Large Granular Lymphocyte Lymphocytosis Research Articles

Expansion of large granular lymphocytes after autologous hematopoietic stem cell transplantation

Expansion of large granular lymphocytes (LGLs) is sometimes observed in allogeneic hematopoietic stem cell transplantation (HSCT) recipients, and is reported to be associated with a favorable transplant outcome. LGLs are also observed after autologous HSCT, but their clinical implications have not been well investigated. We retrospectively reviewed peripheral blood smears of consecutive autologous HSCT recipients. LGL lymphocytosis was defined as the observation of LGLs in the peripheral blood (> 20% white blood cells) in at least two consecutive blood tests. We evaluated the clinical impact of LGL lymphocytosis on autologous HSCT recipients. LGL lymphocytosis was observed in 18 of 197 patients (9.1%) who received autologous HSCT, at a median of 49 days after transplantation, with a median duration of 120.5 days. Incidence of cytomegalovirus reactivation was significantly higher in patients with LGL lymphocytosis than those without (16.7% vs. 3.3%, p = 0.038). No significant difference in survival rates was observed between groups (3 year OS 90.9% vs. 90.5%, p = 0.793 for lymphoma; 100 vs. 92.4%, p = 0.328 for myeloma). LGL lymphocytosis was observed in almost 10% of autologous HSCT recipients. In contrast to allogeneic HSCT, the duration of LGL was shorter and no significant improvement in survival was observed.

Germline Mutations in CTLA4 and Spectrum of Bone Marrow Features Overlapping with Aplastic Anemia and Large Granular Lymphocyte Lymphocytosis

Introduction: CTLA4 haploinsufficiency due to germline heterozygous mutations in the cytotoxic T-lymphocyte antigen-4 gene (CTLA4) results in severe systemic immune dysregulation; infiltrative lymphocytic lesions of lungs (e.g. lymphocytic pneumonitis), GI tract (e.g.lymphocytic colitis), and CNS; and a spectrum of autoimmunity, immunodeficiency and peripheral blood cytopenias. We describe the hematologic, bone marrow (BM), and flow cytometry (FC) features of CTLA4 haploinsufficiency of a cohort of 14 patients, which show overlapping morphologic features with acquired aplastic anemia (acAA), and large granular lymphocyte lymphocytosis (LGL) in a subset of patients. We compared the findings to those in a separate cohort of 20 age matched patients diagnosed with acAA. There were significant differences between acAA and CTLA4 deficiency based on analysis of BM lymphocyte subsets and marrow lymphocyte infiltration patterns.Materials and Methods: Peripheral blood (PB), BM and FC analysis of patients with germline mutations in CTLA4, and acAA was performed along with review of clinical, molecular and laboratory data. Statistical comparisons were made using Mann Whitney test, with p < 0.05 for significance.Results: Median age at initial evaluation for patients with CTLA4 haploinsufficiency was 23.5 years (range 11-71 years), with a male-to-female ratio of 11:3. 11 out of 14 patients had cytopenias. Of those with cytopenias 2/11 had pancytopenia, 4/11 had bicytopenia and 5/11 had unilineage cytopenia. Hypogammaglobulinemia was common (13/14). In some cases cytopenias were recognized as autoimmune in etiology with immune thrombocytopenic purpura and autoimmune hemolytic anemia, among the most common hematologic manifestations. Three patients met criteria for a diagnosis of moderate AA, 1 for severe AA, and 1 for LGL. Overall bone marrow biopsies were hypocellular for age (10/14) with a subset (29% [4/14]) showing severe trilineage hypoplasia with ≤20% cellularity. Atypical interstitial CD8-positive T-cell infiltrates with prominent non-paratrabecular CD4-positive T-cell aggregates were evident by immunohistochemistry in most cases (10/14), accompanied by a near absence of B-cells. T-cell clonality was assessed by T-cell receptor (TCR) gamma PCR and showed abnormal oligoclonal T-cell populations in 3/8, and one case with a clonal T-cell receptor gene rearrangement. B-cell IgH PCR reactions were polyclonal. In all cases, cytogenetic studies were either negative or showed only minor abnormalities (i.e. loss of Y chromosome). Given the morphologic overlap of a subset of CTLA4 cases with acAA, we compared bone marrow flow cytometry analysis of 8 CTLA4 cases with 20 acAA cases. CTLA4 patients showed >5 fold reduction in the median percentage of B-cells in comparison to acAA (p=0.0005). While acAA cases demonstrated relative preservation the B-cell compartment with intact B-cell maturation sequence; in contrast CTLA4 cases showed abnormal B-cell maturation patterns in half of cases characterized by few detectable early B-cell precursors, and progressive loss of intermediate and mature forms. T-cell populations in CTLA4 showed inverted CD4:CD8 ratios with relatively increased CD57+ T-cells, and atypical dim expression of CD20 on T-cells. Morphologically, when the markedly hypocellular CTLA4 cases were compared to acAA on core biopsies, little difference in marrow cellularity was seen; however, the CTLA4 cases showed multiple large atypical T-cell aggregates and near total absence of B-cells, which was not seen in acAA; acAA cases showed scattered background T-cells and B-cells.Conclusions: CTLA-4 deficiency presents with cytopenias, autoimmunity, and immunodeficiency with a subset of patients that have features overlapping with bone marrow failure, specifically AA and LGL. Herein we demonstrate a combination of abnormal BM findings, previously unreported, comprising hypocellularity, CD8-positive interstitial T-cell infiltrates with atypical CD4-positive T-cell aggregates, marked B-cell lymphopenia, increased LGLs and abnormal T-cell clonality patterns by PCR. These features may help identify patients harboring CTLA4 mutations for early detection and optimal therapeutic management. DisclosuresYoung:Novartis/GSK to institute: Research Funding. Townsley:Novartis/GSK to institute: Research Funding.

