Large Granular Lymphocytes Research Articles

Long-Term Experience with Large Granular Lymphocytic Leukemia Evolving after Solid Organ and Hematopoietic Stem Cell Transplantation

T-cell large granular lymphocyte leukemia (T-LGLL) is a clonal proliferation of cytotoxic T lymphocytes (CTL). T-LGLL mainly manifest in elderly and is associated with autoimmune diseases including rheumatoid arthritis (RA), B cell dyscrasias, non-hematologic cancers and immunodeficiency (e.g., hypogammaglobulinemia). LGL manifestations often resemble reactive immune processes leading to the dilemmas that LGLs act like CTL expansion during viral infections (for example EBV associated infectious mononucleosis). While studying a cohort of 246 adult patients with T-LGLL seen at Cleveland Clinic over the past 10 years, we encountered 15 cases of overt T-LGLL following transplantation of solid organs (SOT; n=8) and hematopoietic stem cell transplantation (HSCT; n=7). Although early studies reported on the occurrence of LGL post-transplant, these studies focused on the analysis of oligoclonality skewed reactive CTL responses rather than frank T-LGLL. We aimed to characterize post-transplantation T-LGLL in SOT and HSCT simultaneously and compare them to a control group of 231 de novo T-LGLL (cases with no history of SOT or HSCT). To characterize an unambiguous "WHO-defined T-LGLL" we applied stringent and uniform criteria. All cases were diagnosed if 3 out of 4 criteria were fulfilled, including: 1) LGL count >500/µL in blood for more than 6 months; 2) abnormal CTLs expressing CD3, CD8 and CD57 by flow cytometry; 3) preferential usage of a TCR Vβ family by flow cytometry; 4) TCR gene rearrangement by PCR. In addition, targeted deep sequencing for STAT3 mutations was performed and charts of bone marrow biopsies were reviewed to exclude other possible conditions. Diagnosis was made 0.2-27 yrs post-transplantation (median: 4 yrs). At the time of T-LGLL diagnosis, relative lymphocytosis (15-91%), T lymphocytosis (49-99%) and elevated absolute LGL counts (>500 /µL; 93%) were also seen. Post-transplantation T-LGLL were significantly younger than de novo T-LGLL, (median age: 48 vs. 61 yr; P<.0001). Sixty% of post-transplantation T-LGLL patients were males. Fifteen% of patients had more cytogenetic abnormalities compared to de novo T-LGLL, had a lower absolute LGL count (median: 4.5 vs. 8.5 k/µL) and had less frequent neutropenia, thrombocytopenia and anemia (27 vs. 43%, 33 vs. 35% and 20% vs. 55%; P=.01). TCR Vb analysis identified clonal expansion of ≥1 of the Vb proteins in 60% (n=9) of the patients; the remaining 40% (n=6) of the cases had either a clonal process involving a Vb protein not tested in the panel (20%; n=3) or no clear expansion (20%; n=3). Signs of rejection were observed in 20% (n=3/15) and GvHD in 13% (n=2/15) of the patients. Post-transplantation, 27% of cases presented with neutropenia (absolute neutrophil count <1.5 x109/L; n=4), 33% with thrombocytopenia (platelet count <150 x109/L; n=5) and 25% with anemia (hemoglobin <10 g/dL; n=3). T-LGLL evolved in 10 patients (67%; 10/15) despite IST including cyclosporine (n=5), tacrolimus (n=4), mycophenolate mofetil (n=5), cyclophosphamide (n=1), anti-thymocyte globulin (n=1), and corticosteroids (n=6). Lymphadenopathy and splenomegaly were seen in 13% (n=2) and 33% (n=5) of the patients. Other conditions observed were MGUS (20%; n=3) and RA (7%; n=1). Conventional cytogenetic showed normal karyotype in 89% (n=11, tested individuals 13/15). Somatic STAT3 mutations were identified in 2 patients. Sixty% of cases (n=9) were seropositive for EBV when tested at different time points after transplant. Similarly, 53% (n=8) were seropositive for CMV, of which, 5 were positive post-transplantation and 3 pre-/post-transplantation. The complexity of T-LGLL expansion post-transplantation might be due to several mechanisms including active viral infections, latent oncogenic viral reactivation and graft allo-antigenic stimulation. However, in our cohort graft rejection or GvHD was encountered in a few patients (2 allo-HSCT recipients). Autoimmune conditions were present in 50% of SOT recipients (n=4/ 8, including RA, ulcerative colitis, systemic lupus erythematosus). Some of our patients also had low immunoglobulin levels. Overt EBV (post-transplant lymphoproliferative disorder) and CMV reactivation was diagnosed in only 27% (4/15) of the patients. In sum we report the long term follow up of a cohort of T-LGLL and emphasize the expansion of T-LGLL post-transplant highlighting the difficulty in assigning one unique origin of LGLL. Disclosures Hill: Genentech: Consultancy, Research Funding; Takeda: Research Funding; Celegene: Consultancy, Honoraria, Research Funding; Kite: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; TG therapeutics: Research Funding; AstraZeneca: Consultancy, Honoraria. Majhail:Atara Bio: Consultancy; Mallinckrodt: Honoraria; Nkarta: Consultancy; Anthem, Inc.: Consultancy; Incyte: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Aberrant SYK Expression As a Potential Therapeutic Target in T/NK Cell Neoplasms

