Large Granular Lymphocyte Leukemia
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《肿瘤学家》
LEARNING OBJECTIVES
After completing this course, the reader will be able to:
Discuss the basic principles of molecular and cellular biology of LGL leukemia.
Describe distinct clinical entities among disorders of LGLs.
Discuss the diagnostic criteria for T-cell LGL leukemia.
Discuss the therapeutic algorithm of LGL leukemia.
ABSTRACT
Clonal disorders of large granular lymphocytes (LGLs) represent a spectrum of biologically distinct lymphoproliferative diseases originating either from mature T cells (CD3+) or natural killer (NK) cells (CD3–). Both subtypes, T-cell and NK-cell LGL leukemia, can manifest as indolent or aggressive disorders. The majority of patients with T-cell LGL leukemia have a clinically indolent course with a median survival time >10 years. Immunosuppressive therapy with low-dose methotrexate, cyclophosphamide, or cyclosporine A can control symptoms and cytopenias in more than 50% of patients, but this approach is not curative. Several cases of an aggressive variant (CD3+CD56+) of T-cell LGL leukemia with a poor prognosis have also been reported. Aggressive NK-cell LGL leukemia is usually a rapidly progressive disorder associated with Epstein-Barr virus (EBV), with a higher prevalence in Asia and South America. This disease is usually refractory to conventional chemotherapy, with a median survival time of 2 months. Chronic NK-cell leukemia/lymphocytosis is a rare EBV-negative disorder with an indolent clinical course. The malignant origin of this subtype is uncertain because clonality is difficult to determine in LGLs of NK-cell origin.
DEFINITION AND CLASSIFICATION
Large granular lymphocytes (LGLs) represent 10%–15% of the total peripheral blood mononuclear cells in normal adults [1]. The majority of these cells (85%) are derived from the CD3– natural killer (NK)–cell lineage, and a minority are derived from the CD3+ T-cell lineage (15%). Cytologically, LGLs are medium to large cells with eccentric nuclei and abundant cytoplasm with coarse azurophilic granules (Fig. 1). T-cell LGLs are post-thymic, antigen-primed cytotoxic CD8+ T lymphocytes. NK-cell LGLs belong to the innate immune system with the capability of non-major histocompatibility complex (MHC)–restricted cytotoxicity.
LGL leukemia was initially described in 1985 as a clonal disorder involving blood, marrow, and spleen [2]. In 1993, we proposed two LGL disorders based on either T-cell or NK-cell lineage, which was subsequently adopted by all pathology classification systems [3]. Clonal disorders of LGLs represent a biologically heterogeneous spectrum of lymphoid malignancies (Fig. 2) [4, 5]. Both the T-cell and NK-cell subtypes can clinically present as an indolent or aggressive diseases (Table 1). World Health Organization (WHO) classification of lymphoid malignancies includes T-cell LGL leukemia and aggressive NK-cell leukemia as two separate entities among T-cell/NK-cell lymphomas/leukemias [6]. Since only rare cases of the aggressive variant of T-cell LGL leukemia have been described [7], this subtype was not given separate status in the WHO classification. Transient (<6 months) and chronic (>6 month) expansions of LGLs are two benign conditions in the spectrum of disorders of LGLs [8]. Transient reactive populations of LGLs have been detected in patients with viral infections, autoimmune diseases, and malignancies, and in patients after solid organ transplantation [9–13]. The reactive LGLs are polyclonal with expression of the T-cell (CD3+) immunophenotype in the majority of cases. LGL count normalizes spontaneously or with therapy of underlying condition, usually within 6 months. Since it is difficult to determine clonality for NK cells, it is uncertain whether chronic NK-cell lymphocytocis represents a chronic NK-cell leukemia. In the absence of a clonal marker, clinical presentation is the most important factor available for the differential diagnosis of these two conditions. Patients with systemic symptoms or infiltration of the spleen, liver, or bone marrow could be appropriately classified in the category of chronic NK-cell leukemia. Asymptomatic patients might be better given a diagnosis of benign chronic NK-cell lymphocytosis. In this review we focus mainly on diseases of LGLs included in the WHO classification.
EPIDEMIOLOGY
LGL leukemia comprises 2%–5% of all T-cell/NK-cell malignancies, with only 400 cases reported in the literature [8]. Indolent T-cell LGL leukemia is the most common subtype, representing approximately 85% of all cases diagnosed in Western countries. The male-to-female ratio is approximately equal to one. This entity is more frequently diagnosed in older individuals, with a median age at diagnosis of 60 years (Table 1). The aggressive type of NK-cell LGL leukemia typically occurs in younger individuals, with median age at diagnosis of 39 years and with a higher prevalence in Asia and South America [3, 14, 15]. Fewer than 100 cases have been described in literature [16] (Table 1).
ETIOPATHOGENESIS
The etiology of LGL leukemia is not known. Chronic activation of T cells with autoantigen or viral antigen has been suggested as an initial stimulus leading to an expansion of LGLs [17, 18]. Whether a second molecular event is necessary to establish the full malignant phenotype is not clear. It has also been suggested that T-cell LGL leukemia could represent an autoimmune disorder caused by chronic antigenic stimulation leading to extreme expansion of only one clone of CD8+ cytotoxic T cells [19, 20]. An association of T-cell LGL leukemia with several different autoimmune conditions supports this hypothesis. Human T-cell leukemia virus II (HTLV-II) sequences have been detected in two patients with indolent T-cell LGL leukemia [21, 22]. Seroindeterminate reactivity against HTLV-I envelope (env) epitope BA21 has been described in approximately 50% of patients with CD3+ and 73% of patients with CD3– LGL leukemia [22, 23]. Detailed amino acid analysis of BA21 revealed that a 10–amino acid peptide, PP10, was responsible for the seroreactivity [24]. This peptide shared high amino acid homology only with HTLV-I env protein and not with any known human proteins. However, most patients with LGL leukemia are not infected with prototypical members of the HTLV family, including HTLV-I, HTLV-II, or bovine leukemia virus (BLV) [25, 26]. Epstein-Barr virus (EBV) is implicated in the pathogenesis of aggressive NK-cell leukemia [27]. It has been suggested that this condition is a leukemic variant of a more common NK-cell/T-cell lymphoma, the nasal type.