Interim Results of the Canadian Tyrosine Kinase Inhibitor Discontinuation Trial for 2nd Attempt of Treatment Free Remission: Treatment Free Remission Accomplished By Dasatinib (TRAD)

Interim Results of the Canadian Tyrosine Kinase Inhibitor Discontinuation Trial for 2nd Attempt of Treatment Free Remission: Treatment Free Remission Accomplished By Dasatinib (TRAD)

Dasatinib-induced anti-leukemia cellular immunity through a novel subset of CD57 positive helper/cytotoxic CD4 T cells in chronic myelogenous leukemia patients.

Dasatinib induces lymphocytosis of large granular lymphocytes (LGLs) in a proportion of patients with chronic myelogenous leukemia (CML), and is associated with better clinical outcomes. LGLs consist of cytotoxic T lymphocytes and natural killer cells; however, the context and phenotypic/functional features of each type of LGL are unknown. To better define features of these LGLs, we investigated lymphocytosis in CML patients treated with dasatinib. D57-positive and CD4-positive type I T-helper (Th) cells (CD57+ Th cells) rarely occur in CML patients without lymphocytosis and in healthy individuals; however, a substantial increase in the proportion of CD57+ Th cells was observed in CML patients treated with dasatinib. In addition, these cells showed appreciable levels of cytocidal activity via cytotoxic degranulation. Analysis of T-cell receptor α and β sequences showed a skewed T-cell repertoire in the CD57+ Th cells. Furthermore, patients with LGLs and CD57+ Th lymphocytosis achieved stronger molecular responses than did those without lymphocytosis. While further studies are warranted, our observations suggest that dasatinib induces the expansion of CD57+ Th-LGLs, which may play a crucial role in the dasatinib-induced response against Philadelphia chromosome-positive leukemia.

Distinctive clinical characteristics and favorable outcomes in patients with large granular lymphocytosis after allo‐HCT: 12‐year follow‐up data

An increase in large granular lymphocytes (LGL) is frequently seen in patients following allogeneic hematopoietic cell transplantation (allo-HCT) and it has been associated with better outcomes in some reports. We assessed 826 consecutive patients at our institution with over 12years of follow-up for the occurrence of LGL lymphocytosis after allo-HCT. The 3-year cumulative incidence of LGL lymphocytosis was 14.5% with a median duration of over 3.5years. The development of LGL lymphocytosis was strongly correlated with CMV viremia and GVHD. The clinical course of patients with LGL lymphocytosis after allo-HCT was indolent, with the majority of these patients not displaying any clinical signs or symptoms related to the LGL proliferation. LGL lymphocytosis was associated with better outcomes, including higher overall survival (OS 86.6% vs 44.7% at 3years), lower non-relapse mortality (NRM 5.5% vs 30.4% at 3years), and lower risk of relapse (8.9% vs 22.9% at 3years). A time-dependent multivariable analysis confirmed the favorable impact of LGL lymphocytosis on OS and NRM, but not on the risk of relapse. In multivariable analysis, a longer duration of LGL lymphocytosis was associated with better OS and NRM. Improved immunomodulatory properties of these cells, regulating GVHD and infections, may explain the observed favorable outcomes of patients who developed LGL lymphocytosis following allo-HCT.