Background: During T cell development spleen tyrosine kinase (SYK) is highly expressed at pre-TCR signaling stages, and gradually becomes undetectable at the mature stage. We have previously reported aberrant expression of SYK in peripheral T-cell lymphoma (PTCL). Additionally, ITK-SYK fusion protein detected in a subset of PTCL, mimics a constitutively active TCR signal and drives oncogenesis in mouse models of PTCL. Multiple SYK inhibitors are currently under active investigation in clinical trials for patients with B cell lymphoma and acute myeloid leukemia, and show promising results. Multiparameter flow cytometry can quantitate protein levels at single cell resolution in immunophenotypically defined neoplastic populations. In this study, we employ multiparameter flow cytometry to quantitate the SYK expression in T and NK cell malignancies and to guide the clinical trial design and as potential tool to evaluate the therapeutic responses. Methods: Patients with T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), T-cell large granular lymphocyte leukemia (T-LGL), HTLV-1+ adult T-cell leukemia/lymphoma (ATLL), T-cell prolymphocytic leukemia (T-PLL), angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), mixed phenotype acute leukemia (MPAL) and natural killer cell large granular lymphocyte leukemia (NK-LGL) were included. Abnormal samples included the peripheral blood (46 cases), bone marrow aspirates (10 cases) or tissue (9 cases) from 65 patients diagnosed with T or NK cell malignancies, including AITL (8 cases), ATLL (5 cases), ALCL (2 cases), CTCL (25 cases), PTCL-NOS (5 cases), T-PLL (7 cases), T-LGL (7 cases), NK-LGL (1 case), MEITL (1 case), MAPL (3 cases) and T-ALL (1 case). Normal controls included peripheral blood from healthy donors (15 cases), or patient derived- peripheral blood (6 cases), bone marrow aspirates (13 cases) and lymph node (16 cases) with diagnostic normal immunophenotype. Results: SYK was highly expressed in all the B and most NK cells from peripheral blood, bone marrow and lymph node of normal controls and there was no difference in the percentage of positive cells or intensity of staining in different specimen types. In normal T cell subsets SYK was expressed in a small proportion of T cells and at very low level. Only 3.9% (95% CI: 3.3% - 6.3%) of the CD4+ T cells from peripheral blood, 2.6% (1.5% - 6.6%) from bone marrow, and 1.9% (1.4% - 5.8%) from lymph node were positive for SYK. Similar to CD4+ T cells, SYK was expressed in 5.4% (95% CI: 4.3% - 7.8%) of the CD8+ T cells from peripheral blood. These data suggest SYK expression is nearly undetectable in the normal mature T cells which are part of adaptive immunity. In contrast, lymphoid cells of innate immunity such as NK cells express high levels of SYK. Similar to normal T-cell subsets, the neoplastic T-cells of most PTCL believed to originate from cells of adaptive immunity (AITL, ATLL, ALCL, CTCL, PTCL-NOS and T-PLL) showed low levels of SYK expression (median: 5.8%; 95% CI: 5.8% -10.4%) with rare exceptions (Figure 1). In contrast, the T and NK cell neoplasms of innate immunity origin (NK-LGL, MEITL) and immature precursor T-cell origin (MPAL and T-ALL) showed high levels of SYK expression (median 91.1%, 95% CI as 97.1% - 99.6%). SYK expression in T-LGL was more modest and variable (median 43.6%, 95% CI as 43.6% - 78.0% (Figure 1). Conclusion: SYK is highly expressed in the neoplastic lymphoid cells of innate immunity such as NK-LGL, MEITL and T-LGL cases, and immature hematopoietic neoplasms with T-cell differentiation. The expression of SYK in neoplastic cells from patients with mature T cell lymphoma of adaptive immunity remains low. Given the significant physiological role of SYK in innate immunity, SYK expression in NK/T-cell neoplasms is likely to have an oncogenic role, suggesting that SYK might be a good therapy target for these tumors. Disclosures Roshal: Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Dogan:Roche: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Seattle Genetics: Consultancy; Corvus Pharmaceuticals: Consultancy.

Overexpression and Targeted Activation of the Protein Phosphatases SHP-1 Abrogates Survival Pathways in Large Granular Lymphocyte Leukemia (LGLL)

Introduction: Aberrant phosphorylation-mediated signaling is a well-established condition involved in the onset and progression of virtually all types of cancer. That not only depends on the over-activation of protein kinases, but also on the lack of the proper counterbalance of protein phosphatases, because of their decreased expression or activity. In turn, this is likely to be due to genetic and epigenetic alterations, or interactions with cellular inhibitors and inhibitory post-translational modifications, respectively. In Large Granular Lymphocyte Leukemia (LGLL), a rare lymphoproliferative disorder characterized by clonal expansion of either cytotoxic T lymphocytes (T-LGLL) or natural killer cells (chronic lymphoproliferative disorder of NK cells, CLPD-NK), constitutive activation of phosphorylation-regulated survival pathways, especially the JAK2/STAT3 axis, but also PI3K/Akt and NF-κB pathways among others, has been found to account for the molecular mechanisms sustaining monoclonal cell proliferation and resistance to apoptosis. Interestingly, since such pathways are modulated by the tyrosine phosphatase SHP-1 under normal conditions, it is conceivable that putative alterations of this phosphatase, and consequent inability to disrupt hyperactive pathways, play a role in LGLL pathogenesis. In this study, we assessed the ability of small molecules inducing the expression and stimulating the activity of SHP-1 to abrogate the survival pathways in LGL by counteracting aberrant signals generated by constitutively activated protein kinases. Methods: Peripheral blood specimens were collected from untreated patients with LGLL (both T-LGLL and CLPD-NK). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Sigma Aldrich) gradient separation. LGLs were further separated from PBMCs by using immunomagnetic beads (Miltenyi Biotec). LGLs were incubated either with acetyl-11-keto-β-boswellic acid (AKBA), a pentacyclic triterpene capable of inducing SHP-1 expression, or SC-78, a regorafenib-like derivative specifically stimulating SHP-1 activity, or both at increasing concentrations and discrete time points. After such treatments, LGLs underwent annexin V-PI flow cytometry to assess the extent of apoptosis or were lysed to monitor the abundance of SHP-1 and the phosphatase activity thereof. Moreover, the phosphorylation/activation status and the protein level of factors directly involved in the constitutively activated survival pathways of LGLs were evaluated by Western blot analysis. Results: Both AKBA and SC-78 proved effective at eliciting a significant level of apoptosis in the low micromolar range, exhibiting an additive effect when used in combination. Moreover, both compounds activated SHP-1 activity, as demonstrated by in-vitro phosphatase assays. As to the phosphorylation-dependent signals affected by SHP-1 activation, we observed a marked decrease in the phosphorylation status of STAT3, resulting in the downregulation of the STAT3-induced expression of downstream target genes (Mcl-1, survivin, Cyclin D and S1P5), as well as the dephosphorylation of Akt and p65, which confirmed the role of SHP-1 as a functional antagonist of survival pathways in LGLs. Conclusion: Taken together, our results support the newly emerging evidence that the targeted activation of SHP-1, which is already recognized as a tumor suppressor in other tumor cells, may serve as a mechanism countering the aberrant pro-survival signals, thus opening new options for the treatment of LGLL. Disclosures Zambello: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Canine non-B, non-T NK lymphocytes have a potential antibody-dependent cellular cytotoxicity function against antibody-coated tumor cells