IMMUNOPHENOTYPE
LGL leukemia cells have a mature T- or NK-cell immunophenotype [3]. The most common immunophenotypes for each subtype of LGL leukemia are described in Table 1. CD57 is a 110-kDa glycoprotein found on NK cells and activated, effector CD8+ T cells. It is a characteristic marker for LGL leukemia. It was suggested that LGLs in T-cell LGL leukemia originate in a CD57– memory T-cell compartment that continually produces CD57+ (effector cell) progeny [28]. Rare immunophenotypic variants that are CD3+ T-cell receptor (TCR)-ßCD4+CD8+, CD3+TCR-ßCD4–CD8–, and CD3+TCR-CD4–CD8– have been reported in T-cell LGL leukemia [1, 29]. Aberrant expression of pan T-cell markers, including CD5 and/or CD7, can be useful in differentiating the malignant T-cell LGL population from normal T lymphocytes.
The NK-cell marker CD56 is typically detected in aggressive NK-cell LGL leukemia [30]. The immunophenotype of this subtype is similar to that of the nasal type of NK-cell/T-cell lymphoma that is CD2+CD56+CD3+ [31]. In the differential diagnosis, this aggressive NK-cell malignancy must be distinguished from indolent chronic NK-cell leukemia and benign chronic NK-cell lymphocytosis, which are not associated with EBV (Fig. 2) [3, 32].
CYTOGENETICS
Indolent T-cell LGL leukemia cells most frequently have a normal karyotype. Less than 10% of patients display distinct chromosomal aberrations, including inversion of 12p and 14q, deletion of 5q, and trisomy of 3, 8, and 14 chromosomes [2, 33, 34]. The most frequent clonal chromosomal abnormality in patients with aggressive NK-cell LGL leukemia is the deletion of the 6q chromosome, but cases with complex karyotypes have also been reported [35, 36].
CLONALITY
LGL leukemia is a disorder of mature T or NK cells. T-cell ontogenesis is associated with TCR gene rearrangement resulting in a unique molecular fingerprint for each T lymphocyte. Thus, the malignant LGL population, which arises from a single cell, has the same TCR gene rearrangement pattern. Southern blotting and polymerase chain reaction (PCR) are the two methods most commonly used for confirmation of clonality [37]. Recently, a panel of monoclonal antibodies was used against the variable domain of the ß chain as a new clonality technique in T-cell malignancies [38]. Since available antibodies recognize approximately 75% of the Vß repertoire of T cells, this technique has not yet achieved widespread use in clinical practice. NK cells have TCR genes in a nonrearranged germline position. In the past, cytogenetic abnormalities and clonality studies based on the X-chromosome inactivation pattern (XCIP) were the only methods available for confirmation of the clonal origin of the NK-cell population [36]. Recently, a new class of NK-cell receptors, killer cell immunoglobulin-like receptors (KIRs), has been detected on the surface of NK cells and a subset of T cells [39]. Aberrant expression of these receptors has been reported in some patients with LGL leukemia [39]. In particular, we demonstrated that patients’ NK cells had high levels of activating receptors and a loss of inhibitory receptors [40]. These findings suggest a potential use of KIR expression as clonality markers but will require validation in a larger study.
CLINICAL PRESENTATION
Approximately two thirds of the patients with indolent T-cell LGL leukemia develop cytopenias, recurrent bacterial infections, autoimmune disorders, and/or splenomegaly during the course of their disease (Table 1) [1]. Recurrent infections resulting from neutropenia initially manifest in 20%–40% of patients. B symptoms—including fever, night sweats, and weight loss—occur in 20%–40% of patients. Mild-to-moderate splenomegaly is found in 20%–50% of patients, and hepatomegaly is found in 10%–20% of patients. Diffuse infiltration of red splenic pulp with preservation of sinuses and white pulp cords is characteristic of T-cell LGL leukemia [41]. Lymphadenopathy is an uncommon presentation of this disease. Bone marrow biopsy typically reveals discrete interstitial and sinusoidal infiltration with T lymphocytes [8]. Rheumatoid arthritis is the most common autoimmune disorder, manifesting in up to 30% of patients with T-cell LGL leukemia [8].
Clinical manifestations of the aggressive variant of T-cell LGL leukemia closely resemble those of aggressive NK-cell LGL leukemia (Table 1). This condition is very rare, with only several well-described cases in literature [7].
Aggressive NK-cell LGL leukemia typically presents as an acute illness, with B symptoms, lymphocytosis, hepatosplenomegaly, lymphadenopathy, severe anemia and thrombocytopenia, and hemophagocytic syndrome [3, 16]. Chronic NK-cell lymphocytosis usually is an indolent disorder with a good prognosis. Rare cases present with cytopenias, cutaneous vasculitis, peripheral neuropathy, and splenomegaly (Table 1) [42].
HEMATOLOGIC FEATURES
The normal range of LGLs in peripheral blood is 0.2–0.4 x109/l [1]. Evaluation of peripheral blood film is invaluable if a diagnosis of LGL leukemia is suspected. A previous study suggested that >90% of patients with T-cell LGL leukemia present with an LGL count in excess of 1.0 x 109/l [1]. In our original paper, diagnostic criteria for T-cell LGL leukemia required an absolute LGL count >2 x 109/l [3]. More recent studies revealed that 25%–30% of newly diagnosed patients present with an absolute neutrophil count (ANC) <0.5 x 109/l [43]. The advent of flow cytometry and progress in immunohistochemistry and molecular clonality studies has allowed the detection of much smaller populations of clonal LGLs in a background of normal polyclonal hematopoiesis [44]. Flow cytometry can also uncover rare cases of LGL leukemia with the absence of typical LGL morphology but with the typical immunophenotype [45]. Detection of a smaller clonal LGL population in patients with systemic symptoms or cytopenias does not require a waiting period of 6 months to establish the diagnosis [8, 43]. However, in cases with chronic NK-cell lymphocytosis and in patients with small oligoclonal or polyclonal populations of T-cell LGLs, a repeat study in 6 months can help to differentiate reactive lymphocytosis from the malignant condition [3, 8]. Evaluation of bone marrow aspirate and biopsy is not required for the diagnosis of LGL leukemia [3]. Recently, however, we and others found that this test can be useful, especially in patients with small circulating LGL subpopulations [8]. A careful review of the bone marrow biopsy supported by immunohistochemistry may reveal infiltration with LGLs in up to 85% of patients [8]. The linear arrays of intravascular CD8+, T-cell intracellular antigen–1+ (TIA-1+), granzyme B+ lymphocytes observed with immunohistochemistry appear specific for LGL leukemia [46]. There is not a good correlation between the extent of marrow infiltration with LGL leukemic cells and the degree of cytopenias or severity of systemic symptoms [1, 3]. Since the majority of T-cell LGL leukemia cells are in the G0/G1 phase, it is reasonable to believe that LGL lymphocytosis is caused by the accumulation of leukemic cells resulting from a block in apoptosis [1]. We have suggested that increased resistance of unstimulated T-cell LGL leukemic cells to apoptosis triggered via the Fas/Fas ligand (FasL) pathway could play a critical role in the pathogenesis of this disorder [47]. However, no causative somatic mutations in Fas or FasL genes have been found.