Large granular lymphocytes are universally increased in human, macaque, and feline lentiviral infection

Large granular lymphocytes (LGLs) have only been anecdotally reported in HIV infection. We previously reported an LGL lymphocytosis in FIV-infected cats associated with a rise in FIV proviral loads and a marked neutropenia that persisted during chronic infection. Extensive immunophenotyping of peripheral blood mononuclear cells in cats chronically infected with FIV were identified LGLs as CD8lo+FAS+; this cell population expanded commensurate with viral load. CD8lo+FAS+ cells expressed similar levels of interferon-γ compared to CD8lo+FAS+ cells from FIV-naive control animals, yet CD3ɛ expression, which was increased on total CD8+ T cells in FIV-infected cats, was decreased on CD8lo+FAS+ cells. Down-modulation of CD3 expression was reversed after culturing PBMC for 3 days in culture with ConA/IL-2. We identified CD8lo+FAS+ LGLs to be polyclonal T cells lacking CD56 expression. Blood smears from HIV-infected individuals and SIVmac239-infected rhesus macaques revealed increased LGLs compared to HIV/SIV negative counterparts. In humans, there was no correlation with viral load or treatment and in macaques the LGLs arose in acute SIV infection with increases in viremia. This is the first report describing and partially characterizing LGL lymphocytosis in association with lentiviral infections in three different species.

AB0438 Vasculitis associated with large granular lymphocyte leukemia : presentation and treatment outcomes

BackgroundLarge granular lymphocyte (LGL) leukemia represent ∼5–10% peripheral blood mononuclear cells. These include mainly cytotoxic T cells and natural killer (NK) cells. LGL disease has four forms: chronic or transient...

Large granular lymphocytosis and its impact on long-term clinical outcomes following allo-SCT

A total of 418 patients receiving hematopoietic SCT and surviving beyond day 100 were examined for the occurrence of large granular lymphocytes (LGLs). LGL lymphocytosis was defined as the presence of at least two of the following criteria: (1) sustained lymphocytosis above 3.0 × 10(9)/L observed in at least three consecutive determinations over a time frame of 2-3 months, (2) predominance (>30%) of LGLs in peripheral blood, (3) confirmation of monoclonality by T-cell receptor analysis using PCR 77 patients developed LGL lymphocytosis during their post-transplant course with a median onset of 312 days from transplant. The cumulative incidence at 1-, 2- and 3-years was 12.3±1.8, 20.8±2.4 and 23.6±2.7%. Patients with LGL lymphocytosis showed an OS advantage (86.2 vs 53.8%, P<0.001), lower non-relapse mortality (NRM; 3.2 vs 27.3%, P<0.001) and lower relapse incidence (9.6 vs 29.4%, P<0.001). Three clinical factors were associated with the development of LGL lymphocytosis: (1) CMV seropositive recipients (CMV-R(+)) compared with CMV seronegative recipients (CMV-R(-); P<0.001) regardless of CMV serostatus of donor; (2) CMV reactivation (P<0.001); (3) chronic GVHD (P=0.007). In conclusion, the incidence of LGL lymphocytosis following allogeneic hematopoietic SCT was detected in ~20% of recipients and is associated with favorable outcomes.

A comparative study of flow cytometric T cell receptor Vβ repertoire and T cell receptor gene rearrangement in the diagnosis of large granular lymphocytic lymphoproliferation

Large granular lymphocytes (LGLs) are medium- to large-sized lymphocytes with azurophilic cytoplasmic granules. Reactive vs. neoplastic LGLs are usually morphologically indistinguishable. We investigated 25 consecutive cases of LGL lymphoproliferation using flow cytometric T cell receptor Vβ (FC-Vβ) repertoire and T cell receptor gene rearrangement (TCR-GR) in detecting clonality. Seventeen patients (68%) were T-LGL leukemia (T-LGLL) with a male predominance, a median age of 67, and a median absolute LGL count of 2.592 × 10(9) /L. All cases were clonal using the FC-Vβ analysis, and all but one (94%) was clonal by TCR-GR. Eight patients (32%) had reactive LGL lymphoproliferation. Two had EBV-associated infectious mononucleosis; one was clonal by both FC-Vβ and TCR-GR; and the other was clonal only by TCR-GR. The remaining six cases were polyclonal by both assays. Patients with reactive LGL lymphoproliferation were more frequently associated with an underlying/concurrent malignancy than those with T-LGLL (4/8 cases vs. 1/17; P = 0.023, Fisher's exact test). We compared the demographic, hemogram, and clonality data between these two groups and found that the only significant difference was the lower median platelet count in the LGL lymphocytosis group (201 × 10(9) /L vs. 223 × 10(9) /L; P = 0.031; Student's t-test). A literature review including the current study showed a high sensitivity of FC-Vβ analysis for T-LGLL (97.2%; 107/110 cases). FC-Vβ analysis was slightly more sensitive than TCR-GR for the detection of clonal T cell lymphoproliferation. However, we must interpret the laboratory findings with clinical context as clonal T cell lymphoproliferation may occur in patients with viral infection.