BackgroundThe antibody-dependent cellular cytotoxicity (ADCC) is a cell-mediated immune defense mechanism in which effector immune cells actively lyse antibody-coated target cells. The ADCC of tumor cells is employed in the treatment of various cancers overexpressing unique antigens, and only natural killer (NK) cells are known to be major effectors of antibody mediated ADCC activity. Canine NK cells are still defined as non-B, non-T large granular lymphocytes because of the lack of information regarding the NK cell-restricted specific marker in dogs, and it has never been demonstrated that canine NK cells have ADCC ability against tumor cells. In the present study, we investigated whether canine non-B, non-T NK cells have ADCC ability against target antibody-coated tumor cells, using cetuximab and trastuzumab, the only human antibodies reported binding to canine cancer cells.ResultsActivated canine non-B, non-T NK cells (CD3−CD21−CD5−TCRαβ−TCRγδ−) for 13~17 days ex vivo showed ADCC ability against trastuzumab- or cetuximab-coated target tumor cells expressing various levels of human epidermal growth factor receptor 2 (HER-2) and epidermal growth factor receptor (EGFR). Trastuzumab and cetuximab induced significant ADCC responses of canine NK cells even in CMT-U334 and CF41.Mg cells expressing low levels of HER-2 and/or EGFR, as well as in SKBR3 and DU145 cells overexpressing HER-2 and/or EGFR. The trastuzumab-mediated ADCC activity of NK cells was significantly enhanced by treatment with rcIL-21.ConclusionsThe results of this study suggest that canine non-B, non-T NK lymphocytes have a potential ADCC function and that combinational strategies of monoclonal antibodies with either cytokines, which activate NK cells in vivo, or adoptive transfer of NK cells may be a feasible method for amplifying the efficacy of immunotherapy against malignant cancers even with very low expression of target molecules in dogs.

Natural Killer Cells in Myeloid Malignancies: Immune Surveillance, NK Cell Dysfunction, and Pharmacological Opportunities to Bolster the Endogenous NK Cells.

Natural killer (NK) cells are large granular lymphocytes involved in our defense against certain virus-infected and malignant cells. In contrast to T cells, NK cells elicit rapid anti-tumor responses based on signals from activating and inhibitory cell surface receptors. They also lyse target cells via antibody-dependent cellular cytotoxicity, a critical mode of action of several therapeutic antibodies used to treat cancer. A body of evidence shows that NK cells can exhibit potent anti-tumor activity against chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and myelodysplastic syndromes (MDS). However, disease-associated mechanisms often restrain the proper functions of endogenous NK cells, leading to inadequate tumor control and risk for disease progression. Although allogeneic NK cells can prevent leukemia relapse in certain settings of stem cell transplantation, not all patients are eligible for this type of therapy. Moreover, remissions induced by adoptively infused NK cells are only transient and require subsequent therapy to maintain durable responses. Hence, new strategies are needed to trigger full and durable anti-leukemia responses by NK cells in patients with myeloid malignancies. To achieve this, we need to better understand the interplay between the malignant cells, their microenvironment, and the NK cells. This review focuses on mechanisms that are involved in suppressing NK cells in patients with myeloid leukemia and MDS, and means to restore their full anti-tumor potential. It also discusses novel molecular targets and approaches, such as bi- and tri-specific antibodies and immune checkpoint inhibitors, to redirect and/or unleash the NK cells against the leukemic cells.

Feline alimentary lymphoma: a guide to cytological and histopathological diagnosis for the general practitioner

Lymphoma is the most common neoplasm in domestic cats and alimentary lymphoma is now the most common anatomical site. This article aims to highlight and explain the shift from FeLV-positive multicentric to FeLV-negative alimentary lymphomas, and to explain the cytological and histopathological methods of diagnosing and classifying alimentary lymphoma, in a way that is useful to the general practitioner. Alimentary lymphoma (AL) in felines can be histologically graded into low-, intermediate- and high-grade lymphoma. Low-grade AL has a fair prognosis, while intermediate- and high-grade have poorer prognoses. Transmural invasion by neoplastic lymphocytes also carries a poor prognosis. Large granular lymphocyte lymphoma is a histological sub-classification determined by cell morphology, can be of any grade, and the prognosis is very poor.

Ultrasonographic honeycomb pattern of the spleen in cats: correlation with pathological diagnosis in 33 cases.

The aim of this study was to evaluate the relationship between the ultrasonographic (US) diffuse honeycomb pattern (HCP) of the spleen and a pathological diagnosis in cats, and to assess the influence of transducer type on HCP visualisation. Abdominal ultrasounds of cats with an HCP were reviewed and splenic size, shape, margination, other parenchymal alterations and splenic hilar lymphadenopathy were recorded. When applicable, images acquired with high-frequency linear and curvilinear transducers were compared to determine if an HCP was more frequently demonstrated on high-resolution images. A retrospective review of the corresponding splenic cytohistopathological samples was also performed. Thirty-three cats met the inclusion criteria. Five cases were diagnosed by histology and 28 by cytology, confirmed by PCR for antigen receptor rearrangements (PARR) in uncertain cases. There were 15 cases of lymphoid hyperplasia, eight cases of lymphoma (four B cell, three T cell and one large granular lymphocytes), six cats with splenitis, three with extramedullary haematopoiesis and one with histiocytic sarcoma. The prevalence of lymphoma in cats with an HCP of the spleen was 24%. Splenomegaly was the most frequent US feature associated with an HCP and was observed in all lymphoma cases. In the images obtained from both high-frequency linear and micro-convex transducers the visualisation of an HCP was enabled in all cases (24/24) and in 62.5% (15/24), respectively. Based on our findings, a US HCP of the spleen in cats can be associated with benign and malignant disorders and is infrequently associated with lymphoma in comparison with dogs. Cytological or histological examination, possibly supplemented by PARR, should always be performed for diagnostic support. Use of high-frequency linear transducers is recommended to properly recognise an HCP or subtle changes in splenic parenchyma.

