were of varying sizes and were at least partially composed of rheumatoid factor^ref. The binding of immune complexes to neutrophils leads to several important physiologic sequelae. First, neutrophils become activated after binding immune complexes, and the degree of activation appears to be dependent on the type of immune complex encountered. When neutrophil activation is measured by chemiluminescence, activation is most intense after incubation with precipitated immune complex followed by soluble immune complex with monomeric (non-complexed) IgG providing the least activation. Not surprisingly then, neutrophils incubated with sera from patients with FS showed greater activation than with sera from patients with RA and normal controls^ref. Neutrophil activation results in cellular changes that account for neutropenia. Neutrophils incubated with sera from patients with FS, but not from patients with RA, display increased adherence to endothelial cells in vitro^ref. This mechanism of increased endothelial adherence may explain the occurrence of transient neutropenia in mice after injection with sera from patients with FS and to a lesser degree with sera from RA patients, while injection of sera from normal controls results in transient elevations of the neutrophil counts. Necropsy of mice following injection with FS sera confirmed that the increased endothelial adherence occurs in vivo, showing increased neutrophil margination with deposition of IgG, IgA, and IgM within vascular beds^ref. These observations suggest that immune complexes may induce neutropenia by altering the distribution of neutrophils into the marginating pool. Finally, some types of immune complexes appear to induce neutrophil apoptosis. The characteristics of the immune complexes appear to be critical in this role, as precipitated immune complexes induce apoptosis while soluble immune complexes decrease apoptosis. Neutrophils can bind immune complexes by means of Fc-receptors, of which FcgR-II (CD32)

but not FcgR-III (CD16)