Rapid and sustained increase of large granular lymphocytes and rare cytomegalovirus reactivation during dasatinib treatment in chronic myelogenous leukemia patients

We retrospectively investigated increases in large granular lymphocytes (LGL) in peripheral blood during dasatinib treatment in 25 chronic myelogenous leukemia patients. Fifteen of 25 patients (60%) showed an increase in LGL. All 15 of these patients also showed an increase in NK cells, and 11 showed an increase in CD8(+) T cells. High frequencies of clonal rearrangements of TCR-β, -γ, and -δ genes were observed in LGL (+) patients, and at lower frequencies in LGL (-) patients as well. Clinical responses were favorable for all. With respect to their newly obtained complete molecular response after dasatinib treatment, LGL (+) patients showed higher response rates than did LGL (-) patients. In contrast, pleural effusions were more commonly observed in LGL (+) patients (60%) than in LGL (-) patients (20%). LGL counts significantly increased at 2h after oral intake of dasatinib in all 25 patients. This was not observed in treatment with imatinib or nilotinib. Cytomegalovirus (CMV) C7-HRP tests were negative in all patients. Serum CMV-IgM antibodies were positive in only 2 of 25 patients without symptom of infection. Thus, LGL lymphocytosis during dasatinib treatment may be correlated with favorable molecular response, and with increased incidence of pleural effusions. In the clinical setting, CMV reactivation appears uncommon.

Rapid and Sustained Increase of LGL and Rare CMV-Reactivation During Dasatinib Treatments in CML

Abstract 2752Dasatinib has extensive inhibition profile on multiple kinases. Clonal expansion of large granular lymphocyte (LGL) is a noteworthy off-target phenomenon. In this study, we investigated the increase of LGL in peripheral blood during dasatinib treatments which were changed from imatinib treatments, in 25 chronic myelogenous leukemia (CML) patients in CP or AP. METHODS: The criteria of the LGL-increase was set by “increase of lymphocytes over 3,000/μL, and increase of LGL count over 1,500/μL” was found during the course of the treatment. Flow cytometry analyses were performed during the treatments in all cases, and also before the dasatinib treatments in 17 cases. T-cell receptor (TCR) clonal rearrangements for TCR-β, -γ, and -δ, were examined by PCR-based method during the treatments in all cases. Furthermore, comparison of LGL counts before and 2 hours after oral intake were investigated in all 25 cases. CMV activations were assessed in all cases by serum CMV-IgG, serum CMV-IgM, and by “CMV antigen test Teijin (HRP-C7)” which can detect CMV pp65 antigen in leukocytes. RESULTS: Median follow-up time was 11 months (range 3–28). (1) Fifteen out of the 25 patients (60%) showed increase of LGL, which had not been observed during preceding imatinib treatments. Median time until LGL count reached 1,500/μL was 11 weeks (range 6–44), and increased-LGL sustained as far as dasatinib was continued. In these 15 patients, all showed increase of CD56+, CD16+, CD3- NK-cells, and 11 patients also showed increase of CD3+, CD8+, CD4- T-cells. Evident increase of γδ-T cells was not observed. Clonal rearrangements of TCR-β gene were observed in 13 of 15 (87%), and TCR-γ in 12 (80%), and TCR-δ in 9 (60%) patients. Pleural effusions were observed in 9 out of 15 (60%) patients. Regarding clinical response, 4 cases newly achieved CMR, 7 maintained CMR (totally 73% achieved or maintained CMR), and 3 achieved or maintained MMR. (2) On the other hand, 10 out of 25 patients (40%) did not show increase of LGL. In these 10 patients, clonal rearrangements of TCR-β gene were observed in 4 of 10 (40%), and TCR-γ in 3 (30%), and TCR-Δ in 4 (40%) patients. Three patients showed no clonality for all the three TCR genes. Pleural effusions were observed in 2 out of 10 (20%) patients. Regarding clinical response, no case newly achieved CMR, 5 maintained CMR (totally 50% achieved or maintained CMR), and 4 achieved or maintained MMR. (3) LGL counts increased 2 hours after intake of dasatinib in all patients. Regarding the 15 patients who showed LGL increase during the treatment courses, LGL counts were 1,349±918/μL (mean±SD)(before), and 2,895±2,240/μL (2 hours after), indicating 2.1 times increase (P=0.006); total lymphocyte counts were 2,153±1,129/μL (before), and 4,456±3,219/μL (after), indicating 2.1 times increase (P=0.01). Regarding the 10 patients who did not show LGL increase during the treatment courses, LGL counts were 422±384/μL (before), and 805±419/μL (after), indicating 1.9 times increase (P=0.02); total lymphocyte counts were 1,094±556/μL (before), and 1,859±899/μL (after), indicating 1.7 times increase (P=0.03). (4) In the total 25 patients, serum CMV-IgM were negative in most of the cases (23 of 25; 92%). CMV HRP-C7 were completely negative (meaning undetectable) in most of the cases (23 of 25; 92%), and it was barely positive only in 2 cases (1 cell and 2 cells were positive out of ∼60,000 leukocytes examined, respectively). CONCLUSION: LGL lymphocytosis occurred in 60% of patients, which seems to be related with favorable molecular responses, and with relatively high incidence of pleural effusions. The TCR gene rearrangements were observed in both groups, but higher in the increase-LGL group than the non-increase-LGL group. Approximately 2-times rapid increase of LGL and total lymphocytes were observed 2 hours after intake of dasatinib, in all the patients, irrespective of the LGL-increase during the treatment courses. In the clinical setting, evident immunodeficiency was not observed in terms of CMV-reactivation. Disclosures:No relevant conflicts of interest to declare.

