Expansion Of Large Granular Lymphocytes Research Articles

Expansion of T or B Lymphocytes after Unrelated Cord Blood (UCB) Allogeneic Stem Cell Transplantation in Adults Correlates with CMV Reactivation and Is Associated with a Better Outcome

Introduction: Peripheral lymphocytosis encountered after myeloablative (MAC) or reduced-intensity conditioning (RIC) allogeneic stem cell transplantation (allo-SCT) is an ill-defined feature. Most reports in the literature deal with large granular lymphocytes (LGL) expansions and only seldom of B-cell increases (Bellucci, Blood, 2002). With an incidence of 3 to 18%, LGL proliferations occur generally late after allo-SCT with a median onset of 9 to 16 months. Such expansions can be polyclonal, oligoclonal or monoclonal, arising from either CD3+ T-cells or CD3- NK cells or both. LGL expansion has been frequently linked to CMV reactivation, indolent clinical course and a usually favorable outcome. Most available data were mainly described in the setting of allo-SCT using bone marrow (BM) or peripheral blood (PBSC) as stem cell source. Here, we report data regarding the incidence and features of lymphocyte expansions after unrelated cord blood (UCB) transplantation.Patients and Methods: Ninety-nine UCB allo-SCT performed in adults between October 2005 and October 2014 were considered for the purpose of this study. Most patients received double CB units (n=94) and a RIC regimen (n=89), for various hematological diseases. Whenever detected, we collected the date of onset and termination of peripheral blood lymphocyte expansions (4x109/L) among the 86 UCB-SCT patients alive at 3 months post-transplant. LGL expansion was defined as sustained LGL above 0.5x109/L and/or >40% of LGL in peripheral blood (Zambello, Haematologica, 1998). Concomitant immunophenotypic results, allowed to discriminate expansions of cytotoxic T-cells (CD3+CD8+CD56+), NK-cells (CD3-CD16+/CD56+) and B-cells (CD19+). LGL expansion data were also analyzed with respect to viral reactivation episodes, acute or chronic graft vs host disease, relapse and survival.Results: Lymphocytosis was observed in 21 cases (24%; 10 females and 11 males; median age: 58 y., range: 32-69). Most patients had a myeloid-lineage disease (67%) and were in complete remission at time of UCB-SCT (76%). The median onset of lymphocyte expansion after UCB-SCT was 12.6 months (range, 1.4-49). The median initial lymphocyte count was 4.76x109/L at time of expansion diagnosis. The median duration of expansion was 12 months (range: 1-52). Twenty patients could be further analyzed phenotypically, showing 8 CD8+ T, 1 NK and 1 T-NK LGL expansions. Interestingly, 7 cases of polyclonal B-lymphocytes expansions were also documented while 3 patients presented both T CD8+ and B expansions. Of note, B-cell expansions were CD5+. For 6 patients with T-cell expansion, concomitant DNA from CD3+ sorted cells is available to test clonality.Lymphocyte expansion were from donor origin for 12/14 tested patients. Acute and chronic GVHD developed respectively in 31% and 68% of lymphocytosis patients, and in 57 and 45% of the 65 patients without lymphocyte expansion (P=NS). Comparing these two groups for viral reactivations, the rates were 86% and 76% for HHV-6 (P=NS) and 23% and 39% for EBV (P=NS) respectively. CMV reactivation was significantly more frequent in the group of lymphocytosis patients (76% vs. 29%, P=0.0001). Interestingly, CMV reactivation was significantly higher in the 10 patients of the T or NK group compared to the 7 patients with B cell expansion (100% vs 57%, P=0.05).At time of analysis, 1 patient had relapsed and 4 had died, the causes of death being disease in 1 case and transplant-related mortality in 3. These events were significantly lower than in the group of patients without lymphocytosis (p=0.003 for relapses and p=0.04 for death). Two-year disease-free survival (Fig A) and overall survival (Fig B) were significantly different at respectively 85% vs. 55% (p=0.01) and 85% vs. 63%. (p=0.03).Conclusion: Lymphocyte expansion, at 24%, is not a rare event in adults receiving UCB allo-SCT. These expansions involve equally the T or B-lineages. The latter are often CD5+ suggesting a proliferation of innate B1 cells from the UCB. Lymphocyte expansions are significantly associated with previous reactivation of CMV, but not HHV-6 or EBV. Because these cells were of donor origin, it can be postulated that they represent primo-activation upon encounter with CMV. Finally, both types of lymphocyte expansions are associated with a significant favorable outcome, suggesting a possibly bystander anti-GVL effect. [Display omitted] DisclosuresMoreau:Celgene, Janssen, Takeda, Novartis, Amgen: Membership on an entity's Board of Directors or advisory committees.