appears critical in the pathway of neutrophil apoptosis. Surprisingly, CCR3 (CD11b/CD18) is not essential in precipitated immune complex-induced apoptosis. Moreover, precipitated immune complex-induced apoptosis is independent of the Fas/Fas-L pathway and is instead dependent upon reactive oxygen intermediates, in particular hydrogen peroxide^ref. Cell-mediated mechanisms are of critical importance in the pathogenesis of neutropenia in T-LGL. While the mechanisms responsible for T-LGL cell survival result from inhibition of signaling pathways leading to Fas/FasL-induced apoptosis, the Fas/FasL system is also intimately related to neutropenia in T-LGL. As previously discussed, T-LGL cells constitutively express Fas-ligand (FasL) on their cell surface, whereas normal T and NK cells express FasL only after activation. Moreover, the FasL expressed on T-LGL cells can produce cytotoxicity against W4 cells in vitro, and this cytotoxicity appears to be independent of the perforin pathway of cytotoxicity^ref. The FasL receptor, Fas (CD95), is expressed ubiquitously on a variety of cells including normal granulocytes. Neutrophils express higher levels of Fas than eosinophils or monocytes. It therefore follows that neutrophils are more susceptible to Fas-mediated apoptosis than eosinophils or monocytes when treated with Fas-activating antibody (CH-11)^ref. Others have noted that proinflammatory cytokines such as FasL and INF- may inhibit myeloid progenitors in patients with idiopathic chronic neutropenia^ref. While FasL is constitutively expressed on the surface of T-LGL cells, MMPs can cleave this protein and generate soluble FasL (sFasL). In support of this process is marked elevation of sFasL in the sera of patients with T-LGL, NK-LGL, and NK-lymphomas but not other leukemias^ref. Indeed, sera from patients with T-LGL with elevated sFasL induce neutrophil apoptosis in vitro in a similar fashion to Fas-activating antibody (CH-11). Furthermore, clinical response to therapy in patients with T-LGL is associated with a decrease in serum sFasL^ref. It remains unclear, however, what significance sFasL plays in the mechanism of neutropenia in vivo as disorders such as NK-LGL and NK-lymphomas also exhibit high levels of sFasL but are characterized by severe pancytopenia and hepatic dysfunction rather than the isolated neutropenia observed in T-LGL. Furthermore, mice injected with high levels of sFasL experience hepatic necrosis, which is uncharacteristic of T-LGL^ref. As such, the pattern of neoplastic cell infiltrate better correlates with the distribution of tissue dysfunction in these disorders, suggesting bound FasL and perhaps the local paracrine effects of sFasL contribute to neutropenia in T-LGL and FS, while the diluted serum sFasL represents a useful test in monitoring disease activity. In summary, the mechanisms of neutropenia can be attributed to problems with production, distribution, and destruction. Unlike many of the congenital neutropenias described elsewhere in this section, neutrophil production defects are not typical in T-LGL/FS. Examination of the bone marrow myeloid elements in patients with T-LGL usually reveals mild hypercellularity with left-shifted myeloid maturation^ref1,
ref2. This assessment would suggest that neutropenia is at least in part a result of peripheral destruction. However, bone marrow coculture experiments with the lymphocytes from some patients with FS results in diminished granulocyte colony growth^ref. It appears likely then that both peripheral and intramedullary destruction of neutrophils are responsible for neutropenia in T-LGL. Neutrophil destruction may occur by means of immune complex induction via FcgR-II (CD32) activation leading to death by reactive oxygen intermediates. Fas-mediated apoptosis resulting from direct contact of T-LGL cells expressing FasL or local paracrine effects of sFasL released by the functions of MMPs also are important in pathogenesis of neutropenia. Finally, measured neutropenia may result from increased margination as a result of precipitated immune complex activation of neutrophils. While these studies have approached the mechanisms of neutropenia in T-LGL and FS independently, it should be clear that the variability in diagnostic criteria utilized renders these distinctions meaningless. Furthermore, it is likely that any or all the above mechanisms are involved in the pathogenesis of neutropenia in a given patient with T-LGL. In patients with active rheumatologic disease, immune complexes may play a dominant role in inducing neutrophil margination and apoptosis via reactive oxygen intermediates. In such patients, only small populations of clonal cytotoxic T cells may exist and their contribution to neutropenia may be less pronounced. Alternatively, patients with marked expansions of clonal FasL-bearing T-LGL cells may experience neutropenia as a result of Fas-mediated apoptosis in the absence of active rheumatological disease. Still, patients with relatively inactive RA with only moderate numbers of clonal T-LGL cells may experience severe neutropenia, and the mechanism of this cytopenia is probably a result of the combined mechanisms described herein. The mechanism of adult onset cyclic neutropenia in T-LGL remains unknown.
Mechanisms of cytopenia are not clearly defined. Inhibition of erythropoiesis in patients with pure red cell aplasia appears to be mediated directly by leukemic LGL. In one patient with LGL of phenotype, it was demonstrated that leukemic LGL expressing killer receptors were cytotoxic for red cell progenitors lacking HLA class I^ref. As there may be clinical overlap with LGL leukemia, MDS, and aplastic anemia, it is conceivable that hematologic suppression in these diseases occurs through a similar pathway involving activated CD8⁺ T cells producing inhibitors belonging to the TNF family. For example, Fas ligand plays a role in mediating neutropenia in LGL leukemia (Liu JH, Wei S, Lamy T, et al. Chronic neutropenia mediated by Fas ligand. Blood. 2000;95:3119–3222). Autoimmune features are prominent in LGL leukemia. Frequent serologic abnormalities include positive tests for rheumatoid factor and/or ANA, high levels of circulating immune complexes, polyclonal hypergammaglobulinemia, and high levels of ß₂-microglobulin^ref. Autoimmune diseases are also a characteristic finding in LGL leukemia. Clinical, immunologic, molecular, and genetic data indicate that patients with LGL leukemia and rheumatoid arthritis (RA) and patients with Felty's syndrome are part of the spectrum of the same disorder. Up to one third of neutropenic patients with RA have clonal LGL expansions^ref1,
ref2. In addition, oligoclonal CD3⁺8⁺57⁺T cells have been demonstrated in RA patients (Hingorani R, Monteiro J, Pergolizzi R, Furie R, Chartsah E, Gergersen PK. CDR3 length restriction of T-cell receptor ß chains in CD8+ T-cells of rheumatoid arthritis patients. American New York Of Academic Sciences. 1995;756:179–182). Increased numbers of cells with a phenotype similar to leukemic LGL have been observed in blood or synovial fluid of RA patients^ref. The identical MHC locus association with HLA-DRß *0401 is found in patients with LGL/RA as well as those with Felty's syndrome^ref. It has been recently recognized that clonal expansion of CD28^- T cells with many phenotypical and functional characteristics of LGL, including CD57 and perforin expression as well as TcR-mediated cytotoxicity, is a characteristic finding in RA^ref. These data suggest a common pathogenetic link between LGL leukemia and RA. Distinctive pathologic findings in LGL leukemia are found in bone marrow, spleen, and liver (Agnarsson BA, Loughran TP, Jr, Starkebaum G, Kadin ME. The pathology of large granular lymphocyte leukemia. Hum. Pathol. 1989;20:643–651). Unlike other T cell malignancies, skin and lymph node involvement is uncommon. It is important to emphasize that it is difficult to recognize LGL in tissue sections; consequently morphologic findings may be indistinguishable from other indolent lymphoproliferative disorders. Correlation with immunophenotyping studies are therefore very helpful for diagnostic evaluation. In contrast to our initial description of frequent lymphoid aggregates in marrow sections, an interstitial diffuse infiltration appears more common. This finding is particularly highlighted by use of immunostaining for T cells in marrow sections. Typical findings in the spleen include red pulp infiltration by leukemic cells and often reactive follicular hyperplasia of the white pulp. This pattern of red pulp infiltration needs to be distinguished from hairy cell leukemia, in which red pulp infiltration is especially characteristic. Liver biopsies from LGL leukemia patients show prominent intrasinusoidal infiltration. In cases where infiltration is marked, portal areas are sometimes involved.
Symptoms & signs : infection and fever