Prevalence of Large Granular Lymphocyte Proliferation in Chronic Myeloid Leukemia (CML) Patients Treated with Dasatinib.

Abstract 1111Poster Board I-133Dasatinib is a potent inhibitor of the BCR-ABL tyrosine kinase which is effective in the treatment of imatinib refractory CML. While hematologic toxicities of neutropenia and thrombocytopenia are well known, large granular lymphocytosis has been reported in only a small number of patients without prior allogeneic stem cell transplant treated with dasatinib for CML. During routine follow up of leukocyte counts in 15 consecutive patients (age range 27-77 years) treated with dasatinib, 4 patients (2 chronic-phase, 1 accelerated phase with clonal cytogenetic progression, 1 blast-phase) developed a lymphocytosis (> 3800/mm3). Peripheral blood smear and peripheral blood flow cytometry revealed a population of large granular lymphocytes (LGLs) expressing CD3, CD8, CD57, and variable expression of CD56. Lymphocytosis was first noted between 1 and 9 months after initiation of dasatinib and has persisted in 3 of the patients with a median follow up of 33 months from the onset of lymphocytosis. Peak absolute lymphocyte count ranged from 5000/mm3 to 6900/mm3 and approximately 40 to 60% of the lymphocytes were LGLs by flow cytometry with the remainder being predominantly T lymphocytes. These 4 patients with LGL lymphocytosis have all have major molecular responses and the patient with blast-phase has remained in a complete cytogenetic remission with a major molecular response 44 months after initiation of dasatinib. The 11 other patients (6 chronic-phase, 2 accelerated-phase, 3 blast-phase) treated with dasatinib for CML have not developed lymphocytosis. These patients have been followed for a median of 25 months (range 3-50 months) although some were treated with dasatinib for a relatively short period of time because of poor response of their advanced CML. Review of the peripheral blood smears from 3 of the 6 chronic phase patients without lymphocytosis who remain on dasatinib treatment did not reveal any LGLs. All of these 6 patients have had complete, sustained cytogenetic responses. A persistent pleural effusion developed in the blast phase patient with lymphocytosis approximately 12 months after lymphocytosis developed; no significant side effects were noted in the other 3 patients although one remains thrombocytopenic. Pleural effusions developed in 2 of the 6 patients without lymphocytosis who remain on dasatinib treatment. Previous reports have suggested an increased incidence of “inflammatory” type side effects such as pleural effusions and pneumonitis in patients with dasatinib related LGL proliferation, although the small number of patients in this series precludes analysis of this association. In summary, LGL proliferation was detected in a minimum of 27% of dasatinib recipients and may be associated with a beneficial response. While the mechanism of LGL proliferation has not been fully explained, it has been suggested that dasatinib mediated inhibition of immunoregulatory kinases such as Src is permissive of LGL proliferation. Further evaluation of the frequency and clinical impact of this phenomenon in the large clinical trials of patients treated with dasatinib is warranted. DisclosuresSchiffer:Bristol Myers: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Temporal association of large granular lymphocytosis, neutropenia, proviral load, and FasL mRNA in cats with acute feline immunodeficiency virus infection

During acute feline immunodeficiency virus-C PGammar (FIV-C-PG) infection, we observed that cats develop large granular lymphocyte (LGL) lymphocytosis concurrent with a marked neutropenia that is temporally associated with the rise and fall of FIV-C-PG proviral loads. LGLs, generally considered to be analogous to natural killer (NK) cells, can also be highly cytolytic CD8/CD57 T cells. Neutropenia has been reported during acute human immunodeficiency virus (HIV-1) infection, but there is a paucity of information describing the pathogenesis of this condition. During HIV-1 infection, LGLs have been shown to be both CD16 + NK cells and CD8 +/CD57 + T cells, but an association with neutropenia has not been described. However, neutropenia with concurrent LGL lymphocytosis has been demonstrated in both LGL leukemia and common variable immunodeficiency syndrome in people, and in both syndromes, an increase in soluble Fas ligand (FasL) has been associated with neutrophil apoptosis leading to neutropenia. Flow cytometric analysis demonstrated increases in CD56 and CD8 peripheral blood cell surface expression during acute FIV-C-PG infection. Expression of FasL mRNA was increased at the same time points as these peripheral hematologic abnormalities, and also decreased as FIV-C-PG proviral load reached set point. We describe an interesting temporal association between innate immune responses and viral load during acute FIV-C-PG infection, which has similarities to HIV-1 infection and other immune dyscrasias of people, and which may contribute to the neutropenia and LGL lymphocytosis during FIV-C-PG infection.