Phenotypic Heterogeneity of Chronic Lymphoproliferative Disorder of NK Cells

BackgroundNK cells represent a subset of lymphocytes belonging to the innate immunity branch typically expressing CD16 and CD56 associated to CD3 negativity. Two major subtypes of NK cells can be distinguished through CD16 and CD56 expression: CD56high/CD16dim/neg NK cells with low cytotoxic function and CD56dim/CD16high NK cells with high cytotoxic function. Recently a subtype of NK cells with memory properties characterized by CD56dim/CD16high/CD57+/CD62L- phenotype has been discovered. Chronic Lymphoproliferative Disorders of Granular Lymphocytes are characterized by the clonal expansion of Large Granular Lymphocyte (LGL) that can be CD3 positive (T-LGLL) or CD3 negative (Chronic Lymphoproliferative Disorder of NK Cell, CLPD-NK). The disease generally has indolent course but some patients develop cytopenia, particularly neutropenia, exposing to potentially lethal bacterial infections. Furthermore, NK-CLPD is usually referred as a more indolent disorder with respect to T-LGLL, with lower incidence of cytopenia and treatment need. CLPD-NK therapy does not differ from that of T-LGLL and is usually represented by an immunosuppressive therapy with low dose cyclophosphamide or methotrexate, with cyclosporine A usually being reserved to refractory patients. Somatic STAT3 mutations represent a new diagnostic marker of these disorders, initially reported in T-LGLL in about 40% oh patients, but also present in CLPD-NK in about 30% of cases.Using flow analysis, the aim of the present study was to identify a subset of CLPD NK patients characterized by a more severe disease requiring a shorter follow-up as compared to patients with a more indolent disease.MethodsIn a cohort of 16 patients affected by CLPD-NK, NK cells were analysed by flow for CD3, CD16, CD56, CD57 and CD62L antigen expression. These patients were studied for the presence of cytopenia and treatment requirement. STAT3 mutation analysis of exon 21 was performed with Sanger sequencing. Finally, p-STAT3 tyr 705 level and total STAT3 level were examined by western blotting.ResultsIn relation to CD16 and CD56 expression, three major NK cells populations can be recognized in CLPD-NK patients: CD56high/CD16neg NK cells, CD56dim/CD16neg NK cells and CD56neg/dim/CD16high. As a consequence, patients can be separated into three groups characterized by the preferentially expansion of one of these populations: 2/16 (13%) with CD56high/CD16neg NK population, 4/16 (25%) with CD56dim/CD16neg NK population and 10/16 (62%) with CD56neg/dim/CD16high NK population. Furthermore, patients with predominance of this last NK cells subset were studied for CD57 and CD62L expression to identify NK cytotoxic subset (CD57-/CD62Llow/neg) and NK memory subset (CD57+/CD62Llow/neg); a NK cytotoxic/memory ratio (C/M ratio) was then calculated. 4 of 10 CD56neg/dim/CD16high patients (40%) were characterized by prevalence of NK cytotoxic cells expansion and high C/M ratio (≥3) while the remaining 6/10 patients were characterized by NK memory cells expansion with low C/M ratio (≤1.6).We then evaluated the presence of cytopenia, in particular neutropenia, in our patients' cohort. Neutropenia was shown in 7/16 (44%) patients with 4/16 (25%) experiencing severe neutropenia. Anemia and thrombocytopenia were less frequent (19% and 6% respectively). Interestingly, 6 out of 7 (86%) neutropenic patients were in the CD56neg/dim/CD16high subset and all patients with severe neutropenia belonged to the high C/M ratio subset. Interestingly, 3 out of 4 patients (75%) of this subset required therapy during the natural history of the disease. Concerning STAT3 mutation analysis, no one mutated patient was found in this setting. By western blot analysis, patients with high C/M ratio presented higher p-STAT3 levels than other patients and normal NK cells.SummaryAlthough CLPD-NK represents an extreme heterogeneous disorder, discrete subtypes of disease characterized by different NK cells population expansion can be identified by flow analysis. Interestingly, this splitting allows to identify a subset of patients with prevalence of CD56neg/dim/CD16high NK cells with high C/M ratio that are characterized by high level of p-STAT3, high frequency of severe neutropenia and treatment requirement. DisclosuresNo relevant conflicts of interest to declare.

Second-Generation Relative Donor for T-Replete Haplo-Identical Allogeneic Stem Cell Transplantation with High-Dose Post-Transplant Cyclophosphamide: Towards Disappearance of the HLA Barrier

Introduction: Allogeneic stem cell transplantations (allo-SCT) using alternative donors are increasingly used in patients lacking suitable matched sibling or unrelated donor. Recently, the use of post-transplant cyclophosphamide (CY) has allowed to re-consider first-generation relatives (brother, sister, father, mother, son or daughter) as haplo-identical donors for allo-SCT. Indeed, the incidence of severe acute GVHD is lowered by post-transplant administration of CY, due to the early destruction of putative alloreactive T cells. Still, some patients may not have a suitable relative donor for various reasons such as older age of the parents, no siblings nor children, no cord blood available, or because use of a graft from a first-generation haplo-identical relatives is contra-indicated. The probability of having a haplo-identical donor among siblings is 50% while it is almost 100% when considering both biological parents and offspring. When considering as a potential donor the child of a matched or haplo-identical sibling, the probabilities for the donor to be haplo-identical with the recipient remain 50% and 25%, respectively. Thus, second-generation relative donor (i.e. nephew or niece) may be finally considered as a source of stem cell graft.Methods: Here we report the case of a 61-year old man who received a T-replete haplo-identical allo-SCT with high-dose post-transplant CY from his second-generation relative nephew. In 2010, the patient was diagnosed with ALK- CD30+ anaplastic T-cell lymphoma and received 8 cycles of CHOD allowing to obtain morphologic and metabolic partial response. The patient received an allo-SCT from a sibling matched donor (brother) on November 2010 after an FB2A2 reduced-intensity conditioning regimen but secondary graft failure was rapidly documented in spite of donor lymphocyte infusions. Relapse occurred in September 2014 and brentuximab vedotin was administered each 21 days for seven cycles allowing to obtain a complete morphologic and metabolic response. It was decided to perform a second allograft using a different donor. The patient was childless and no unrelated donor or cord blood units were available. It was thus chosen to ask for donor the son of the matched brother, who was haplo-identical and had no antibodies directed against the patient's HLA specificities. This nephew had a O- blood group and negative CMV serology while the recipient was O+ and CMV+.Results: The second allograft was performed on April 2nd 2015 using the Baltimore (Luznik, BBMT 2008) conditioning regimen with 2 days of post-transplant CY. A megadose of peripheral blood stem cell was administered (14.16x106 CD34+ cells/Kg) at day 0. Neutrophils and platelets recovery (>50 Giga/L) were achieved as early as days +18 and +33, respectively, with full donor whole blood chimerism (99.8%) at day +30, persisting on day +60 and +100 (both whole blood 99.9%, CD3+ T cells: 99.9%).Grade 2 acute cutaneous GVHD occurred at day +21. The evolution was favorable after initiating corticosteroids at 2 mg/kg/day with tapering thereafter. Not surprisingly, CMV reactivation was documented at day+22, controlled by ganciclovir treatment. Immune reconstitution evaluated at days +30, +60 and +100 showed normal monocyte counts while B lymphocytes were undetectable. NK cells increased from 42/mm3 at day+30 to 453/mm3 at day +60 and 665/mm3 at day +100. Interestingly, after profound lymphopenia within the first 100 days post-transplant, CD8+ T large granular lymphocytes expansion was documented at day + 110. At four months post-transplant, the patient is alive in persistent metabolic remission with no active acute or chronic GVHD nor active infection. His outcomes will be updated for the meeting.Conclusion: T-replete haplo-identical allo-SCT with high-dose post-transplant CY using a second-generation relative donor was feasible allowing for full engraftment and moderate acute GVHD in our patient. This result expands the possibility to find a donor for a selected patient, meanwhile paving the way for using unrelated haplo-identical donor, which could be of interest when considering solid transplantation. Indeed, combining solid organ transplantation and allo-SCT from the same haplo-identical donor may open the door to withdraw immunosuppression in this particular setting, because stable tolerance may be induced, as it has been already reported for kidney transplant (Kawai, NEJM 2013). DisclosuresMoreau:Celgene: Honoraria, Other: Adboard; Amgen: Other: Adboard; Takeda: Other: Adboard; Janssen: Other: Adboard; Novartis: Other: Adboard.