are the presenting features in 20–40% of patients. As the infection is related to neutropenia, common locations of infection are the skin, oropharynx, and perirectal regions. B symptoms (fever, night sweats, and weight loss) occur in 20–30% of patients, while about 1/3 are asymptomatic. Organomegaly involving the spleen (20–50%) and liver (10–20%) is typical, while lymphadenopathy and skin involvement are uncharacteristic.
Laboratory examinations :

severe chronic neutropenia (most commonly)
mild to moderate anemia is not an infrequent finding
less commonly, adult onset cyclic neutropenia, pure red cell aplasia, and immune-mediated thrombocytopenia may be the presenting hematologic feature.
typical bone marrow findings demonstrate increased numbers of CTLs in an interstitial and sinusoidal distribution^{ref1,
ref2,
ref3,
ref4}.
cytopenia
immunophenotype of peripheral blood : CD3⁺8⁺16⁺56⁺57⁺, TcRab⁺
clonal TCR gene rearrangement at peripheral blood PCR : circulating, clonal CD3⁺ LGL counts > 3 standard deviations above the number of T cells coexpressing CD3 and CD57, an LGL marker (the normal values for CD3⁺, CD57⁺ cells are 22 ± 99/µl^ref, so some consider values above approximately 520/µl as being abnormal). Clonality is best determined by utilization of TcR gene rearrangement studies, either using Southern blotting or PCR. It has been recognized that patients may have a clonal T cell population with even normal LGL counts, and such cases would be compatible with the diagnosis of LGL leukemia^ref. Since the total lymphocyte count may not be elevated, it is recommended that the peripheral blood smear be carefully evaluated for LGL in patients with hematologic and/or autoimmune abnormalities suggestive of LGL leukemia.

Differential diagnosis :

	LGL leukemia	hairy cell leukemia	B-cell chronic lymphocytic leukemia (B-CLL)	follicular lymphoma (FL)	reactive lymphocytosis
cytology (peripheral blood)	LGL	hairy cells	small lymphocytes	cleaved lymphocytes	atypical lymphocytes and LGL
liver	sinus and portal infiltrates	sinus and portal infiltrates	sinus and portal infiltrates	portal infiltrates infiltrates; hepatocyte injury	sinus and portal
spleen	red pulp infiltrate plasmacytosis follicular hyperplasia of white pulp	red pulp infiltrate red cell "lakes" reduction of white pulp	primary expansion of white pulp frequent infiltration of red pulp	white pulp infiltrate	red pulp infiltrate (immunoblasts, neutrophils)
bone marrow	diffuse or nodular pattern usually nonparatra- becular often subtle involvement maturation arrest	diffuse pattern frequent "dry tap"	diffuse or nodular pattern usually nonparatrabecular	usually nodular pattern	usually diffuse pattern plasmacytosis paratrabecular sometimes granulomas