Clonal expansion of T/NK-cells during tyrosine kinase inhibitor dasatinib therapy

Dasatinib, a broad-spectrum tyrosine kinase inhibitor (TKI), predominantly targets BCR-ABL and SRC oncoproteins and also inhibits off-target kinases, which may result in unexpected drug responses. We identified 22 patients with marked lymphoproliferation in blood while on dasatinib therapy. Clonality and immunophenotype were analyzed and related clinical information was collected. An abrupt lymphocytosis (peak count range 4-20 x 10(9)/l) with large granular lymphocyte (LGL) morphology was observed after a median of 3 months from the start of therapy and it persisted throughout the therapy. Fifteen patients had a cytotoxic T-cell and seven patients had an NK-cell phenotype. All T-cell expansions were clonal. Adverse effects, such as colitis and pleuritis, were common (18 of 22 patients) and were preceded by LGL lymphocytosis. Accumulation of identical cytotoxic T cells was also detected in pleural effusion and colon biopsy samples. Responses to dasatinib were good and included complete, unexpectedly long-lasting remissions in patients with advanced leukemia. In a phase II clinical study on 46 Philadelphia chromosome-positive acute lymphoblastic leukemia, patients with lymphocytosis had superior survival compared with patients without lymphocytosis. By inhibiting immunoregulatory kinases, dasatinib may induce a reversible state of aberrant immune reactivity associated with good clinical responses and a distinct adverse effect profile.

Natural killer or natural killer/T cell lineage large granular lymphocytosis associated with dasatinib therapy for Philadelphia chromosome positive leukemia

Dasatinib, a dual tyrosine kinase inhibitor, is known to modulate or suppress T-cell activation and proliferation. We report a series of 8 patients who developed chronic peripheral lymphocytosis, identified as natural killer cells or natural killer/T-cells based on their large granular lymphocyte morphologies and CD16(+), CD56(+), CD3(-) or CD3(+) immunophenotypic profiles, out of 18 patients receiving dasatinib therapy. All cases that developed large granular lymphocyte lymphocytosis achieved optimal molecular response (8/8 in large granular lymphocyte(+) patients vs. 3/10 in large granular lymphocyte(-) patients, p=0.002). A (51)Cr release assay demonstrated that natural killer cell cytotoxicity has been enhanced in a case of large granular lymphocyte lymphocytosis compared to normal healthy donors, and that natural killer cell cytotoxicity in dasatinib-responders was superior to that in non-responders. In summary, the present study suggests that natural killer or natural killer/T cell lineage large granular lymphocyte lymphocytosis develops in association with dasatinib therapy and that large granular lymphocyte might have a therapeutic effect on Ph(+) leukemic cells.

Clonal Expansion of T/NK-Cells during Tyrosine Kinase Inhibitor Dasatinib Therapy