PATHOGENESIS OF CYTOPENIAS
Neutropenia is the most common cytopenia, found in 80% of patients with indolent T-cell LGL leukemia [1, 3]. Approximately 45% develop severe neutropenia (<0.5 x 109/l) associated with infections [8]. Several different cellular and humoral mechanisms have been suggested in the pathogenesis of neutropenia in T-cell LGL leukemia, including the direct destruction of myeloid precursors with infiltrating leukemic cells, dysregulation of the maturation of myeloid cells, antibody- or immune complex–mediated peripheral destruction of neutrophils, and induction of apoptosis of neutrophils as a result of activation of the Fas/FasL pathway [48].
Adult-onset cyclic neutropenia is a rare acquired disorder characterized by periodic neutropenia and bacterial infections. The majority of cases with this rare condition are associated with LGL leukemia [49]. Anemia with a hemoglobin level <11 g/dl is found in approximately 48% of patients with T-cell LGL leukemia [3]. Approximately 20% of patients require transfusions. Coombs-positive autoimmune hemolytic anemia is diagnosed only in rare cases [50]. Pure red cell aplasia (PRCA) characterized by hypoproliferative anemia and the absence of mature erythroid precursors in otherwise normocellular marrow occurs in 8%–19% of patients [3]. T-cell LGL leukemia surpasses thymoma as the leading cause of PRCA [51]. It was suggested that LGL leukemia cells can suppress growth or directly lyse human erythroid progenitor cells in bone marrow [52]. Erythroid progenitors usually lose HLA class I antigens during maturation. T cells or NK cells normally express KIRs, which inhibit cytotoxicity when the target cell expresses the specific HLA class I antigen(s). Loss of these antigens on maturing erythroid progenitors makes them susceptible to destruction by the LGL leukemia cells [52, 53]. Thrombocytopenia (<150 x 109/l) presents in about 20% of patients [1]. However, severe thrombocytopenia requiring transfusions is rare [4]. Possible mechanisms responsible for thrombocytopenia include inhibition of megakaryopoiesis in bone marrow by LGL leukemic cells, antibody-mediated peripheral destruction in cases associated with immune thrombocytopenia (ITP), and splenic sequestration in patients with splenomegaly [8].
AUTOIMMUNE DISORDERS
A significant proportion of patients (40%–60%) with T-cell LGL leukemia present with immunologic abnormalities, including seropositivity for rheumatoid factor (RF) and antinuclear antibodies (ANAs), polyclonal hypergammaglobulinemia, circulating immune complexes, and anti-neutrophil antibodies [1, 3]. Although the dysregulation of B-cell function as a result of the presence of malignant cytotoxic CD8+ T cells has been suggested, the pathobiology of these laboratory findings is not well understood [54].
The most common autoimmune disorder associated with T-cell LGL leukemia is rheumatoid arthritis, which manifests in about 25%–33% of patients [1, 3]. A rare autoimmune clinical entity, Felty syndrome, which is characterized by the triad of rheumatoid arthritis, neutropenia, and splenomegaly, closely resembles T-cell LGL leukemia with rheumatoid arthritis [55]. Both diseases have a significantly higher frequency of expression of the HLA-DR4 haplotype than matched healthy controls, suggesting that they may represent a clinical spectrum of the identical disorder. Therapy with low-dose methotrexate can control both arthritis and LGL leukemia [3, 56]. Case reports of patients with LGL leukemia associated with Sjögren syndrome, systemic lupus erythematosis, and Hashimoto’s thyroiditis have also been reported [1, 11, 57]. No significant association with autoimmune disease has been reported in aggressive NK-cell leukemia [8].
DIAGNOSTIC CRITERIA AND DIFFERENTIAL DIAGNOSIS
Figure 3 provides an algorithm for the diagnosis of T-cell LGL leukemia. Diagnosis is based on the following four criteria.
Criterion A: Sustained Expansion of T-cell LGLs in Peripheral Blood
Lymphocytosis is usually in the range of 2–20 x 109/l; however, 25%–30% patients with T-cell LGL leukemia may display a smaller population of circulating LGLs (<0.5 x 109/l) [1].
Criterion B: Expression of Characteristic Immunophenotype
Expression of the characteristic immunophenotype of T-cell LGL leukemia is assessed by multiparameter flow cytometry (Table 1). Rare immunophenotypic variants might also be associated with indolent T-cell LGL leukemia [3, 8].
Criterion C: Clonality of T-Cell Population
The clonality of the T-cell population should be confirmed either by the detection of a TCR gene rearrangement using PCR or Southern blot or by the detection of a restricted TCR Vß repertoire with flow cytometry and monoclonal antibody panel. Chronic NK-cell leukemia may be more difficult to differentiate from reactive polyclonal expansions of NK cells as a result of the germinal configuration of TCR genes in these cells. In a minority of cases, the clonal origin of NK cells is confirmed with the detection of an aberrant karyotype or using the XCIP in informative female patients [3].
Criterion D: Clinical Presentation
Clinical presentation is an important part of the diagnostic process of T-cell/NK-cell malignancies because a similar morphology of cells and immunophenotype can be associated with diseases of distinct biological behavior. The cytopenias, splenomegaly, and rheumatoid arthritis that are typically found in patients with T-cell LGL leukemia support the diagnosis of this condition (Table 1). The acute onset of illness with B symptoms and organomegaly differentiates aggressive NK-cell leukemia from chronic NK-cell leukemia despite having a similar morphology of cells and immunophenotype.