Dasatinib Expands Pre-Existing, CMV-Associated, Highly Differentiated NK Cells in Ph+ Leukemia

[Background] Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib are key drugs for the treatment of Philadelphia chromosome-positive (Ph+) leukemia. Among these TKIs, only dasatinib expands large granular lymphocytes (LGLs) including NK cells in some patients. This dasatinib-induced lymphocytosis is associated with enhanced therapeutic responses. Recently, it has been suggested that dasatinib-associated LGL expansion is linked to cytomegalovirus (CMV) reactivation. CMV infection induces substantial changes in phenotypic and functional profiles of NK cells. However, characteristics of NK cells in dasatinib-treated Ph+ leukemia patients (DA) and mechanisms by which dasatinib expands NK cells remain unknown.[Method] NK-cell marker expression and cytotoxic activity of NK cells were examined in 31 DA, 15 patients treated with other TKIs (OT) and 15 healthy donors (HD). We mapped NK-cell profiles, using principal component analysis (PCA) of 19 NK-cell markers.[Result] NK cells increased in 18 of 26 CMV-seropositive (CMV+) DA, in contrast to no increase in 5 CMV- DA and 13 CMV+ OT. Expression of an activating receptor NKG2C was enhanced on the increased NK cells. PCA mapped NK-cell profiles according to the degree of differentiation, which correlated with higher expression of NKG2C, CD57 and LIR-1, and lower expression of NKp30 and NKp46, revealing that NK cells in CMV+ DA were highly differentiated compared to those in CMV- DA and HD (Figure 1). NK cells in CMV+ OT were mapped between CMV+ DA and HD. In 10 CMV+ DA, recent CMV reactivation was detected by CMV IgM and/or quantitative PCR. Of these, CMV reactivation was symptomatic only in 4 patients, indicating that dasatinib induces subclinical CMV reactivation in the majority of patients. CD107a degranulation assay showed higher cytotoxic activity of NKG2C+ NK cells, compared to NKG2C- NK cells. Interestingly, some of the CMV+ patients with Ph+ leukemia had NKG2C+ highly differentiated NK cells already at diagnosis, suggesting that CMV has been reactivated before starting TKIs at least in some patients. In fact, we detected CMV genome by nested PCR in PBMCs from some of the pretreatment patients. Moreover, the CMV+ patients with highly differentiated NK cells developed NK-LGL expansion earlier after starting dasatinib, compared to those with less differentiated NK cells.[Discussion] Our data indicate that CMV seropositivity underlies the development of NK-LGL expansion in DA patients. Intriguingly, it appears that CMV has been subclinically reactivated at least in some Ph+ leukemia patients even before starting dasatinib, leading to expansion of "ready-to-go" NKG2C+ highly differentiated NK cells. It is likely that dasatinib further expands such NK cells presumably due to chronic stimulation by reactivated CMV. The NKG2C+ NK cells have high cytotoxic activity against leukemic cells, which might contribute to enhanced therapeutic responses in DA patients. The phenotypic profile of NK cells at diagnosis may be a strong predictor of NK-LGL expansion during dasatinib therapy. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 2587: Dasatinib-induced reduction of tumor growth is accompanied by the changes in the immune profile in melanoma B16.OVA mouse model

Abstract Dasatinib, a tyrosine kinase inhibitor used in the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia, has been shown to exert immunomodulatory effects in addition to direct oncokinase inhibition. Recently, we observed that dasatinib induces a rapid (within one hour after oral administration) and marked mobilization of lymphocytes (up to 8-fold increase in lymphocyte count), which closely follows the drug plasma concentration. In addition, in a subgroup of patients a clonal expansion of large granular lymphocytes occurs, which is correlated with good therapy response. As dasatinib-induced immunomodulatory effects are not leukemia specific, we aimed to exploit this activity to slow down or inhibit the growth of a syngeneic murine melanoma model and to characterize in detail the anti-tumor immune responses. Ovalbumin expressing murine melanoma cell line B16.OVA was grown under the skin of immunocompetent mice. The mice (n=6/group) were treated daily i.g. either with 30 mg/kg dasatinib or vehicle only. Blood was collected before tumor transplantation, before treatment, and on treatment days 4, 7 and 10. Tumor volumes were measured manually and specific growth rate was calculated based on the first and the last day of the treatment. In addition to white blood cell differential counts, immunophenotyping of blood and tumor homogenate was done by flow cytometry using antibodies against CD45.1, CD3, CD4, CD8b and NK1.1, and SIINFEKL-pentamer to detect B16.OVA-specific cells. To assess the functional properties of lymphocytes, we used antibodies against immune checkpoint inhibitors CTLA-4 and PD-1, and a degranulation marker CD107. On the 10th day of treatment, the tumor volumes were smaller in dasatinib group compared to control group, and there was a significant decrease in the tumor growth rate (0.06 vs. 0.18, p=0.01). Furthermore, dasatinib treated mice had increased proportion of CD8+ cells in the circulation (21% vs. 14%, p=0.04) and the CD4/CD8 ratio was significantly decreased (1.39 vs. 1.52, p= 0.04). During the tumor growth the mean CTLA-4 expression on CD8+ cells increased from 1.2% to 9% in the control group, whereas, in dasatinib group the increase was more modest (1.2% to 5.7%). Interestingly, 80% of tumor infiltrating CD8+ cells expressed PD1 antigen compared to <5% of PD1 positive CD8+ cells in the peripheral blood suggesting lymphocyte anergy or exhaustion induced by tumor cells. To conclude, dasatinib treatment slowed down the tumor growth in B16.OVA mouse model, which could be associated with the immunomodulatory effects of dasatinib. However, combinatorial treatment regimens may be needed as dasatinib is not able to fully break the immune cell anergy induced by tumor cells. Citation Format: Can Hekim, Mette Ilander, Markus Vähä-Koskela, Paula Savola, Siri Tähtinen, Akseli Hemminki, Kimmo Porkka, Satu Mustjoki. Dasatinib-induced reduction of tumor growth is accompanied by the changes in the immune profile in melanoma B16.OVA mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2587. doi:10.1158/1538-7445.AM2014-2587

Characteristics of patients with development of large granular lymphocyte expansion among dasatinib-treated patients with relapsed Philadelphia chromosome-positive acute lymphoblastic leukemia after allogeneic stem cell transplantation.