Leukemic LGL represent antigen-driven cytotoxic T lymphocytes (CTL). Data supporting this contention include :

the unique subset of normal CD3⁺ LGL may represent in vivo primed CTL directed against viral targets^ref
the CD3⁺8⁺57⁺DR⁺ phenotype of leukemic LGL and the rapid triggering of non-MHC-restricted killing via CD3/CD16 pathway^ref1,
ref2
structural analysis of rearranged TCR genes as well as TCR Vß repertoire analyses in some LGL cases have provided evidence for antigenic selection^ref1,
ref2

Perhaps the most compelling data come from analyses of molecules involved in the cytotoxic process, perforin and Fas ligand. These proteins are only expressed in CTL after activation. Leukemic LGL constitutively express high levels of both perforin and Fas ligand^ref1,
ref2. High expression of other cytotoxic molecules, such as granzyme and calpain, was found in leukemic LGL compared to normal CD8⁺ cells, using microarray analyses. Constitutive expression of such proteins suggests that leukemic LGL are constantly exposed to some antigen in vivo. Although these data suggest that leukemic LGL are antigen driven CTL, the antigen specificity of these leukemic clones is not known. It is interesting to note that increased numbers of LGL can be seen after infection with viruses such as CMV and HIV^ref (Zambello R, Trentin L, Agostini C, et al. Persistent polyclonal lymphocytosis in human immunodeficiency virus-1-infected patients. Blood. 1993;11:3015–3021). CTL responding to these infections have been characterized as memory or effector CD8+ populations based on expression pattern of antigens such as CD28, CD45, CD62L, and perforin. Using Vß antibodies to identify the leukemic clone, we have determined that leukemic LGL are CD3⁺8⁺57⁺45RA⁺45RO^-25^-62L^-, and CD28^-. These data show that leukemic LGL are effector CTL. Others have detected the clonal abnormality in both memory and effector CD8⁺ cells^ref. This effector cytotoxic phenotype of the leukemic clone is similar to that of CTL generated after HIV infection^ref. Retroviral infection is also characterized by production of pro-inflammatory chemokines such as RANTES, MIP1-a, and MIP1-ß^ref. A similar pattern of chemokine expression is observed in patients with LGL leukemia (unpublished observations). Taken together, these findings suggest that retroviral infection may be a stimulus for LGL activation. LGL leukemia patients are HIV negative; a few patients have been infected with HTLV-I or HTLV-II^ref1,
ref2. Although most patients are not infected with prototypical HTLV, sera from these patients do show reactivity against gag p24 and env p21e of HTLV-I^ref. Epitope mapping studies have shown that reactivity against env p21e is directed at the BA21 epitope of HTLV-I^ref. We hypothesize that a cellular or retroviral protein having homology to BA21 may be important in the pathogenesis of LGL leukemia. Dysregulated apoptosis is a characteristic feature of LGL leukemia. Normal CTL recognizing viral peptide in the correct MHC context upregulate Fas ligand. Virally infected target cells expressing Fas are then eliminated through apoptosis. Deletion of such antigen-activated T cells occur through the same process of Fas-mediated apoptosis^ref1,
ref2. Leukemic LGL express high levels of both Fas and Fas ligand (Lamy T, Liu JH, Landowski T, et al. Dysregulation of CD95/CD95 Ligand-apoptotic pathway in CD3+ LGL leukemia. Blood. 1998;12:4771–4777). Ilness in LGL leukemia such as neutropenia, anemia, or RA may be caused in part by constitutive production of Fas ligand. Accumulation of the leukemic clone is due to inhibition of the Fas apoptotic pathway in the leukemic LGL. The mechanism causing neutropenia in LGL leukemia has not been defined. Normal neutrophils undergo apoptosis through Fas triggering^ref. High levels of circulating Fas ligand were found in sera from 39 of 44 patients with LGL leukemia. Serum from patients caused apoptosis of normal neutrophils that depended partly on the Fas pathway. Resolution of neutropenia was associated with disappearance or marked reduction in Fas ligand levels in 10 of 11 treated patients (Liu JH, Wei S, Lamy T, et al. Chronic neutropenia mediated by Fas ligand. Blood. 2000;95:3119–3222). These data suggest that neutropenia in LGL leukemia is mediated by Fas ligand. Decreased levels of Fas ligand have also been observed in a few LGL leukemia patients who had correction of anemia on therapy (Liu JH, Wei S, Lamy T, et al. Chronic neutropenia mediated by Fas ligand. Blood. 2000;95:3119–3222; Saitoh T, Karasawa M, Sakuraya M, et al. Improvement of extrathymic T cell type of large granular lymphocyte (LGL) leukemia by cyclosporin A: the serum level of Fas ligand is a marker of LGL leukemia activity. Eur J Haematol. 200;65:272-275). Leukemic LGL are resistant to Fas-mediated death in vitro, despite expressing high levels of both Fas and Fas ligand (Lamy T, Liu JH, Landowski T, et al. Dysregulation of CD95/CD95 Ligand-apoptotic pathway in CD3+ LGL leukemia. Blood. 1998;12:4771–4777). Fas resistance can not be explained on the basis of Fas mutations^ref, unlike children with autoimmune lymphoproliferative syndrome^ref. Resistance to Fas mediated death can be overcome in vitro (Lamy T, Liu JH, Landowski T, et al. Dysregulation of CD95/CD95 Ligand-apoptotic pathway in CD3+ LGL leukemia. Blood. 1998;12:4771–4777)^ref. These data suggest that resistance is due to inhibition of Fas signaling pathway in leukemic LGL. Leukemic LGL displayed high levels of activated STAT3. Inhibition of STAT signaling with either AG-490, a JAK-selective tyrosine kinase inhibitor, or STAT3 antisense reversed apoptotic resistance in leukemic LGL. AG-490-induced apoptosis was independent of Bcl-XL or Bcl-2 expression. In contrast, levels of Mcl-1 expression decreased in leukemic LGL after AG-490 treatment and that Mcl-1 is a STAT-3 regulated gene. STAT3 activation contributes to the accumulation of the leukemic LGL clones, possibly through upregulation of the anti-apoptotic protein Mcl-1^ref. These results identify the STAT3 signaling pathway as molecular targets for drug discovery in LGL leukemia
Therapy : T-LGL is an indolent disorder that responds well to immunosuppressive therapies. Indications for therapy of T-LGL include severe neutropenia