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL fusion protein are an effective therapy for Philadelphia chromosome positive (Ph+) leukemia. Dasatinib, a 2nd generation pan-TKI, also inhibits wild-type kinases, which may result in unexpected clinical responses. Recent data suggest that dasatinib has a potent immunosuppressive effect on T- and NK-cells in vitro. In contrast, we have noticed a marked expansion of lymphocytes in blood in a subset of dasatinib patients, but its clinical significance and molecular mechanisms are unclear. In this multicenter study, 19 Ph+ leukemia patients with marked lymphoproliferation in blood while on dasatinib (9 CML CP, 2 CML AP, 2 CML BC, 6 Ph+ALL) were identified. Clonality, immunophenotype, and intracellular signaling was analyzed and related clinical information was collected. In addition, prevalence and prognostic significance of the phenomenon was retrospectively assessed in a Phase II clinical study on 46 Ph+ ALL patients. An abrupt lymphocytosis (peak count range 4–20×109/L) with large granular lymphocyte (LGL) morphology was observed after a median of 3 months from the start of therapy (range 1–8 months). In most patients, LGL lymphocytosis was long-lasting and continued throughout dasatinib therapy, albeit with marked fluctuations in absolute lymphocyte count (Fig. 1a). In all evaluable patients (n=4), lymphocyte counts normalized after drug discontinuation. All patients were previously exposed to imatinib without similar changes in lymphocyte count or morphology. By immunophenotyping, 14 patients had a cytotoxic T-cell and 5 patients a NK-cell phenotype. After initiation of dasatinib therapy, the CD4/CD8 ratio shifted, expression of activation antigens HLA-DR and CD57 increased, expression of homing antigen CD62L decreased and relative numbers of regulatory T-cells were significantly decreased (p<0.001). All patients with T-LGL phenotype had mono/oligoclonal rearrangements in TCR g/d genes. Quantitative allele-specific oligonucleotide PCR analysis from samples before and during dasatinib therapy indicated that LGL lymphocytosis results from expansion of a pre-existing terminal memory cell clone (CD8+CD62Llow). By using single-cell profiling of nodal phosphoproteins (ERK1/2, STAT1, STAT3, STAT5, STAT6) by multi-color FACS, we showed that during dasatinib therapy LGL patients were more responsive to cytokine or growth factor stimuli (IL-2, IL-4, IL-6, IFNs, GM-CSF), when compared to dasatinib patients without lymphocytosis or to healthy controls. This indicates alternative signaling pathway usage not inhibited by dasatinib and/or cytokine hypersensitivity. Dasatinib-related severe adverse effects were common in patients with LGL lymphocytosis. Pleural effusions and/or pulmonary infiltrates were seen in 13/19 patients, modest CMV reactivation in 9/19 patients, colitis in 11/19 patients and long lasting mild fever in 14/19 patients. Accumulation of pheno- and genotypically identical cytotoxic T-cells was also detected in pleural effusion and colitis biopsy samples. Adverse effects were temporally related to dasatinib therapy and emerged after the appearance of LGL lymphocytosis. All patients with relapsed poor prognosis leukemia (Ph+ ALL n=5, blastic phase CML n=2) achieved durable complete molecular responses (CMR) during dasatinib therapy, CMR still ongoing in 3 patients (follow-up 18+ months). In the confirmatory Phase II dasatinib study in Ph+ ALL (START-L), patients with lymphocytosis had superior survival compared to patients without lymphocytosis (Fig. 1b). In conclusion, we propose that by inhibiting immunoregulatory kinases, dasatinib may induce a reversible state of aberrant immune reactivity in a distinct subset of patients and result in anti-leukemia and anti-host responses driven by cytotoxic T/NK LGL cells. Further studies pinpointing the mechanisms, such as the target antigens on the malignant cells and activation pathways are ongoing. [Display omitted]

Natural Killer (NK) or NK/T Cell Lineage Large Granular Lymphocytosis Associated with Dasatinib Therapy for Philadelphia Chromosome Positive Leukemia.

Dasatinib, a dual tyrosine kinase inhibitor, is known to modulate or suppress T-cell activation and proliferation. We report a series of patients of chronic peripheral lymphocytosis development, identified as natural killer (NK) cells or NK/T-cells based on their large granular lymphocyte (LGL) morphologies and CD16+CD56+CD3− or CD3+ immunophenotypic profiles during dasatinib therapy. All cases that developed LGL lymphocytosis achieved optimal molecular response (8/8 in LGL+ patients vs 3/10 in LGL− patients, p=0.002). A 51Cr release assay demonstrated that NK cell cytotoxicity has been enhanced in a case of LGL lymphocytosis compared to normal healthy donors (Figure 1), and that NK cell cytotoxicity in dasatinib-responders was superior to that in non-responders (Figure 2). In summary, the present study suggests that NK or NK/T cell lineage LGL lymphocytosis develops associated with dasatinib therapy and that LGL might have a therapeutic effect on Ph+ leukemic cells. [Display omitted] [Display omitted]

Evaluation of functional single nucleotide polymorphisms of different genes coding for the immunoregulatory molecules in patients with monoclonal large granular lymphocyte lymphocytosis

TCR alpha beta+/CD4+ T-large granular lymphocyte (LGL) lymphocytosis is a subgroup of monoclonal T-LGL lymphoproliferative disorders that are different from the CD8+ TCR alpha beta T-LGL. An increasing evidence supports the involvement of a common antigen-driven mechanism in the etiology of TCR alpha beta+/CD4+ T-LGL. In this study, we tested several polymorphic markers associated with chronic viral infections and autoimmune diseases, including cytotoxic T-lymphocyte antigen-4 (CTLA-4), tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), interferon-gamma (IFN-gamma), RANTES, IL-1 alpha, FAS, FAS-ligand (FASL), and NKG2D, to investigate the potential association of these immunogenetic factors with the development of T-LGL. Overall, 38 patients with CD4+ T-LGL were analyzed and compared with a group of both CD8+/TCR alpha beta+ T-LGL patients (n = 43) and a group of control subjects (n = 176). Our results did not show any clear association between the different single nucleotide polymorphisms (SNPs) analyzed and the development of CD4+/TCR alpha beta T-LGL. An increase in the frequency of -380 (AA/GA) TNF-alpha genotype associated with a greater production of this cytokine was found among CD8+ T LGL patients in comparison to the CD4+LGL patients and the control group. Our results suggest that the frequency of SNP of the genes coding for the studied immunoregulatory molecules are not associated with the development of CD4+/TCR alpha beta+ T-LGL.