Widespread use of tyrosine kinase inhibitors (TKIs) in combination with chemotherapy and allogeneic hematopoietic stem cell transplantation (allo-SCT) has totally changed the existing treatment strategies for Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL). However, the prognosis after relapse after allo-SCT is still dismal. We analyzed the clinical outcome of therapy using dasatinib, a second-generation TKI, in 9 patients with relapsed Ph(+)ALL after allo-SCT. Dasatinib was initiated at a median time of 168 days after allo-SCT at dosages ranging from 20 mg to 100 mg daily. Six of 9 patients manifested a marked increase in large granular lymphocytes (LGLs), but all 6 patients discontinued dasatinib because of adverse events (AEs) such as pleural effusion. Four of 6 patients resumed dasatinib, and 3 of them have been alive with molecular complete remission and a persistent increase of LGLs. Our results demonstrated that dasatinib therapy can induce LGL expansion accompanied by AEs, but this phenomenon can be associated with long-term survival benefit in a proportion of relapsed Ph(+)ALL patients after allo-SCT.

LGL Disorders: From An Inflammatory-Mediated To a Self-Maintaining Proliferation

T large granular lymphocytes leukemia (T-LGLL) and NK-type chronic lymphoproliferative disorder (CLPD-NK) are rare diseases characterized by the abnormal expansion of large granular lymphocytes (LGLs) with cytotoxic activity, belonging to T and NK lineage, respectively. Currently, the etiology of these diseases is still largely unknown. Several data support the hypothesis that the inciting event is represented by the persistence of antigenic stimulation, maintained by the abnormal release of cytokines (mainly IL-6 and IL-15), establishing an inflammation status not achieving resolution. Recently, we showed that IL-6 and soluble IL-6Rα were highly expressed and released by patients' LGL-depleted peripheral blood mononuclear cells (PBMC), accounting for a trans-signaling process. IL-6 trans-signaling is critically involved in inflammatory disease and promotes the transition from acute to chronic inflammation. Additionally, LGL proliferation is maintained for an impairment of the apoptotic machinery due to the activation of many survival signaling pathways, including JAK/STAT and RAS/MEK/ERK pathways. In some patients (both T-LGLL and CLPD-NK) STAT3 hot-spot mutations, inducing STAT3 activation, have been demonstrated.With this as a background, we investigated the IL-15 contribution to sustain IL-6 trans-signaling and in turn inflammation. We analyzed the relationships between STAT3 mutations, IL-6 and IL-15 in disease progression to assess the hypothesis that these findings characterize different stages of LGL disease. Thirty T-LGLL and 15 CLPD-NK patients were included in this study. Patients were subdivided according to the percentage of LGLs in PBMCs (LGL range: 35-90%). By ELISA in patients' plasma, we showed that IL-6 concentrations were significantly higher in patients characterized by a disease with less than 60% circulating LGLs (35.7 ± 11.4 pg/ml with respect to patients with LGLs >60%: 9.1 ± 2.7 pg/ml; p <0.05). Considering that IL-6 is a mediator of inflammation, we suggest that these low burden disease patients (LGLs <60%) are mostly characterized by an active inflammatory background. By Real Time-PCR, we observed that IL-15 mediated IL-6 expression in patients PBMCs (IL-6 increased 4.7 fold after IL-15) while inhibited IL-6Rα in leukemic LGLs (by 2-fold down), indicating that IL-15 favors IL-6 trans-signalling. By western blotting analysis we showed that both IL-6 and IL-15 were able to activate STAT3 and ERK, then sustaining LGL survival. Interestingly, in the patients showing STAT3 mutations (20% of patients) we demonstrated a significantly lower level of plasma IL-6 (8.9 pg/ml). This feature was found to be associated to a high proliferative disease, with more than 60% circulating LGLs.These results suggest that an initial step along the development of disease characterized by a low lymphocytosis (LGLs <60% of total PBMCs) is mostly sustained by extrinsic factors contributing to the relevant inflammatory background. A subsequent stage is characterized by a high lymphocytosis (LGLs >60% of total PBMCs) in which LGL disease goes on independently from exogenous stimuli, likely becoming self-maintaining due to the contribution of the emerging STAT3 mutations. It is suggested that these phases can represent two sequential steps in the progression of disease. Disclosures:No relevant conflicts of interest to declare.

Ex vivo expansion of canine cytotoxic large granular lymphocytes exhibiting characteristics of natural killer cells

Canine NK cells still are not well-characterized due to the lack of information concerning specific NK cell markers and the fact that NK cells are not an abundant cell population. In this study, we selectively expanded the canine cytotoxic large granular lymphocytes (CLGLs) that exhibit morphologic, genetic, and functional characteristics of NK cells from normal donor PBMCs. The cultured CLGLs were characterized by a high proportion of CD5(dim) expressing cells, of which the majority of cells co-expressed CD3 and CD8, but did not express TCRαβ and TCRγδ. The phenotype of the majority of the CLGLs was CD5(dim)CD3+CD8+ TCRαβ−TCRγδ−CD4−CD21−CD11c+/−CD11d+/−CD44+. The expression of mRNAs for NK cell-associated receptors (NKG2D, NKp30, NKp44, Ly49, perforin, and granzyme B) were highly upregulated in cultured CLGLs. Specifically, NKp46 was remarkably upregulated in the cultured CLGLs compared to PBMCs. The mRNAs for the NKT-associated iTCRα gene in CLGLs was present at a basal level. The cytotoxic activity of the CLGLs against canine NK cell-sensitive CTAC cells was remarkably elevated in a dose-dependent manner, and the CLGLs produced large amounts of IFN-γ. The antitumor activity of CLGLs extended to different types of canine tumor cells (CF41.Mg and K9TCC-pu-AXC) without specific antigen recognition. These results are consistent with prior reports, and strongly suggest that the selectively expanded CLGLs represent a population of canine NK cells. The results of this study will contribute to future research on canine NK cells as well as NK cell-based immunotherapy.