< 500/mL or recurrent infections due to chronic, less severe neutropenia. Symptomatic or transfusion

-dependent anemia are other reasons for initiating treatment^ref. The majority of patients with T-LGL (75%) will require therapy over the course of their disease, although rare cases will spontaneously remit. Methotrexate

10 mg/m²/week orally induces complete remission in 50% of patients; however, it is likely that indefinite treatment is required to prevent relapse. It is important to note that several months of therapy are generally required before counts improve. Cyclosporine A

represents an alternative therapy to methotrexate. In a study of 25 patients, 50% had response to therapy, with 24% gaining complete remission. It is interesting to note that therapeutic response to cyclosporine A is related to HLA-DR4 haplotype, which is common in patients with FS and T-LGL with RA^ref. In addition, patients with concurrent T-LGL and myelodysplasia

have a lower response to cyclosporine A than patients with T-LGL alone^ref. Cyclophosphamide

has been used orally to treat T-LGL with good response^ref. Prednisone

in combination with cyclophosphamide appears to increase the duration of response compared to prednisone alone. Overall response to therapy with prednisone and cyclophosphamide is 66% with a median duration of 32 months. 2 prospective therapeutic trials are currently underway. The Eastern Cooperative Oncology Group (ECOG) is evaluating the efficacy of oral methotrexate 10 mg/m²/week with crossover to cyclophosphamide in non-responders. The Cancer and Leukemia Group B (CALGB) is testing cyclosporine A orally 2 mg/kg/q12 hours as front-line therapy. Indications for both trials are neutropenia or symptomatic or transfusion-dependent anemia. Because immunosuppressive therapies slowly correct neutropenia, hematopoietic growth factors such as GM-CSF