All of the first three criteria (A, B, C) are required for the diagnosis of T-cell LGL leukemia. Asymptomatic patients with only a small clonal T-cell LGL subpopulation (<0.5 x 109/l) may benefit from bone marrow evaluation. The infiltration of marrow with clonal LGLs supports the diagnosis. A small clonal T-cell LGL population in symptomatic patient is sufficient to establish the diagnosis.
Differential Diagnosis
Transient, or chronic, polyclonal T-cell or NK-cell lymphocytosis is occasionally seen in normal individuals with viral infections and autoimmune disorders (Fig. 2) [8, 13]. Oligoclonal and small clonal populations of LGLs have been observed in healthy elderly individuals [12]. Clonal CD3+ LGL populations have also been observed in patients after organ transplantation [10]. While repeat CBCs, clonality studies, and detailed clinical evaluations after 6 months can successfully differentiate among most of these conditions of T-cell lineage, it is more difficult to separate similar conditions of NK-cell lineage, for which no clonality marker is easily available [8].
THERAPEUTIC PRINCIPLES
The principles of therapy for LGL leukemia are based on case reports and retrospective cohorts of patients treated in single institutions. Although 60%–70% of patients with T-cell LGL leukemia require therapy during the course of their disease, 30% of patients need only watchful follow-up [3, 58]. The most common indications for therapy include life-threatening infections, severe neutropenia, symptomatic anemia or thrombocytopenia, and severe B symptoms [8]. First-line, single-agent therapy with low-dose methotrexate (10 mg/m2 per week), cyclophosphamide (50–100 mg orally daily), and cyclosporine A (5–10 mg/kg per day) is effective in approximately 50%–60% of patients (Fig. 4) [59–61]. It is recommended that 4 months of treatment be given before a decision is made to change to an alternative agent because of no response. Responders usually require indefinite therapy to prevent relapses; however, in some patients, durable remission can be obtained with only a short course of treatment. The mechanism of molecular action of these agents is not well understood. In the majority of treated patients, the improvements in cytopenias or systemic symptoms do not correlate with the number of circulating or bone marrow–infiltrating T-cell LGL leukemic cells. This phenomenon suggests that the main effect of low-dose chemotherapy is not cytotoxic but immunosuppressive or immunomodulatory. Therapeutic responses have been correlated with lowered levels of circulating FasL [62]. Although cyclosporine A is used by some investigators as an initial therapy, toxicities of long-term therapy are greater, especially in older patients, compared with the side effects of methotrexate or cyclophosphamide. After normalization of the neutrophil count or achievement of the best response, the dose of cyclosporine A should be tapered down to obtain the lowest effective maintenance dose.
Monotherapy with prednisone (1 mg/kg per day) may improve cytopenias and relieve systemic symptoms; however, it usually does not induce durable remission. We combine corticosteroids with low-dose methotrexate and cyclophosphamide to accelerate the onset of response [8]. In this situation, prednisone is tapered and subsequently discontinued in the second month of therapy.
Patients who fail first-line therapy may benefit from second-line monotherapy with nucleoside analogues, including fludarabine, 2'-deoxycoformycine, and 2-chloroxyadenosine, or targeted therapy with anti-CD52 antibodies (alemtuzumab, Campath®; Berlex Inc., Wayne, NJ) [63–66].
Hematopoietic growth factors (G-CSF, GM-CSF) have also been used successfully as first-line monotherapy for neutropenia or as a supportive treatment together with immunosuppressive therapy [67–69].
Administration of antithymocyte globulin (ATG) with prednisone and cyclosporine can also be effective in the treatment of refractory cytopenias in patients with T-cell LGL leukemia, but experience with this approach is limited to case reports [70, 71].
Splenectomy is usually rarely used as a front-line therapeutic modality. Rare case reports describe the resolution of Coombs-positive hemolytic anemia and ITP associated with T-cell LGL leukemia [50, 72].
Since LGL leukemia is a rare condition, it is important to treat patients in the context of clinical studies if possible. An Eastern Cooperative Oncology Group protocol is evaluating oral methotrexate (10 mg/m2 per week) for 4 months, with crossover to cyclophosphamide in nonresponders. A phase II study for untreated patients with tipifarnib (ZarnestraTM, Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Raritan, NJ) should be open in the near future. That clinical study is based on work showing that constitutively activated Ras is a survival pathway in leukemic LGLs [73]. A phase I study with an anti-CD2 monoclonal antibody (siplizumab, Medi-507; Medimmune, Inc., Gaithersburg, MD) is enrolling patients who failed at least one prior therapy. Both autologous and allogeneic hematopoietic stem cell transplantation have been used rarely in younger patients with T-cell LGL leukemia refractory to conventional therapy [74].
Aggressive T-cell/NK-cell LGL leukemia usually has a rapid progressive course with a short survival duration. Therapy with CHOP-like chemotherapy regimens has been found to be ineffective [16]. Induction chemotherapy with an intensive acute lymphoblastic leukemia (ALL)–like regimen followed by allogeneic stem cell transplantation has been used with curative intent in a limited number of cases (Fig. 5) [75, 76]. Chronic NK-cell leukemia/lymphocytosis usually has an indolent course. Patients who require therapy can be treated according to the algorithm for T-cell LGL leukemia (Fig. 4).
CONCLUSION
Since the first definition of LGL leukemia as a clonal disease 20 years ago [2], progress in molecular and cellular biology and clinical investigation has resulted in a better characterization and understanding of the biology and prognosis of this rare condition. LGL leukemia represents a spectrum of distinct clinicopathologic presentations. Since aggressive and indolent conditions can manifest with similar cell morphologies and immunophenotypes, hematologists should play an integral role in the process of diagnosing this disease. Stratification of patients into prognostic groups (aggressive, indolent) results in more accurate classification and management. We believe that elucidation of the etiopathogenesis, resulting in the discovery of new specific molecular targets in all subgroups of LGL leukemia patients, will lead to the development of more sophisticated and less toxic therapy. To further accelerate progress in the laboratory and clinical research of this rare condition, the participation of clinicians and patients in experimental and clinical studies is invaluable. A national LGL leukemia registry has been established at the Penn State Cancer Institute in order to elucidate molecular pathogenesis and prognosis of this disease.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.