Availability of NK cell expansion agent combined with recombinant IL‑2 and IL‑15 stimulation on the expansion and high‑purity of NK cells in patients with immune‑related pancytopenia invitro.

Natural killer (NK) cells are a group of large granular lymphocytes that play an important regulatory role in innate immunity and adaptive immunity. Immune‑related pancytopenia (IRP) is a type of pancytopenia resulting from bone marrow hematopoietic cells that were destroyed or suppressed by auto‑antibodies. The specific mechanism of IRP is not clear. In the present study, it was identified that the percentage of NK cells in peripheral blood lymphocytes was decreased in patients with IRP. Subsequently, high purity NK cells were extracted from 6 untreated patients with IRP using the immunomagnetic beads sorting, magnetic‑activated cell‑sorting method, which were then cultured then in RPMI‑1640 medium containing 20%FBS. NK cell expansion agents, with or without recombinant interleukin (IL)‑15, were used to amplify high‑purity NK cells on the basic of recombinant IL‑2. Expression of the activated receptors NKG2‑D typeII integral membrane protein (NKG2D) and natural killer cell p46‑related protein (NKp46), and the inhibitory receptors CD158a and NKG2‑A/NKG2‑B typeII integral membrane protein (NKG2A), in CD56+ NK cells were detected by flow cytometry before and after cell culture. It was observed that treatment with an NK cell expansion agent combined with the stimulation of recombinant IL‑2 and recombinant IL‑15 could increase the number whilst maintaining the purity of NK cells. There were no significant changes in the expression of NKG2D, NKp46, NKG2A and CD158a in patients with IRP before and after NK cell culture. This new amplification method lays a foundation for clinical NK cell immunotherapy and anti‑tumor applications.

Identification of a miR-146b-Fas ligand axis in the development of neutropenia in T large granular lymphocyte leukemia.

Tlarge granular lymphocyte leukemia (T-LGLL) is characterized by the expansion of several large granular lymphocyte clones, among which a subset of large granular lymphocytes showing constitutively activated STAT3, a specific CD8+/CD4− phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNAs has not been evaluated in T-LGLL patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. The expression level of 756 mature miRNA was assessed on purified T large granular lymphocytes (T-LGLs) by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-LGLs. Remarkably, CD8 T-LGLs exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of Fas ligand (FasL), that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-LGLs occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-LGLs lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-LGLL.

THE DISTRIBUTION OF MOLECULES OF THE INTERCELLULAR ADHESION ON BLOOD LEUKOCYTES DEPENDING ON THEIR MORPHOLOGY

Physiological understanding of the processes of migration of immunocompetent cells, as well as the conditions under which migration can be activated or slowed down, is especially important for the interpretation of the reserves of stability and preservation of homeostasis. Migration of leukocytes through the endothelium of the microcirculation is regulated by adhesion molecules. The aim of the study was to study the distribution of intercellular adhesion molecules on leukocytes depending on their morphology. An examination of 50 healthy individuals aged 20 to 60 years with no history of chronic disease was carried out. Venous blood for the study was taken in the morning on «empty» stomach. On a flow cytometer FC-500 company Beckman Coulter (USA) determined the number of neutrophils, monocytes and lymphocytes of venous blood containing molecule L-selectin (CD62L), LFA-1 (CD11a), LFA-3 (CD58), ICAM-1 (CD54), PECAM-1 (CD31), while differentiated them based on morphology. The results is shown that more than 50% of neutrophils and 42% of monocytes are ready to enter the slip phase, while the number of lymphocytes entering this phase is relatively small - 11%. In the phase of strong adhesion and transmigration more than 90% of neutrophils and monocytes were ready to enter, while the number of lymphocytes in blood did not exceed 13%. Correlation analysis showed that in the absence of antigenic stimulation the greatest readiness for the implementation of the slip phase, strong adhesion and the transmigration was observed in neutrophils with 5-segments in the nucleus and polymorphonuclear monocytes. Willingness to transmigrate lymphocytes was relatively small and concerned mainly of medium size lymphocytes and large granular lymphocytes.

PB1871 ONCOGENIC MIRNAS CONTROL PROLIFERATION AND APOPTOSIS IN TCRαβ+ CD8+ LARGE GRANULAR LYMPHOCYTE (LGL) LEUKEMIA CELLS