or G-CSF

may induce more rapid correction of the neutrophil count in patients with severe neutropenia.
Active agents for treatment of this disease are drugs which are categorized as immunosuppressants and include oral low-dose methotrexate (10 mg/m² po once weekly), cyclosporine (2 mg/kg po q12 hours), oral cyclophosphamide (100 mg po daily), and prednisone (1 mg/kg po daily)^{ref1,
ref2,
ref3,
ref4}. Prednisone alone is not recommended as cytopenias almost always recur as the dose of prednisone is tapered^ref. Low dose methotrexate has been used by us primarily for treatment of severe neutropenia. In a small series we documented a complete remission in 50% of cases^ref. This favorable response was also observed in a group of patients who had both LGL leukemia and rheumatoid arthritis^ref. Cyclosporine has also been used effectively for correcting neutropenia in some patients^ref. LGL leukemia was identified as the most common cause of pure red cell aplasia in two large single institution studies^ref1,
ref2. Immunosuppressive therapy typically given to patients with pure red cell aplasia was effective in the cases associated with LGL leukemia^ref. A small proportion of patients with LGL leukemia will present with a more aggressive clinical course^ref1,
ref2. In such cases, lymphoma type regimens do not appear particularly effective although reported experience is quite limited^ref1,
ref2. This resistance might be explained on the basis of leukemic LGL expressing high levels of multidrug resistant genes such as P-glycoprotein and lung resistant protein^ref. Some of these patients failing combination chemotherapy have had sustained clinical responses to low dose methotrexate and prednisone^ref. The mechanisms of therapeutic response in LGL leukemia are not well understood. Retrospective analyses of patients treated primarily with methotrexate did show that responses were associated with decreased levels of Fas ligand. It is likely that a similar mechanism occurs in patients treated with cyclosporine, as resolution of neutropenia occurs despite persistence of the leukemic clone^ref. Indeed reduction of Fas ligand levels on cyclosporine treatment have been observed in a case report (Saitoh T, Karasawa M, Sakuraya M, et al. Improvement of extrathymic T cell type of large granular lymphocyte (LGL) leukemia by cyclosporin A: the serum level of Fas ligand is a marker of LGL leukemia activity. Eur J Haematol. 200;65:272-275). It is likely that methotrexate has additional mechanisms of action. Prolonged methotrexate therapy of 1-2 years duration does result in complete remission and disappearance of the leukemic clone in some patients, in contrast to cyclosporine therapy^ref. Methotrexate treatment leads to reversal of apoptotic resistance seen in leukemic LGL. Preliminary studies from our lab indicate that methotrexate induces apoptosis of activated T cells through a mitochondrial-dependent pathway. We are currently investigating whether regulation of Mcl-1 is involved in the mitochondrial pathway triggered by methotrexate.
Clinical trials : enrollment into clinical trials should be encouraged as there are only limited treatment data available, as noted above. 2 trials are currently being conducted in the cooperative group setting. Both of these trials involve correlative laboratory studies designed to examine mechanisms of treatment response. ECOG is studying initial treatment of LGL leukemia with methotrexate (TP Loughran, PI) whereas CALGB is investigating cyclosporine (Maria Baer, PI, Roswell Park). Indications for treatment in both studies include either neutropenia (ANC < 500 or neutropenia with recurrent infections) or anemia (ECOG: symptomatic or transfusion-dependent anemia: CALGB: hemoglobin < 9g/dL). A national registry for LGL leukemia is established at Moffitt Cancer Center in order to define the natural history and prognosis of this disease.
Prognosis : in the largest series of 68 patients reported from a single institution, the median survival was superior to 10 years^ref. In an earlier smaller series we had reported that actuarial survival at 5 years was 67% (Loughran TP Jr. Clinical course of LGL leukemia. Fundamental and Clinical Immunology. 1994;2:147–151). However, the majority of patients in both series eventually needed treatment for symptoms resulting from neutropenia or anemia (69% and 89%, respectively).

LGL leukemia

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