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After completing this course, the reader will be able to:
Discuss the basic principles of molecular and cellular biology of LGL leukemia.
Describe distinct clinical entities among disorders of LGLs.
Discuss the diagnostic criteria for T-cell LGL leukemia.
Discuss the therapeutic algorithm of LGL leukemia.
ABSTRACT
Clonal disorders of large granular lymphocytes (LGLs) represent a spectrum of biologically distinct lymphoproliferative diseases originating either from mature T cells (CD3+) or natural killer (NK) cells (CD3–). Both subtypes, T-cell and NK-cell LGL leukemia, can manifest as indolent or aggressive disorders. The majority of patients with T-cell LGL leukemia have a clinically indolent course with a median survival time >10 years. Immunosuppressive therapy with low-dose methotrexate, cyclophosphamide, or cyclosporine A can control symptoms and cytopenias in more than 50% of patients, but this approach is not curative. Several cases of an aggressive variant (CD3+CD56+) of T-cell LGL leukemia with a poor prognosis have also been reported. Aggressive NK-cell LGL leukemia is usually a rapidly progressive disorder associated with Epstein-Barr virus (EBV), with a higher prevalence in Asia and South America. This disease is usually refractory to conventional chemotherapy, with a median survival time of 2 months. Chronic NK-cell leukemia/lymphocytosis is a rare EBV-negative disorder with an indolent clinical course. The malignant origin of this subtype is uncertain because clonality is difficult to determine in LGLs of NK-cell origin.
DEFINITION AND CLASSIFICATION
Large granular lymphocytes (LGLs) represent 10%–15% of the total peripheral blood mononuclear cells in normal adults [1]. The majority of these cells (85%) are derived from the CD3– natural killer (NK)–cell lineage, and a minority are derived from the CD3+ T-cell lineage (15%). Cytologically, LGLs are medium to large cells with eccentric nuclei and abundant cytoplasm with coarse azurophilic granules (Fig. 1). T-cell LGLs are post-thymic, antigen-primed cytotoxic CD8+ T lymphocytes. NK-cell LGLs belong to the innate immune system with the capability of non-major histocompatibility complex (MHC)–restricted cytotoxicity.
LGL leukemia was initially described in 1985 as a clonal disorder involving blood, marrow, and spleen [2]. In 1993, we proposed two LGL disorders based on either T-cell or NK-cell lineage, which was subsequently adopted by all pathology classification systems [3]. Clonal disorders of LGLs represent a biologically heterogeneous spectrum of lymphoid malignancies (Fig. 2) [4, 5]. Both the T-cell and NK-cell subtypes can clinically present as an indolent or aggressive diseases (Table 1). World Health Organization (WHO) classification of lymphoid malignancies includes T-cell LGL leukemia and aggressive NK-cell leukemia as two separate entities among T-cell/NK-cell lymphomas/leukemias [6]. Since only rare cases of the aggressive variant of T-cell LGL leukemia have been described [7], this subtype was not given separate status in the WHO classification. Transient (<6 months) and chronic (>6 month) expansions of LGLs are two benign conditions in the spectrum of disorders of LGLs [8]. Transient reactive populations of LGLs have been detected in patients with viral infections, autoimmune diseases, and malignancies, and in patients after solid organ transplantation [9–13]. The reactive LGLs are polyclonal with expression of the T-cell (CD3+) immunophenotype in the majority of cases. LGL count normalizes spontaneously or with therapy of underlying condition, usually within 6 months. Since it is difficult to determine clonality for NK cells, it is uncertain whether chronic NK-cell lymphocytocis represents a chronic NK-cell leukemia. In the absence of a clonal marker, clinical presentation is the most important factor available for the differential diagnosis of these two conditions. Patients with systemic symptoms or infiltration of the spleen, liver, or bone marrow could be appropriately classified in the category of chronic NK-cell leukemia. Asymptomatic patients might be better given a diagnosis of benign chronic NK-cell lymphocytosis. In this review we focus mainly on diseases of LGLs included in the WHO classification.
EPIDEMIOLOGY
LGL leukemia comprises 2%–5% of all T-cell/NK-cell malignancies, with only 400 cases reported in the literature [8]. Indolent T-cell LGL leukemia is the most common subtype, representing approximately 85% of all cases diagnosed in Western countries. The male-to-female ratio is approximately equal to one. This entity is more frequently diagnosed in older individuals, with a median age at diagnosis of 60 years (Table 1). The aggressive type of NK-cell LGL leukemia typically occurs in younger individuals, with median age at diagnosis of 39 years and with a higher prevalence in Asia and South America [3, 14, 15]. Fewer than 100 cases have been described in literature [16] (Table 1).
ETIOPATHOGENESIS
The etiology of LGL leukemia is not known. Chronic activation of T cells with autoantigen or viral antigen has been suggested as an initial stimulus leading to an expansion of LGLs [17, 18]. Whether a second molecular event is necessary to establish the full malignant phenotype is not clear. It has also been suggested that T-cell LGL leukemia could represent an autoimmune disorder caused by chronic antigenic stimulation leading to extreme expansion of only one clone of CD8+ cytotoxic T cells [19, 20]. An association of T-cell LGL leukemia with several different autoimmune conditions supports this hypothesis. Human T-cell leukemia virus II (HTLV-II) sequences have been detected in two patients with indolent T-cell LGL leukemia [21, 22]. Seroindeterminate reactivity against HTLV-I envelope (env) epitope BA21 has been described in approximately 50% of patients with CD3+ and 73% of patients with CD3– LGL leukemia [22, 23]. Detailed amino acid analysis of BA21 revealed that a 10–amino acid peptide, PP10, was responsible for the seroreactivity [24]. This peptide shared high amino acid homology only with HTLV-I env protein and not with any known human proteins. However, most patients with LGL leukemia are not infected with prototypical members of the HTLV family, including HTLV-I, HTLV-II, or bovine leukemia virus (BLV) [25, 26]. Epstein-Barr virus (EBV) is implicated in the pathogenesis of aggressive NK-cell leukemia [27]. It has been suggested that this condition is a leukemic variant of a more common NK-cell/T-cell lymphoma, the nasal type.