Background:TCRαβ+ CD8+ large granular lymphocyte (LGL) leukemia is a rare heterogeneous hematological disorder that has a chronic disease course and mostly affects the elderly. LGL leukemia is estimated to account for 2‐5% of all chronic lymphoproliferative disorders and most commonly TCRαβ+ CD8+ T cells are affected. Although the precise disease etiology is largely unknown and disease course is heterogeneous, it is generally accepted that LGL cells arise through chronic antigenic stimulation and inflammation and thrive as a consequence of dysregulation of proliferation and apoptosis pathways. One level of dysregulation of these pathways could involve miRNAs. miRNAs are a class of endogenous noncoding RNAs (19‐25 nt) that are involved in regulation of gene expression. miRNAs primarily act at the post‐transcriptional level and are involved in various cellular processes such as proliferation and apoptosis. Moreover, dysregulated expression of miRNAs that hold tumor suppressor or oncogenic capacity has been associated with the pathogenesis of human cancers and leukemias. Here, we explored the miRNAome of TCRαβ+ CD8+ LGL leukemic cells by performing small noncoding RNA sequencing of sorted TCRαβ+ CD8+ LGL cells (n = 7) and sorted age‐matched healthy control TCRαβ+ CD8+ TEMRA effector T cells (n = 6). Furthermore, we evaluated whether differentially expressed miRNAs could potentially drive the dysregulation of proliferation and apoptosis pathways in LGL leukemia.Aims:To determine the miRNAome of TCRαβ+ CD8+ LGL cells and to elucidate whether the aberrant expression of miRNAs drives the dysregulation of proliferation and apoptosis pathways in LGL cells.Methods:Purified LGL leukemia cells and healthy control cell subsets were sorted with FACS Aria III. Cells were sorted directly in Trizol reagent and RNA was isolated according to manufacturer's protocol. Libraries were prepared with cleantag small RNA library prep kit and SMART‐seq v4 ultra low input RNA kit for sequencing according to manufacturer's protocol.Results:Using next generation sequencing, we determined differential expression of a subset of miRNAs between LGL leukemia cells and healthy control T cell subsets. Principle component analysis of miRNA expression profiles clearly showed LGL leukemia cells clustering away from healthy control T cells, with more than 100 miRNAs significantly upregulated or downregulated between patient cells and control cells. In silico pathway analysis of top differentially expressed miRNAs indicated that these miRNAs could be involved in cell cycle regulation through repression of cyclin dependent kinases CDKs, the NF‐κB pathway and potentially drive apoptosis resistance through inhibition of the p53 and BCL2 pathways. Currently we are exploiting the RNAome by RNA sequencing to validate the involvement of these pathways in LGL leukemia. By integrating both the miRNA and RNA datasets the association of top candidates in these pathways will be further investigated.Summary/Conclusion:Collectively our data implicate miRNAs as potential drivers in the maintenance of established LGL leukemic cells. Likewise, in silico prediction tools imply that long term survival of these cells is a result of miRNAs that enhance proliferation and inhibit apoptosis.

PB1906 FROM ACADEMY TO REAL WORLD: THE GRAY AREA OF THE LARGE GRANULAR LYMPHOCYTES LEUKEMIA

Background:Large granular lymphocytes (LGLs) are defined as large white blood cells (WBC) that are morphologically characterized by an abundant cytoplasm with azurophilic granulation. LGLs belong to CD3+CD56− cytotoxic T cells (CT) or natural killer (NK) cells. The NK cells are roughly defined by a lack of the T cell marker CD3 and a presence of CD56 (or NCAM). LGL leukaemia represents a chronic lymphoproliferative disorder of CTLs, a malignancy that involves lymphocyte infiltration of multiple organs, including the BM, liver, and spleen.Aims:The aim of this retrospective study was to achieve information about LGL and the association between LGL and clinical characteristics.Methods:A retrospective study analyzing the global diagnosis of LGL leukaemia and factors influencing the disease evolution at the district hospital and included cases with the previous or recent diagnosis of LGL leukaemia between January 2015 and December 2018. Special emphasis was taken on the results of bone marrow examination, flow cytometry, immunophenotype, and results of molecular studies to assess for T‐cell receptor (TCR) gene rearrangements. A total of 26 patients were confirmed as having LGL leukaemia, and they fulfilled the WHO classification criteria. May‐Grünwald‐Giemsa–stained peripheral blood smears were examined for the presence of large granular lymphocytes. Bone marrow trephine biopsy and aspirate specimens (when was applicable) were obtained. Bone marrow cellularity and the pattern of lymphocyte infiltration were determined on core biopsy or aspirate clot specimens stained with H&E. Statistic The Fisher exact test was used to analyze differences between the T‐LGL and T‐LGL/other LPD groups. The median time from completion of last treatment with rituximab to the diagnosis of LGL disorder was 30 months.Results:LGL leukaemia or T‐LGL expansion were identified in the global group. Near total of the patient 21 (80.7%) had a direct history of a clonal B‐cell lymphocytosis, monoclonal B‐cell lymphocytosis (MBL), or clonal B‐cell disorder concomitantly with or shortly after the LGL diagnosis. The other predominant diagnosis was rheumatoid conditions in 9 patients (34.6%) (including Felty syndrome) and monoclonal gammopathy of unknown significance (MGUS) 7 patients (26.9%). The median age at the time of diagnosis was 68.5 years (range, 20‐86 years). B‐symptoms were reported by 4 (15.38%) patients, as well as Lymphadenopathy/splenomegaly was identified in 5 (19.23%). 4 individuals received treatment prior to the diagnosis of LGL, and the predominant treatment was methotrexate/rituximab. The favourite regimens in our daily practice were cyclosporin, rituximab or intermittent G‐CSF. The overall response rate at 3 months and complete response rate for the various treatments were as follows: steroids (11% and 3%), methotrexate (55% and 21%), rituximab (64% and 47%), cyclosporine (19% and 3%), respectively. All patients responded to rituximab after methotrexate treatment had failed. The mean number of treatments was 3.4 (range, 1‐5).Summary/Conclusion:Whether or not the T‐LGL and T‐LGL/other LPD groups are correlated with specific clinical‐biological features remains uncertain and is the subject of ongoing research. We don’t perform routinely in our clinical practice an investigation for the Constitutive activation of STAT3. In our series, we have not reported direct LGL‐related deaths.