Expansion of a Clonal CD8+CD57+ Large Granular Lymphocyte Population After Autologous Stem Cell Transplant in Multiple Myeloma

Clonal expansions of large granular lymphocytes (LGLs) have been identified in patients following stem cell transplants and may represent posttransplant LGL leukemias or reactive immune responses. To differentiate between these 2 possibilities, we assessed peripheral blood and bone marrow of patients with myeloma after autologous stem cell transplant. All patients examined shortly after autologous stem cell transplant had significant increases in the LGLs in the peripheral blood and bone marrow (71% of lymphocytes) as compared with controls (39%). This increase was detectable years after transplant. The LGLs had a reproducible immunophenotype of CD8+CD57+ T cells without phenotypic abnormalities in 19 of 20 patients. Sixty-five percent of the post-autologous stem cell transplant patients had clonal T-cell receptor gene rearrangements in the bone marrow, yet no patients had neutropenia or splenomegaly. Although the LGL expansions were clonal and persistent, the lack of clinical sequelae suggests the clonal LGL expansion is a reactive, potentially beneficial, immune response to autologous stem cell transplant.

Combined SNP Microarray Based Genomic Mapping and Next Generation Transcriptome Sequencing Confirms the Presence of Somatic STAT3 Mutations and Identifies Novel Chromosomal Imbalances and Candidate Genes in T Large Granular Lymphocytic Leukemia (T-LGL)

AbstractAbstract 3512 IntroductionT-cell large granular lymphocytic leukemia (T-LGL) is a rare lymphoproliferative disease characterized by an expansion of large granular lymphocytes involving blood, bone marrow, spleen and liver. T-LGL cells are mature CD3, CD8 and T-cell receptor (TCR) αβ positive cells exhibiting the immunophenotype of activated cytotoxic T lymphocytes (CTLs). CD4 or TCR γδ positive variants occur rarely. T-LGL affects adults at a median age of 55–60 years and arises commonly in patients with a preexisting autoimmune disorder. Many patients remain asymptomatic for years and do not require treatment. Palliative therapy with immunosuppressant agents such as low dose methotrexate, ciclosporin and fludarabine is used for the correction of severe immune-mediated cytopenias, which often complicates the course of the disease. The molecular pathogenesis of T-LGL remains unclear. No recurrent karyotypic anomalities but several numeric and structural chromosomal alterations have been identified. Recently, activating somatic mutations in the signal transducer and activator of transcription 3 gene (STAT3) have been described by Koskela et al. in approximately 40% of T-LGL patients. STAT3 mutations lead to an increased transcriptional activity and were more prevalent in patients with neutropenia and rheumatoid arthritis than in patients without these conditions. As these findings only explain part of the pathogenesis in the fraction of patients affected by STAT3 mutations, we here aimed to identify novel mutations which may help to better understand the mechanisms of disease development. MethodsWe sorted tumor- and non-tumor cells from peripheral blood samples of T-LGL patients by using fluorescence activated cell sorting (FACSDiva®, Becton Dickinson) to perform single nucleotide polymorphism (SNP) chip analysis and next-generation RNA sequencing. SNP chips were analyzed in 10 patients (Affymetrix, Mapping 250K Sty Array®). To identify somatic mutations in patients with T-LGL, we compared CD8/CD57 positive tumor cells with non-tumor cells as germline control. Sample libraries for RNA sequencing of 5 patients were generated with NuGEN Encore®, sequencing was performed on Illumina HiSeq 2000® yielding 100 million 100 basepair single reads, and alignment was realized on TopHat2 against hg19 as reference genome. For quantification and analysis of variants Partek GS 6.6 was used. ResultsHigh resolution copy number determination employing SNP chips in 10 patients revealed both gains and losses on different chromosomes, among others 1q, 7q, 14q and chromosome X. The affected chromosomal regions included genes with potential relevance to the disease process such as WNT and RASSF gene family members in deleted regions and PIM3 and MAPK family members in gained regions. However, in line with previous reports no recurrent chromosomal aberrations were detected. Preliminary analysis of RNA sequencing data revealed activating STAT3 Y640F mutations in 2 out of 5 patients tested (40%). Interestingly, one of the STAT3 mutated T-LGL clones also exhibited an inactivating mutation of the NFKB inhibitory gene TNFAIP3 (A20), which has been reported to play an important role in the molecular pathogenesis of different B cell lymphomas but has as yet not been described in T-LGL. Detailed analysis of sequencing data is currently ongoing and further results will be presented at the conference. In conclusion, combined RNA sequencing and molecular cytogenetic profiling identified novel specific chromosomal loci and genes that could help to better understand the molecular pathogenesis of T-LGL and develop novel targeted treatment modalities for this disease. Disclosures:No relevant conflicts of interest to declare.

Relative Increase of Lymphocytes As Early As 1 Month After Initiation of Dasatinib Is a Reliable Predictor for Achieving Complete Molecular Response At 12 Months in Chronic Phase CML Patients Treated with Dasatinib