IMMUNOPHENOTYPE
LGL leukemia cells have a mature T- or NK-cell immunophenotype [3]. The most common immunophenotypes for each subtype of LGL leukemia are described in Table 1. CD57 is a 110-kDa glycoprotein found on NK cells and activated, effector CD8+ T cells. It is a characteristic marker for LGL leukemia. It was suggested that LGLs in T-cell LGL leukemia originate in a CD57– memory T-cell compartment that continually produces CD57+ (effector cell) progeny [28]. Rare immunophenotypic variants that are CD3+ T-cell receptor (TCR)-ßCD4+CD8+, CD3+TCR-ßCD4–CD8–, and CD3+TCR-CD4–CD8– have been reported in T-cell LGL leukemia [1, 29]. Aberrant expression of pan T-cell markers, including CD5 and/or CD7, can be useful in differentiating the malignant T-cell LGL population from normal T lymphocytes.
The NK-cell marker CD56 is typically detected in aggressive NK-cell LGL leukemia [30]. The immunophenotype of this subtype is similar to that of the nasal type of NK-cell/T-cell lymphoma that is CD2+CD56+CD3+ [31]. In the differential diagnosis, this aggressive NK-cell malignancy must be distinguished from indolent chronic NK-cell leukemia and benign chronic NK-cell lymphocytosis, which are not associated with EBV (Fig. 2) [3, 32].
CYTOGENETICS
Indolent T-cell LGL leukemia cells most frequently have a normal karyotype. Less than 10% of patients display distinct chromosomal aberrations, including inversion of 12p and 14q, deletion of 5q, and trisomy of 3, 8, and 14 chromosomes [2, 33, 34]. The most frequent clonal chromosomal abnormality in patients with aggressive NK-cell LGL leukemia is the deletion of the 6q chromosome, but cases with complex karyotypes have also been reported [35, 36].
CLONALITY
LGL leukemia is a disorder of mature T or NK cells. T-cell ontogenesis is associated with TCR gene rearrangement resulting in a unique molecular fingerprint for each T lymphocyte. Thus, the malignant LGL population, which arises from a single cell, has the same TCR gene rearrangement pattern. Southern blotting and polymerase chain reaction (PCR) are the two methods most commonly used for confirmation of clonality [37]. Recently, a panel of monoclonal antibodies was used against the variable domain of the ß chain as a new clonality technique in T-cell malignancies [38]. Since available antibodies recognize approximately 75% of the Vß repertoire of T cells, this technique has not yet achieved widespread use in clinical practice. NK cells have TCR genes in a nonrearranged germline position. In the past, cytogenetic abnormalities and clonality studies based on the X-chromosome inactivation pattern (XCIP) were the only methods available for confirmation of the clonal origin of the NK-cell population [36]. Recently, a new class of NK-cell receptors, killer cell immunoglobulin-like receptors (KIRs), has been detected on the surface of NK cells and a subset of T cells [39]. Aberrant expression of these receptors has been reported in some patients with LGL leukemia [39]. In particular, we demonstrated that patients’ NK cells had high levels of activating receptors and a loss of inhibitory receptors [40]. These findings suggest a potential use of KIR expression as clonality markers but will require validation in a larger study.
CLINICAL PRESENTATION
Approximately two thirds of the patients with indolent T-cell LGL leukemia develop cytopenias, recurrent bacterial infections, autoimmune disorders, and/or splenomegaly during the course of their disease (Table 1) [1]. Recurrent infections resulting from neutropenia initially manifest in 20%–40% of patients. B symptoms—including fever, night sweats, and weight loss—occur in 20%–40% of patients. Mild-to-moderate splenomegaly is found in 20%–50% of patients, and hepatomegaly is found in 10%–20% of patients. Diffuse infiltration of red splenic pulp with preservation of sinuses and white pulp cords is characteristic of T-cell LGL leukemia [41]. Lymphadenopathy is an uncommon presentation of this disease. Bone marrow biopsy typically reveals discrete interstitial and sinusoidal infiltration with T lymphocytes [8]. Rheumatoid arthritis is the most common autoimmune disorder, manifesting in up to 30% of patients with T-cell LGL leukemia [8].
Clinical manifestations of the aggressive variant of T-cell LGL leukemia closely resemble those of aggressive NK-cell LGL leukemia (Table 1). This condition is very rare, with only several well-described cases in literature [7].
Aggressive NK-cell LGL leukemia typically presents as an acute illness, with B symptoms, lymphocytosis, hepatosplenomegaly, lymphadenopathy, severe anemia and thrombocytopenia, and hemophagocytic syndrome [3, 16]. Chronic NK-cell lymphocytosis usually is an indolent disorder with a good prognosis. Rare cases present with cytopenias, cutaneous vasculitis, peripheral neuropathy, and splenomegaly (Table 1) [42].
HEMATOLOGIC FEATURES
The normal range of LGLs in peripheral blood is 0.2–0.4 x109/l [1]. Evaluation of peripheral blood film is invaluable if a diagnosis of LGL leukemia is suspected. A previous study suggested that >90% of patients with T-cell LGL leukemia present with an LGL count in excess of 1.0 x 109/l [1]. In our original paper, diagnostic criteria for T-cell LGL leukemia required an absolute LGL count >2 x 109/l [3]. More recent studies revealed that 25%–30% of newly diagnosed patients present with an absolute neutrophil count (ANC) <0.5 x 109/l [43]. The advent of flow cytometry and progress in immunohistochemistry and molecular clonality studies has allowed the detection of much smaller populations of clonal LGLs in a background of normal polyclonal hematopoiesis [44]. Flow cytometry can also uncover rare cases of LGL leukemia with the absence of typical LGL morphology but with the typical immunophenotype [45]. Detection of a smaller clonal LGL population in patients with systemic symptoms or cytopenias does not require a waiting period of 6 months to establish the diagnosis [8, 43]. However, in cases with chronic NK-cell lymphocytosis and in patients with small oligoclonal or polyclonal populations of T-cell LGLs, a repeat study in 6 months can help to differentiate reactive lymphocytosis from the malignant condition [3, 8]. Evaluation of bone marrow aspirate and biopsy is not required for the diagnosis of LGL leukemia [3]. Recently, however, we and others found that this test can be useful, especially in patients with small circulating LGL subpopulations [8]. A careful review of the bone marrow biopsy supported by immunohistochemistry may reveal infiltration with LGLs in up to 85% of patients [8]. The linear arrays of intravascular CD8+, T-cell intracellular antigen–1+ (TIA-1+), granzyme B+ lymphocytes observed with immunohistochemistry appear specific for LGL leukemia [46]. There is not a good correlation between the extent of marrow infiltration with LGL leukemic cells and the degree of cytopenias or severity of systemic symptoms [1, 3]. Since the majority of T-cell LGL leukemia cells are in the G0/G1 phase, it is reasonable to believe that LGL lymphocytosis is caused by the accumulation of leukemic cells resulting from a block in apoptosis [1]. We have suggested that increased resistance of unstimulated T-cell LGL leukemic cells to apoptosis triggered via the Fas/Fas ligand (FasL) pathway could play a critical role in the pathogenesis of this disorder [47]. However, no causative somatic mutations in Fas or FasL genes have been found.