PF367 MOLECULAR EVIDENCE THAT ATOVAQUONE IS A NEW STAT3‐INHIBITOR COUNTERBALANCING LEUKEMIC CELL PRO‐SURVIVAL IN LARGE GRANULAR LYMPHOCYTE LEUKEMIA

Background:Large Granular Lymphocyte Leukemia (LGLL) is a rare heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. The hallmark of leukemic LGLs is a constitutive activation of STAT3 leading to enhanced triggering of many pro‐survival genes. Moreover, 30–40% of patients carry mutations in STAT3 gene bringing to the constitutive activation of the malignant clone.No specific treatment is now available for the patients affected by LGLL and the in‐use therapy is based on low doses of immunosuppressive drugs, i.e. Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporin A (CyA). Anyway, these therapies show limited efficacy, induce many side effects with the Overall Response Rate (ORR) being around 50% and a high rate of relapse in the responding patients.The JAK/STAT signaling pathway, which is central to LGL leukemia pathogenesis, includes several players that provide the basis for new targeted treatments. This offers the rationale for using Atovaquone (ATQ, Mepron™), a recently FDA‐approved anti‐microbial agent characterized by a low adverse effect profile that has been recently demonstrated (Xiang et al., 2016) to work as a powerful STAT3 inhibitor with anti‐cancer efficacy in both animal models and human cell lines.Aims:Our study focuses on in vitro tests to evaluate the effects of ATQ as inhibitor of STAT3 phosphorylation and of LGLs survival, with the goal to develop a targeted approach to LGLL, likely devoid of adverse effects.Methods:Patients’ Peripheral Blood Mononuclear Cells (PBMC) were purified to perform cultures to test serial drug concentrations. Using in vitro cells culture, the inhibition of STAT3 protein phosphorylation and the expression of its target proteins, MCL1 and BCL2, were evaluated by Western Blot assay (WB). In addition, the cytotoxicity and the apoptotic selectivity of ATQ was investigated by Annexin V test; a staining with anti‐CD57, CD56 or CD16 was used to identify leukemic LGLs from PBMCs. Finally, by RT‐PCR, STAT3 activity was analyzed along its transcriptional levels and target genes.Results:Through in vitro experiments we initially tested the best concentration inhibiting cell viability and STAT3 activation on patients’ PBMCs. We observed that ATQ was efficiently able to inhibit STAT3 phosphorylation at a concentration of 25 μM. The results of the apoptosis analysis showed that ATQ has a dose and time dependent cytotoxic effect. The IC50 was reached at a dose of 25 μM after 24 h of culture. ATQ demonstrated a selective cytotoxicity against leukemic LGLs (reaching 3.9 ± 0.7 fold increase of apoptosis vs untreated condition after 48 h of culture) sparing non‐leukemic cells (ATQ treated apoptosis vs untreated in non‐LGL PBMC = 1.18 ± 0.3 fold change). By comparing the in vitro cell effect of ATQ with that of the commonly used therapies, ATQ showed a cytotoxic effect consistent with CTX and CyA, but higher than MTX (p < 0.01). Concerning anti‐apoptotic factors under the STAT3 control, after 6 h ATQ treatment the expression of MCL1 and BCL2 showed only minimal changes, thereby suggesting that different mechanisms of action take place.Summary/Conclusion:These in vitro data indicate that ATQ has a promising action on the inhibition of the JAK/STAT pro‐survival pathway in leukemic LGLs, resulting in a selective cytotoxic effect against LGLs through MCL1‐ and BCL2‐independent mechanisms.

PS1261 CLINICAL CHARACTERISTICS AND PROGNOSTIC FACTORS OF 33 PATIENTS WITH SEZARY SYNDROME AT MOFFITT CANCER CENTER: A SINGLE‐INSTITUTION COHORT

Background:Sezary syndrome (SS) is a rare aggressive leukemia variant of cutaneous T‐cell lymphoma (CTCL) characterized by erythroderma and Sezary cells in peripheral blood and lymphoadenopathy. It has a poor prognosis with reported median overall survival ranging from 2–4 years. Limited studies exist to describe the SS cohorts in modern era of biological therapy.Aims:To analyze clinical and laboratory characteristics such as age, lesion characteristics, hematological parameters to determine their impact on survival in patients with SS.Methods:A retrospective chart review was conducted to identify patients with SS seen at Moffitt Cancer Center, Tampa, FL from 2016 to 2018. Kaplan Meier estimates were calculated for overall survival. Log rank test and cox proportional hazard model were used to evaluate categorical and continuous factors associated with overall survival respectively.Results:In total, 33 patients were identified with a clinicopathologic diagnosis of SS. 76% were white, 21% African American and 3% Hispanic; 67% were male and 33% female (2:1 ratio). Mean age was 67.3 years (range: 43–89). 49% had primary SS and 51% had a previous diagnosis of mycosis fungoides (MF). The average time to progression to SS was 6.4 years. The majority presented with advanced stage (III/IV). Seven patients were known to be deceased with a median time to death of 2.4 years. Other 26 patients had a median follow up time of 3.8 years. OS was 68.5% and 42.8% at 1 and 5 years, respectively. Prior diagnosis of MF indicated a poor prognosis (p = 0.005). Age, race, the presence of erythroderma, absolute Sezary cell count, LDH level, CD4/CD8 ratio, ferritin level, and lymph node involvement at the time of diagnosis did not correlate with OS. There was a trend to favorable survival in the group of patients <60 years old at diagnosis and with the presence of large granular lymphocytes (LGL) proliferations.Sezary cells of the immunophenotype of CD3+CD4+CD7‐CD26‐ were present in 78% patients. TCR gene was clonally rearranged in 30/31 tested patients. 28 (90.3%) was TCRβ+TCRγ+; 1 TCRβ+TCRγ‐; 1 TCRβ+TCRγ‐. Median Sezary cell count is 1.81x109 cells/L.Foundation Medicine Hematology gene panel was done in selected patients, which tested 437 genes that are known to be somatically altered in human hematologic malignancies, sarcomas and pediatric cancers and that unambiguous driver of oncogenesis based on current knowledge. 21 mutations were detected in 4 patients: CCND3, MKI67, CDKN2A, EP300, MAPK1, BCOR, RHOA in cell cycle regulation and cell proliferation; TET2, ASXL1, DNMT in DNA methylation; STED2, ARID2 in DNA repair; CBL, DTX1, TNFAIP3 in protein Ubiquination; TP63 (P53 mediated apoptosis), TNFAIP3 (NFkB signaling apoptosis), MPAK1 in Cell Apoptosis; CD 58, BTK and CIITA in immune surveillance; STAT3 in JAK/STAT pathway; TP63 and DTX1 in Notch signaling pathway. GRIN2A, involved in NMDA receptor signaling, was detected with unclear role in oncogenesis. GRIN2A and CDKN2A were detected in 2/4 patients. TET2, DNMT3A, TNFAIP3 were previously reported in CTCL. CCND3, CDKN2A, MAP1, ASXL1, EP300, STAT3 were reported in other T‐cell malignancies.Summary/Conclusion:Our study confirmed overall poor prognosis of SS with OS of 68.5% and 42.8% at 1 and 5 years despite advances in therapy. The preexisting diagnosis of MF was a poor prognostic factor. Younger age at diagnosis and the presence of LGL proliferations are potentially favorable prognostic factors. However, due to limited sample sizes, the results need to be interpreted with caution and verified with a larger cohort.image