AbstractAbstract ▪691▪This icon denotes a clinically relevant abstract Background:Tyrosine kinase inhibitor (TKI) therapy has become the standard treatment for patients with chronic phase chronic myelogenous leukemia (CML-CP). Compared with imatinib, dasatinib is known to induce a faster and better response as a first- and second-line treatment. A unique effect of dasatinib treatment is the induction of clonal lymphocytosis with large granular lymphocyte (LGL) morphology, presumed to be the effect of off-target kinase inhibition. These LGLs are known to possess either cytotoxic T-cell (CTL) or natural-killer (NK) cell phenotype. Several reports have shown that this clonal LGL expansion is associated with a better response to dasatinib treatment.Purpose: To prospectively analyze the immunoprofile of Japanese patients treated with dasatinib, and to correlate the increase of lymphocytes and their subsets (LGL, CTL and NK cells) with the clinical efficacy of dasatinib. Method and Patients:Japanese patients with CML-CP who were resistant or intolerant to first-line imatinib therapy participated in the prospective phase II study assessing the efficacy and safety of dasatinib. Peripheral blood LGL, CTL and NK cell counts before and 2 weeks, 1 month (M), 3M, and 6M after the initiation of treatment were analyzed at the central laboratory (BML Inc.) by flow cytometry. LGL, CTL and NK cells were defined as CD57+/CD14-, CD8+/CD4- and CD56+/CD3- cells, respectively. Lymphocytosis was defined as a lymphocyte count of >3.0×109/L. The clinical efficacy of dasatinib was evaluated at 12M as complete molecular response (CMR) or less than CMR by peripheral blood quantitative RT-PCR (BML Inc.). The full data set was available for 50 out of 65 patients and was included in this analysis. Results:The median age of the patients enrolled was 57 years (range: 16 – 87 years). There were 37 male and 13 female patients. Twenty patients were switched to dasatinib due to intolerance to imatinib and 30 due to resistance. Lymphocytosis was observed in 19 patients (38%) at 3M. Of those 19 patients, 14 (73.7%) had lymphocytosis persisting at 6M. In total, 21 patients (42%) achieved CMR at 12M.The absolute count of lymphocytes, LGL, CTL or NK cells, did not differ significantly between patients who achieved CMR at 12M and those who did not (p>0.05). However, there were significant differences between these two groups in the relative increase of these cell counts. Relative increase of lymphocytes compared to baseline as early as 2 weeks after initiation of treatment was identified by univariate analysis as a significant factor associated with CMR at 12M (p=0.0348). This increase persisted at each time point observed until 6M. As for the different subsets of lymphocytes, the relative increase of CTL at 3 and 6M and LGL at 1, 3 and 6M were also significantly associated with a higher rate of CMR at 12M. When adjusted for possible confounding factors (age, sex, performance status, whether 1st or 2nd CP, resistance or intolerance to imatinib), the relative increase of lymphocytes at 1, 3 and 6M, CTL and LGL at 3 and 6M remained significant (Fig a-c).Patients who showed an increase of lymphocytes of over 1.5-fold at 1M showed a significantly higher rate of CMR at 12M (66.7% vs 25.8%, p=0.0007). Similar differences were seen in patients with increases of over 2-fold in CTL, LGL and NK cells (70.6% vs 22.6%, p=0.0011; 74% vs 21%, p=0.0003; and 57.9% vs 28.6%, p=0.0444). Detailed analysis at 3M showed a further significant difference in the treatment effect. An increase of over 2-fold in lymphocytes (81.3% vs 21.2%, p<0.0001), and of over 5-fold in CTL (85.7% vs 30%, p<0.0001), LGL (66.7% vs 30.3%, p=0.0001) and NK cells (75% vs 31.6%, p<0.0001) compared to baseline was associated with a significantly increased probability of CMR at 12M. Conclusion:Lymphocytosis after the initiation of dasatinib was seen in a substantial proportion of Japanese patients treated with dasatinib. A relative increase of lymphocytes together with LGL, CTL and NK cells, uniformly defined by flow cytometry, at 1 and 3M after the initiation of treatment were associated with a higher rate of CMR at 12M. To our knowledge, this is the first large prospective study analyzing the clinical significance of lymphocytosis associated with dasatinib therapy in Japanese patients with CML-CP using flow cytometry. Our results suggest that a relative increase of lymphocytes as early as 1M after the initiation of dasatinib is a reliable predictive marker for achieving CMR at 12M. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Large Granular Lymphocyte Expansion after Allogeneic Hematopoietic Stem Cell Transplant Is Associated with a Cytomegalovirus Reactivation and Shows an Indolent Outcome

Expansions of CD3+ large granular lymphocytes (LGLs) after allogeneic hematopoietic stem cell transplantation (HSCT) have been described. We sought to evaluate incidence, characteristics, and clinical significance of persistent T cell (T-)LGL after HSCT. Fourteen of 215 recipients (7%) were diagnosed with LGL expansions. Thirteen showed a CD3+/CD8+ immunophenotype, 5 of them with clonal TCR-γ rearrangement. The lymphocytes appeared at a median of 16 months (range, 3-58 months) after HSCT and lasted for a median time of 31 months (range, 2-179 months). Cytomegalovirus (CMV) reactivation (P =.001) and acute graft-versus-host disease (aGVHD) were associated with LGL expansion (P =.02). In the multivariate analysis, only CMV reactivation showed a significant association with T-LGL expansion (relative risk [RR]: 5.063; 95% confidence interval [CI]: 1.586-16.160; P = .006). The observed posttransplantation LGL expansions, even if monoclonal, showed a chronic, indolent course. Our data indicate that such expansions may be considered as an expression of chronic stimulation, triggered by CMV reactivation rather than a malignant transformation.

Clonal Expansion of T-Cell Large Granular Lymphocytes Associated With High-Grade Myelodysplastic Syndromes: Report of Five Cases and Review of the Literature

Clonal Expansion of T-Cell Large Granular Lymphocytes Associated With High-Grade Myelodysplastic Syndromes: Report of Five Cases and Review of the Literature

Enhancement of natural killer cell effector functions against selected lymphoma and leukemia cell lines by dasatinib

As NK cell immunotherapy is still poorly successful, combinations with drugs enhancing NK cell activity are of major interest. NK large granular lymphocyte expansions associated with improved survival have been described under monotherapy with the Bcr-Abl/Src inhibitor dasatinib, which inhibits NK cell functions in vitro. As Src kinases play a major role in inhibitory and activating signaling pathways of NK cells, both outcomes appear plausible. To clarify these contradictory observations and potentially enable the use of dasatinib as adjuvant, we analyzed how clinically relevant doses promote NK cell effector functions. Polyclonal human NK cells were studied ex vivo. Functional outcomes assessed included conjugate formation, calcium flux, receptor regulation, cytokine production, degranulation, cytotoxicity, apoptosis induction and signal transduction. While dasatinib inhibits NK cell effector functions during functional assays, 24 hr pretreatment of NK cells followed by washout of dasatinib, led to dose-dependent enhancement of cytokine production, degranulation marker expression and cytotoxicity against selected lymphoma and leukemia cell lines. Mechanistically, this was neither due to an altered viability of NK cells nor increased NKG2D, LFA-1 or conjugate formation with target cells. Receptor proximal signaling events were inhibited. However, a slight time dependent enhancement of Vav phosphorylation was observed under certain circumstances. The shift in Vav phosphorylation level may be one major mechanism for NK cell activity enhancement induced by dasatinib. Our findings argue for a careful timing and dosing of dasatinib application during leukemia/lymphoma treatment to enhance NK cell immunotherapeutic efforts.