PATHOGENESIS OF CYTOPENIAS
Neutropenia is the most common cytopenia, found in 80% of patients with indolent T-cell LGL leukemia [1, 3]. Approximately 45% develop severe neutropenia (<0.5 x 109/l) associated with infections [8]. Several different cellular and humoral mechanisms have been suggested in the pathogenesis of neutropenia in T-cell LGL leukemia, including the direct destruction of myeloid precursors with infiltrating leukemic cells, dysregulation of the maturation of myeloid cells, antibody- or immune complex–mediated peripheral destruction of neutrophils, and induction of apoptosis of neutrophils as a result of activation of the Fas/FasL pathway [48].
Adult-onset cyclic neutropenia is a rare acquired disorder characterized by periodic neutropenia and bacterial infections. The majority of cases with this rare condition are associated with LGL leukemia [49]. Anemia with a hemoglobin level <11 g/dl is found in approximately 48% of patients with T-cell LGL leukemia [3]. Approximately 20% of patients require transfusions. Coombs-positive autoimmune hemolytic anemia is diagnosed only in rare cases [50]. Pure red cell aplasia (PRCA) characterized by hypoproliferative anemia and the absence of mature erythroid precursors in otherwise normocellular marrow occurs in 8%–19% of patients [3]. T-cell LGL leukemia surpasses thymoma as the leading cause of PRCA [51]. It was suggested that LGL leukemia cells can suppress growth or directly lyse human erythroid progenitor cells in bone marrow [52]. Erythroid progenitors usually lose HLA class I antigens during maturation. T cells or NK cells normally express KIRs, which inhibit cytotoxicity when the target cell expresses the specific HLA class I antigen(s). Loss of these antigens on maturing erythroid progenitors makes them susceptible to destruction by the LGL leukemia cells [52, 53]. Thrombocytopenia (<150 x 109/l) presents in about 20% of patients [1]. However, severe thrombocytopenia requiring transfusions is rare [4]. Possible mechanisms responsible for thrombocytopenia include inhibition of megakaryopoiesis in bone marrow by LGL leukemic cells, antibody-mediated peripheral destruction in cases associated with immune thrombocytopenia (ITP), and splenic sequestration in patients with splenomegaly [8].
AUTOIMMUNE DISORDERS
A significant proportion of patients (40%–60%) with T-cell LGL leukemia present with immunologic abnormalities, including seropositivity for rheumatoid factor (RF) and antinuclear antibodies (ANAs), polyclonal hypergammaglobulinemia, circulating immune complexes, and anti-neutrophil antibodies [1, 3]. Although the dysregulation of B-cell function as a result of the presence of malignant cytotoxic CD8+ T cells has been suggested, the pathobiology of these laboratory findings is not well understood [54].
The most common autoimmune disorder associated with T-cell LGL leukemia is rheumatoid arthritis, which manifests in about 25%–33% of patients [1, 3]. A rare autoimmune clinical entity, Felty syndrome, which is characterized by the triad of rheumatoid arthritis, neutropenia, and splenomegaly, closely resembles T-cell LGL leukemia with rheumatoid arthritis [55]. Both diseases have a significantly higher frequency of expression of the HLA-DR4 haplotype than matched healthy controls, suggesting that they may represent a clinical spectrum of the identical disorder. Therapy with low-dose methotrexate can control both arthritis and LGL leukemia [3, 56]. Case reports of patients with LGL leukemia associated with Sjögren syndrome, systemic lupus erythematosis, and Hashimoto’s thyroiditis have also been reported [1, 11, 57]. No significant association with autoimmune disease has been reported in aggressive NK-cell leukemia [8].
DIAGNOSTIC CRITERIA AND DIFFERENTIAL DIAGNOSIS
Figure 3 provides an algorithm for the diagnosis of T-cell LGL leukemia. Diagnosis is based on the following four criteria.
Criterion A: Sustained Expansion of T-cell LGLs in Peripheral Blood
Lymphocytosis is usually in the range of 2–20 x 109/l; however, 25%–30% patients with T-cell LGL leukemia may display a smaller population of circulating LGLs (<0.5 x 109/l) [1].
Criterion B: Expression of Characteristic Immunophenotype
Expression of the characteristic immunophenotype of T-cell LGL leukemia is assessed by multiparameter flow cytometry (Table 1). Rare immunophenotypic variants might also be associated with indolent T-cell LGL leukemia [3, 8].
Criterion C: Clonality of T-Cell Population
The clonality of the T-cell population should be confirmed either by the detection of a TCR gene rearrangement using PCR or Southern blot or by the detection of a restricted TCR Vß repertoire with flow cytometry and monoclonal antibody panel. Chronic NK-cell leukemia may be more difficult to differentiate from reactive polyclonal expansions of NK cells as a result of the germinal configuration of TCR genes in these cells. In a minority of cases, the clonal origin of NK cells is confirmed with the detection of an aberrant karyotype or using the XCIP in informative female patients [3].
Criterion D: Clinical Presentation
Clinical presentation is an important part of the diagnostic process of T-cell/NK-cell malignancies because a similar morphology of cells and immunophenotype can be associated with diseases of distinct biological behavior. The cytopenias, splenomegaly, and rheumatoid arthritis that are typically found in patients with T-cell LGL leukemia support the diagnosis of this condition (Table 1). The acute onset of illness with B symptoms and organomegaly differentiates aggressive NK-cell leukemia from chronic NK-cell leukemia despite having a similar morphology of cells and immunophenotype.