Scalable optimal Bayesian classification of single-cell trajectories under regulatory model uncertainty

BackgroundSingle-cell gene expression measurements offer opportunities in deriving mechanistic understanding of complex diseases, including cancer. However, due to the complex regulatory machinery of the cell, gene regulatory network (GRN) model inference based on such data still manifests significant uncertainty.ResultsThe goal of this paper is to develop optimal classification of single-cell trajectories accounting for potential model uncertainty. Partially-observed Boolean dynamical systems (POBDS) are used for modeling gene regulatory networks observed through noisy gene-expression data. We derive the exact optimal Bayesian classifier (OBC) for binary classification of single-cell trajectories. The application of the OBC becomes impractical for large GRNs, due to computational and memory requirements. To address this, we introduce a particle-based single-cell classification method that is highly scalable for large GRNs with much lower complexity than the optimal solution.ConclusionThe performance of the proposed particle-based method is demonstrated through numerical experiments using a POBDS model of the well-known T-cell large granular lymphocyte (T-LGL) leukemia network with noisy time-series gene-expression data.

PF479 CLINICAL AND BIOLOGICAL PREDICTORS OF OUTCOME IN LARGE GRANULAR LYMPHOCYTE LEUKEMIA: A SINGLE CENTER EXPERIENCE OF 205 PATIENTS

Background:Large Granular Lymphocyte Leukemia (LGLL) is a rare chronic lymphoproliferative disorder characterized by the expansion of LGLs. Among LGLL, WHO 2016 classification recognizes a CD3+ T‐LGLL and a CD3‐ Chronic Lymphoproliferative disorder of Natural Killer cells (CLPD‐NK), accounting for 85% and 15% of cases respectively. Moreover, according to the T cell receptor rearrangement, the Tαβ and Tγδ subsets can be identified. From a clinical point of view, LGLL is a heterogeneous disease with some patients being asymptomatic and other developing cytopenias, mostly neutropenia. The aetiology is still unknown but a constitutive activation of JAK/STAT pathway is involved in LGL proliferation, supported by the evidence of somatic STAT3 and STAT5b mutations in approximately 40% of patients. We previously demonstrated that in T‐LGLL STAT3 mutations are associated to symptomatic disease.Aims:The aim of this study is to analyse clinical and biological features of a large cohort of LGLL patients to identify prognostic markers affecting patients’ outcome.Methods:From 1992 to 2018, clinical and biological data of 205 patients affected by LGLL have been collected. STAT3 exon 21 mutation analysis and STAT5b exon 16–18 mutation analysis was performed by Sanger sequencing.Results:In our cohort, median age at diagnosis was 58 years. By phenotype, 129/205 (62.9%) patients were affected by Tαβ LGLL, 23/205 (11.2%) by Tγδ LGLL, and 36/205 (17.6%) by CLPD‐NK. Moreover, 17 patients (8.3%) were characterized by a bi‐phenotypical variant, identified by a concomitant T/NK or Tαβ/Tγδ clone. According to CD4 and CD8 expression, Tαβ LGLL was further classified in CD4‐/CD8+ LGLL (CD8+ T‐LGLL, 84/205, 41%) and CD4+/CD8dim/neg LGLL (CD4+ T‐LGLL, 45/205, 21.9%).Neutropenia (Absolute Neutrophil's Count, ANC<1,500/mm3) was the most relevant feature (78/205, 38%), severe neutropenia (ANC<500/mm3) being present in 20.5% of patients. Anemia (Hb<120 g/L, 27/205, 13.2%), severe anemia (Hb<90 g/L, 20/205, 9.8%) and thrombocytopenia (PLTs <100.000/mm3, 12/205, 4.8%) were less recurring. 44 patients (21.5%) were affected by concomitant autoimmune/inflammatory disease, mostly autoimmune hemolytic anemia and rheumatoid arthritis.DNA samples of 166 and 152 patients were available for STAT3 and STAT5b mutations analysis, respectively. STAT3 mutations were detected in 47 patients (28.3%), with higher frequency in CD8+ Tαβ LGLL towards Tγδ LGLL (60% vs 25%, p = 0.0232) and CLPD‐NK (60% vs 5.8%, p < 0.0001). At variance, STAT5b mutations were found in 15 patients (9.8%), mostly in CD4+ Tαβ LGLL (12/35 cases available, 34.3%) and in 3 cases were Tγδ LGLL. Interestingly, STAT3 and STAT5b mutations were mutually exclusive in CD8+ and CD4+ Tαβ LGLL.With a median follow up of 8 years, median overall survival (OS) of our cohort was not reached. Major features associated to reduced OS were ANC<500/mm3 (267 months vs not reached, p = 0.0297), severe anemia (144 months vs not reached, p < 0.0001), treatment requirement (214 vs not reached, p = 0.0011) and presence of STAT3 mutations (267 months vs not reached, p = 0.0113) (Figure 1, Panel A to D). No significant survival differences were found between STAT5b mutated and wild‐type patients (not reached vs 267 months, p = 0.3514).Summary/Conclusion:Our results confirmed the remarkable biological and clinical heterogeneity of LGLL. We identified clinical and biological features associated to reduced OS in LGLL patients and, for the first time, we demonstrated the dismal impact of STAT3 mutations in patients’ survival, suggesting that this marker should be regarded as a potential target of therapy.image