Immunoprofile of Patients with Chronic Myeloid Leukemia Treated with Imatinib, Nilotinib or Dasatinib,

Abstract 3764Recent reports showed that dasatinib induces significant immunostimulation with clonal expansion of large granular lymphocytes (LGL) which, in chronic myeloid leukemia (CML), is related to both better prognosis and to autoimmune-like side effects. It is speculated that lower levels of circulating T-regulatory cells play a partial role in LGL proliferation in patients receiving dasatinib.The immunoprofile was studied using flow cytometry to evaluate lymphocyte subsets and NK-cell reactivity in the peripheral blood of 61 patients in the chronic phase of CML during treatment with a tyrosine kinase inhibitor (TKI) (Median age: 58 years; imatinib 36, nilotinib 9, dasatinib 16). Furthermore, we measured plasma levels of 27 types of cytokines or chemokines in 58 patients in the chronic phase of CML so that a comprehensive comparison could be made of the differences in immunoprofile among the patients receiving these three TKIs.There were no significant differences between the three TKI-treated groups in terms of CD4/8 ratios or the number of T-cells (CD3+CD8+ or CD4+) and NK cells (CD3-CD56+). However, the number of NK-LGL (CD56+CD57+) and T-LGL (CD3+CD57+) increased significantly in the group that received dasatinib. Furthermore, dasatinib significantly enhanced NK-cell reactivity as compared to imatinib and nilotinib. In contrast nilotinib significantly suppressed NK-cell reactivity (E/T ratio =10:1: Median (interquartile range), 8.7% (5.0–16.2), 5.2% (4.8–11.4), 20.8% (13.4–33.3), for imatinib, nilotinib and dasatinib, respectively). In addition, the number of regulatory T-cells (CD4+CD25int-hiCD127low) was similar among the three groups (Median (interquartile range), 36/mm3 (27–53), 48/mm3 (34–60), 39/mm3 (26–53), for imatinib, nilotinib and dasatinib, respectively).Furthermore, in the analysis of cytokines and chemokines, plasma levels of IL-8, IP-10, and MCP-1 were significantly elevated while the level of PDGF-bb was significantly decreased in all three groups compared to those of healthy control. Plasma levels of IL-1 beta, IFN-gamma, and FGF-basic were significantly decreased in only the dasatinib group compared to those of control (P=.02, P=.04, P=.03, respectively). In addition, plasma levels of GM-CSF were significantly elevated in the imatinib and dasatinib groups (Median (interquartile range), 6.1 pg/ml (2.7–11.7) and 7.9 pg/ml (4.5–8.2), P=.02, and P=.03, respectively) but not in the nilotinib group (P=.34) when compared to those of control. In the multiple regression analysis that evaluated the relationship between NK-reactivity and cytokines or chemokines in the patients receiving dasatinib, only plasma levels of GM-CSF were significantly associated with NK-reactivity (P=.03).Notably, in our data, dasatinib and nilotinib exerted opposite effects on NK-cell reactivity, expansion of LGL, and showed different cytokine and chemokine profiles. Based on our results, the activation of NK-cell reactivity induced by dasatinib might be caused by a mechanism other than a decrease in the number of regulatory T-cells. Additionally, in an unphysiological immunological status mediated by dasatinib, GM-CSF might make some contribution to NK-cell reactivity. Disclosures:Nakamae:BMS: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Hino:BMS: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

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.

Dasatinib Has a Dual Mode of Action: Direct BCR-ABL1 Mediated Anti-Leukemic Effects Are Complemented by Promotion of Th1-Type and NK-Cell Mediated Cellular Immune Responses,

Abstract 3760 Background.Tyrosine kinase inhibitors (TKIs; imatinib, dasatinib, nilotinib) have dramatically improved outcome of CML. Besides inhibiting target kinases in leukemic cells, off-target kinases in immune effector cells are also affected. We have previously described that dasatinib therapy induces an oligoclonal expansion and mobilization of large granular lymphocytes (LGLs; CD8+ T-cells or NK-cells) in Ph+ leukemia patients. Importantly, LGL expansion is associated with improved therapy responses, but the actual mechanisms are unknown. In this study, we explored the function and anti-leukemic properties of LGLs. Methods.Peripheral blood samples from CML patients treated with dasatinib (n=10), imatinib (n=4), or nilotinib (n=7), or healthy controls (n=6) were used to analyze the activation and cytotoxicity of T- and NK-cells. Samples were collected before and after drug intake. The number of LGLs was determined from MGG stained blood smears and compared with granzyme B (GrB) positivity analyzed by flow cytometry.Th1-type cytokine (TNF-a, IFN-g) production was measured by flow cytometry after stimulation of mononuclear cells (MNCs) with a-CD3/CD28-antibodies. Unpurified and purified NK cells were cultured with K562 cells, and degranulation (CD107 analysis) and cytotoxicity were measured. Results.As GrB positivity correlated well (r=0.95, p<0.0001, n=17) with the number of LGLs counted from MGG stained blood smears, a GrB specific antibody was used to identify LGLs in further analyses.At diagnosis CML patients had more GrB+CD8+ T-cells than healthy controls (38 % vs. 11%, p=0.028). Also GrB+CD4+ T-cells were slightly increased, but did not differ significantly from healthy controls (3.6% vs. 0.8%, p=0.08). During dasatinib treatment the proportion of GrB+CD4+ (median at 6 months 28.1%, p=0.03) and GrB+CD8+ (70.9%, p=0.03) T cells increased significantly, whereas similar increase was not observed during imatinib (1.2% GrB+CD4+ and 30.0% GrB+CD8+ T-cells) or nilotinib (4.4% and 41.8%, respectively) therapies.In patients on dasatinib therapy, GrB+CD3+cells were more sensitive to CD3/CD28-antibody stimulation and a larger proportion of cells (13.7%) produced Th1-type cytokines (TNF-a+IFN-g) compared to imatinib (2.4%) or nilotinib patients (5.5%) or healthy controls (5%) under same conditions (p=0.015).As Th-1 cytokine-producing T cells are important in promoting cell-mediated immune responses, we next assessed whether dasatinib also enhances the cytolytic activity of NK cells. When MNC fraction was used as effector population (ratio 20:1), the median percentage of dead K562 cells was 18% in samples taken before dasatinib intake and 32% in samples taken 1h after dasatinib intake (p=0.004). Pre-dasatinib killing did not differ significantly from healthy volunteers (p=0.12). No increase in NK-cytotoxicity was observed after imatinib (11% vs. 8%) or nilotinib (10% vs. 10%) intake. Similar results were also obtained with purified NK-cells: the median percentage of dead K562 cells was 12% pre-dasatinib and 29% in post-dasatinib samples (p=0.06), whereas no differences were noticed with imatinib (30% vs. 28%) or nilotinib (14% vs. 15%) patients. The median percentage of dead K562 cells after incubation with pure NK-cells from healthy volunteers was 20%.Interestingly, the cytolytic ability of NK-cells differed significantly among dasatinib treated patients. When the patients were divided into two groups based on therapy response, patients who had achieved CMR within 12 months (n=4) had significantly higher cytotoxic capability compared to patients who had not (n=6): 46% vs. 28% of dead K562 cells in post-dasatinib samples (p=0.02). Conclusions.Dasatinib therapy resulted in increased numbers of GrB+ T-cells and generation of a Th1-type cellular immune response. In addition, 1h dasatinib exposure in vivo improved the cytotoxicity of NK-cells. These data support the dual mode of action of dasatinib: potent BCR-ABL1 inhibition in leukemic cells is accompanied by enhancement of cellular immunity, which likely have implications in be the long term control of Ph+ leukemia. Disclosures:Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria; Novartis: Honoraria.