Clonal Large Granular Lymphocyte (LGL) Expansion Associated with Dasatinib Therapy.

In addition to therapeutic efficacy in imatinib-resistant BCR-ABL positive malignancies, dasatinib may have off-target immunomodulatory effects by inhibiting kinases (e.g. SRCs, c-KIT) in immune effector cells. In a proportion of patients, dasatinib treatment is associated with non-infectious pleural effusions and panniculitis, which may reflect aberrant immune reactivity. Here we describe a marked expansion of clonal LGL lymphocytes in blood among 11 BCR-ABL positive patients on dasatinib therapy (5 CML CP, 1 CML AP, 1 CML BC, 4 Ph+ALL). All patients were previously exposed to imatinib without similar changes in lymphocyte count or morphology. The median time to LGL lymphocytosis was 2 months from the start of dasatinib (range 1–8 months). It developed abruptly, with a peak lymphocyte count of 4–20 ×109/L, and was often preceded by low-grade fever. In all evaluable cases, lymphocyte counts normalized after drug discontinuation. By immunophenotyping, 6 patients had a CD3/CD8+ cytotoxic T-cell phenotype and 4 patients had a CD56+ NK-cell phenotype. The LGL lymphocytes were BCR-ABL negative. All CD3-positive cases had a clonal rearrangement of TCR gamma/delta genes by PCR; clonality was not detected in samples prior to dasatinib therapy (n=3). In TCR beta gene repertoire assay, oligoclonal patterns were seen in selected genes. All patients with HLA data had the A2 allele (n=8). Dasatinib-related complications were common: pleural effusions and/or pulmonary infiltrates (n=8), modest CMV reactivation (n=5), severe colitis (n=5), which developed after appearance of LGL lymphocytosis. Immunophenotyping was available from 2 patients with pleural effusions: in both cases the majority of cells were CD3+CD8+ T-cells and no leukemic cells were detected. TCR gamma assay was done from one patient sample and it showed similar clonal pattern as in peripheral blood. However, despite frequent side-effects, response to dasatinib treatment was very good in all cases, including complete molecular responses in 5 advanced phase patients. Transient clonal LGL lymphocytosis has been described in few patient cases with a primary herpesvirus infection. Our patients share striking pheno/genotypic similarities with LGL leukemia, in which an antigen driven expansion of LGLs precedes the occurrence of oncogenic events. However, in our patients LGL lymphocytosis appeared to be a benign phenomenon. We postulate that by virtue of inhibition of key non-BCR-ABL kinases, dasatinib induces a reversible state of autoimmune-like reactivity upon antigen challenge in a proportion of patients with a distinct genetic and/or environmental background. The aberrant immune response may also result in enhanced anti-leukemic control driven by the cytotoxic T/NK LGL cells. Dasatinib could be a valuable tool in uncovering the pathogenesis of LGL lymphoproliferation, putatively related to mutations in kinases targeted by the drug.

Clinical features of large granular lymphocyte leukemia

The spectrum of large granular lymphocyte (LGL) proliferations consists of four distinct entities: reactive/transient LGL expansion, chronic LGL lymphocytosis, classical indolent LGL leukemia, and aggressive LGL leukemia. LGL leukemias are classified as lymphoid malignancies. They are divided into CD3+/T-cell LGL (85% of cases) and CD3−/natural killer (NK) cell LGL leukemia (15% of cases). Recent progress in the comprehension of the leukemogenesis has shown a dysregulation of survival signals in leukemic cells. Identification of LGL expansion has been improved using T-cell receptor (TCR)β/γ polymerase chain reaction (PCR) analysis and a combination of Vβ and killer cell immunoglobulin-like receptor (KIR)-specific monoclonal antibodies. LGL leukemias are characterized by a clonal LGL infiltration of the bone marrow, spleen, and liver. Monoclonality is recognized by phenotypic, molecular, and karyotypic analysis. T-LGL leukemias affect the elderly and display a relatively indolent behavior. Approximately 60% to 70% of patients are symptomatic: recurrent infections secondary to chronic neutropenia, anemia, and autoimmune disease such as rheumatoid arthritis are the main clinical manifestations. Long-lasting remission can be obtained with low-dose methotrexate, cyclosporine A, or cyclophosphamide. Conversely, NK LGL leukemias behave aggressively, and most patients do not respond to chemotherapy.