All of the first three criteria (A, B, C) are required for the diagnosis of T-cell LGL leukemia. Asymptomatic patients with only a small clonal T-cell LGL subpopulation (<0.5 x 109/l) may benefit from bone marrow evaluation. The infiltration of marrow with clonal LGLs supports the diagnosis. A small clonal T-cell LGL population in symptomatic patient is sufficient to establish the diagnosis.
Differential Diagnosis
Transient, or chronic, polyclonal T-cell or NK-cell lymphocytosis is occasionally seen in normal individuals with viral infections and autoimmune disorders (Fig. 2) [8, 13]. Oligoclonal and small clonal populations of LGLs have been observed in healthy elderly individuals [12]. Clonal CD3+ LGL populations have also been observed in patients after organ transplantation [10]. While repeat CBCs, clonality studies, and detailed clinical evaluations after 6 months can successfully differentiate among most of these conditions of T-cell lineage, it is more difficult to separate similar conditions of NK-cell lineage, for which no clonality marker is easily available [8].
THERAPEUTIC PRINCIPLES
The principles of therapy for LGL leukemia are based on case reports and retrospective cohorts of patients treated in single institutions. Although 60%–70% of patients with T-cell LGL leukemia require therapy during the course of their disease, 30% of patients need only watchful follow-up [3, 58]. The most common indications for therapy include life-threatening infections, severe neutropenia, symptomatic anemia or thrombocytopenia, and severe B symptoms [8]. First-line, single-agent therapy with low-dose methotrexate (10 mg/m2 per week), cyclophosphamide (50–100 mg orally daily), and cyclosporine A (5–10 mg/kg per day) is effective in approximately 50%–60% of patients (Fig. 4) [59–61]. It is recommended that 4 months of treatment be given before a decision is made to change to an alternative agent because of no response. Responders usually require indefinite therapy to prevent relapses; however, in some patients, durable remission can be obtained with only a short course of treatment. The mechanism of molecular action of these agents is not well understood. In the majority of treated patients, the improvements in cytopenias or systemic symptoms do not correlate with the number of circulating or bone marrow–infiltrating T-cell LGL leukemic cells. This phenomenon suggests that the main effect of low-dose chemotherapy is not cytotoxic but immunosuppressive or immunomodulatory. Therapeutic responses have been correlated with lowered levels of circulating FasL [62]. Although cyclosporine A is used by some investigators as an initial therapy, toxicities of long-term therapy are greater, especially in older patients, compared with the side effects of methotrexate or cyclophosphamide. After normalization of the neutrophil count or achievement of the best response, the dose of cyclosporine A should be tapered down to obtain the lowest effective maintenance dose.
Monotherapy with prednisone (1 mg/kg per day) may improve cytopenias and relieve systemic symptoms; however, it usually does not induce durable remission. We combine corticosteroids with low-dose methotrexate and cyclophosphamide to accelerate the onset of response [8]. In this situation, prednisone is tapered and subsequently discontinued in the second month of therapy.
Patients who fail first-line therapy may benefit from second-line monotherapy with nucleoside analogues, including fludarabine, 2'-deoxycoformycine, and 2-chloroxyadenosine, or targeted therapy with anti-CD52 antibodies (alemtuzumab, Campath®; Berlex Inc., Wayne, NJ) [63–66].
Hematopoietic growth factors (G-CSF, GM-CSF) have also been used successfully as first-line monotherapy for neutropenia or as a supportive treatment together with immunosuppressive therapy [67–69].
Administration of antithymocyte globulin (ATG) with prednisone and cyclosporine can also be effective in the treatment of refractory cytopenias in patients with T-cell LGL leukemia, but experience with this approach is limited to case reports [70, 71].
Splenectomy is usually rarely used as a front-line therapeutic modality. Rare case reports describe the resolution of Coombs-positive hemolytic anemia and ITP associated with T-cell LGL leukemia [50, 72].
Since LGL leukemia is a rare condition, it is important to treat patients in the context of clinical studies if possible. An Eastern Cooperative Oncology Group protocol is evaluating oral methotrexate (10 mg/m2 per week) for 4 months, with crossover to cyclophosphamide in nonresponders. A phase II study for untreated patients with tipifarnib (ZarnestraTM, Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Raritan, NJ) should be open in the near future. That clinical study is based on work showing that constitutively activated Ras is a survival pathway in leukemic LGLs [73]. A phase I study with an anti-CD2 monoclonal antibody (siplizumab, Medi-507; Medimmune, Inc., Gaithersburg, MD) is enrolling patients who failed at least one prior therapy. Both autologous and allogeneic hematopoietic stem cell transplantation have been used rarely in younger patients with T-cell LGL leukemia refractory to conventional therapy [74].
Aggressive T-cell/NK-cell LGL leukemia usually has a rapid progressive course with a short survival duration. Therapy with CHOP-like chemotherapy regimens has been found to be ineffective [16]. Induction chemotherapy with an intensive acute lymphoblastic leukemia (ALL)–like regimen followed by allogeneic stem cell transplantation has been used with curative intent in a limited number of cases (Fig. 5) [75, 76]. Chronic NK-cell leukemia/lymphocytosis usually has an indolent course. Patients who require therapy can be treated according to the algorithm for T-cell LGL leukemia (Fig. 4).
CONCLUSION
Since the first definition of LGL leukemia as a clonal disease 20 years ago [2], progress in molecular and cellular biology and clinical investigation has resulted in a better characterization and understanding of the biology and prognosis of this rare condition. LGL leukemia represents a spectrum of distinct clinicopathologic presentations. Since aggressive and indolent conditions can manifest with similar cell morphologies and immunophenotypes, hematologists should play an integral role in the process of diagnosing this disease. Stratification of patients into prognostic groups (aggressive, indolent) results in more accurate classification and management. We believe that elucidation of the etiopathogenesis, resulting in the discovery of new specific molecular targets in all subgroups of LGL leukemia patients, will lead to the development of more sophisticated and less toxic therapy. To further accelerate progress in the laboratory and clinical research of this rare condition, the participation of clinicians and patients in experimental and clinical studies is invaluable. A national LGL leukemia registry has been established at the Penn State Cancer Institute in order to elucidate molecular pathogenesis and prognosis of this disease.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.
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