Expansion of highly differentiated CD8+ T-cells or NK-cells in patients treated with dasatinib is associated with cytomegalovirus reactivation.

The tyrosine kinase inhibitor dasatinib exerts immunosuppressive effects on T-cells and NK-cells in vitro. However, in some dasatinib-treated leukemia patients, clonal lymphocytosis with large granular lymphocyte (LGL) morphology develops, and this is associated with enhanced therapeutic responses. To elucidate the mechanistic basis for this paradoxical observation, we conducted detailed phenotypic and functional analyses of T-cell and NK-cell populations from 25 dasatinib-treated leukemia patients. All tested patients with LGL expansions (15/16) were cytomegalovirus (CMV) immunoglobulin (IgG) seropositive with high frequencies of CMV-specific CD8(+) T-cells; 5/16 LGL patients also experienced symptomatic CMV reactivation during dasatinib therapy. Expanded T-cell and NK-cell populations exhibited late differentiated (CD27(-)CD57(+)) phenotypes; this was associated with a predisposition to apoptosis within the T-cell compartment and impaired NK-cell cytotoxicity. Only 3/9 non-LGL patients were CMV IgG seropositive. Dasatinib inhibited in vitro lymphocyte functions, similarly in LGL patients and controls. Notably, distinct CD8(high) and CD8(low) T-cell subsets were observed in LGL patients; this phenotypic dichotomy was also apparent in CMV-specific CD8(+) T-cell populations, and exhibited features consistent with antigen-driven activation. In addition, plasma levels of IP-10, IL-6, monokine induced by interferon-γ and interleukin-2R were significantly increased in LGL patients. These data provide evidence that dasatinib-associated LGL expansion is linked to CMV reactivation and suggest a potential mechanism for this phenomenon.

A molecular and functional analysis of large granular lymphocyte expansions in patients with chronic myelogenous leukemia treated with tyrosine kinase inhibitors

Tyrosine kinase inhibitor (TKI) therapy has become the standard treatment for chronic myelogenous leukemia (CML). Off-target kinase inhibition has been implicated in the appearance of unique adverse effects, such as colitis and pleural effusions. In addition, some patients present oligoclonal expansions of large granular lymphocytes (LGLs). We sought to further investigate this phenomenon in 64 patients treated with five different TKIs. Clonal expansions of cytotoxic T lymphocytes (CTLs) were identified in all TKI-treated patient groups, but only in dasatinib-treated patients were these expansions characterized as LGLs. Survival factors known to be important in LGL leukemia (interleukin-15 [IL-15] transpresentation, plasma platelet-derived growth factor [PDGF]-BB levels, nuclear factor-κB [NF-κB] and T-bet activation) were found to be associated with TKI-induced LGL expansions. Interestingly, patients with LGL expansions had increased cytotoxicity against non-transformed endothelial cells, which may play a role in observed autoimmune-like side effects. Our results indicate that patients with CML treated with TKIs can develop T cell expansions, which can in certain cases be related to some adverse effects.

Neutropenia associated with rituximab therapy

Several recent studies have reported the occurrence of late-onset neutropenia (LON) following the use of rituximab or rituximab-based therapies. While this phenomenon is typically self-limiting and of no clinical significance, recognizing its existence is important given the expanding use of rituximab in both hematologic and nonhematologic disorders. This review discusses the incidence of LON and explores several hypotheses that have been proposed to explain its occurrence. While the etiology of LON is uncertain and poorly understood, mechanisms that have been suggested include the production of antineutrophil antibodies following rituximab, the expansion of large granular lymphocyte (LGL) populations that may induce neutrophil apoptosis through Fas and Fas-ligand interactions, and aberrant B-cell reconstitution following rituximab leading to immune dyscrasias and the development of neutropenia. We explored an alternative hypothesis that LON following rituximab is caused by perturbations of granulocyte homeostasis, mediated by a complex interaction between B-cell recovery and the chemokine stromal-derived factor-1 (SDF-1). While rituximab has been associated with both early and late neutropenia, LON occurring several weeks to several months after the administration of rituximab is a distinct biologic phenomenon that appears to be related to B-cell recovery. Though it occurs frequently, it is a self-limiting process and is rarely associated with significant clinical sequelae.