PF362 A FIRST HIGH-DEFINITION LANDSCAPE OF SOMATIC MUTATIONS IN CHRONIC LYMPHOPROLIFERATIVE DISORDER OF NK CELLS

Background: Chronic Lymphoproliferative Disorder of NK cells (CLPD-NK) is included in the 2017 WHO classification of mature T- and NK-cell neoplasms as provisional entity. Similarly to T-cell large granular lymphocytes leukemia (T-LGLL), CLPD-NK is characterized by an indolent clinical course and is clearly distinguished from the aggressive NK cell leukemia (ANKL), this latter behaving as an acute severe disease. CLPD-NK and T-LGLL patients mostly present cytopenias when symptomatic. Accumulating evidence on the genomic landscape of T-LGLL pointed to a key role of somatic mutations within JAK-STAT (mostly in STAT3 and STAT5B genes) and RAS-MAPK pathways (Coppe et al. Leukemia 2017) in this form. Recent data highlighted more heterogeneous molecular features in ANKL, involving JAK2 and STAT3 mutations, lesions of epigenetic modifiers, tumour suppressors and a putative role of Epstein Barr Virus (Dufva et al. Nat Comm. 2018). Aims: In our cohort of 57 CLPD-NK patients the frequency of STAT mutations has been found significantly lower (<10% of all cases) compared to literature data (Barilà et al. Blood Cancer J. 2018). The genomic landscape of CLPD-NK patients negative for STAT mutations was thus investigated to better elucidate the molecular basis of the disease. Methods: Ten CLPD-NK patients were recruited according to negativity for STAT mutations and typical immunophenotype (CD16+/CD57±/CD56±). WES profiling (Agilent SureSelect 60 Mbp, Illumina sequencing, paired end reads) of immunomagnetically purified leukemic clone (>95% purity) and of normal granulocytes, as control, was obtained for each patient. Sequencing data were analyzed by an in-house bioinformatic pipeline encompassing data cleaning, read mapping against the reference genome and somatic variant calling with complementary approaches (MuTect, MuTect2 and Strelka2). Variants were next filtered according to population allele frequency (gnomAD <5%), clinical significance (COSMIC and ClinVar) and predicted functional impact on proteins (SnpEff, MetaSVM and MetaLR). Results: High sequencing depth was obtained with a mean coverage of 150 reads. From over 6,000 somatic variants detected in the cohort, 869 SNPs and 14 indels, rare and of high predicted impact were prioritized. Number of variants (88 per patient in average) and mutation spectra were very homogeneous in the cohort. Of note, the high discovery power of our settings disclosed an extensive subclonality of somatic variants, with over 78% with an allele variant frequency (VAF) lower than 5% in the leukemic clone (Figure 1A). Nevertheless, 20 deleterious and high VAF somatic variants occurred in each patient in average (Figure 1B). Very low recurrence of mutated genes in CLPD-NK patients was found. Pathway-derived mutation network analysis (Lovisa et al. Haematologica 2019) revealed functional connections among genes mutated in different patients. A major involvement of JAK-STAT pathway is likely to be ruled out, but all cases presented deleterious high VAF mutations of genes and pathways potentially impacting on cell survival, proliferation and chromatin-remodelling. In addition, somatic lesions in proteins involved in innate immunity and NK-cells activation seem particularly interesting.Summary/Conclusion: Ongoing comparison of genes and pathways mutated in CLPD-NK with available data for T-LGLL and ANKL is providing new clues on biological similarities and differences of these clinical entities. This study contributes to a better definition of the molecular mechanisms accounting for CLPD-NK, thus opening new areas of investigation.

PB1962 THE HIGH INCIDENCE OF MICROCYTOSIS IN THE CONTEXT OF MONOCLONAL PERIPHERAL BLOOD LYMPHOCYTOSIS IN THE LEUKEMIC PHASE OF LYMPHOPROLIFERATIVE DISORDERS

Background:Microcytosis is an established finding in chronic or inflammatory disease. Within this context, it is often described in lymphoid lineage hematologic malignancies, especially Hodgkin's lymphoma. Chronicity or a pro‐inflammatory state, however, are not the only predictors of a microcytic hemogram, with some chronic lymphoproliferative disorders (LPD) ‐ such as chronic lymphocytic leukemia (CLL) – rarely presenting with microcytosis, while the leukemic phase of other LPDs often presents with microcytosis.Aims:To clarify the relationship between monoclonal lymphocytosis in LPD and microcytosis.Methods:We analyzed all samples from adult patients (pts) with lymphocytosis (absolute lymphocyte count (ALC) &gt;4.0 G/L), with analysis of clonality by flow cytometry, over a 4‐year period. A Control group of 50 pts with a LPD but without lymphocytosis and without clonality was selected.Results:We obtained 412 samples (51.4% male); 22.6% of patients had no hematological disease and a reactive polyclonal lymphocytosis (“Reactive”); 8.5% had a known hematologic disease, but polyclonal reactive lymphocytes (“Polyclonal”); and 68.9% had a pathologic monoclonal lymphocytosis (“Monoclonal”). Monoclonal pts had a CLL‐like phenotype in 69.7% of cases, marginal zone (MZL) in 14.8%, and large granular lymphocyte leukemia in 3.2%; follicular (FL), mantle cell (MCL), lymphoplasmacytic (WM), Sézary's, NK cell, and T‐prolymphocytic leukemia phenotypes accounted for &lt;3% of cases, each; the remainder were non‐Hodgkin's Lymphomas, NOS. ALC ranged from 4.0‐141.1 G/L, with a mean of 16.4 ± 23.6 and a median of 6.8. The mean Reactive ALC was 5.0 ± 1.4 G/L, 5.2 ± 1.5 for Polyclonal and 21.4 ± 26.9 for Monoclonal pts (p &lt; 0.001), with 14.4 ± 19.4 for non‐CLL pts (p &lt; 0.001). Controls had an ALC of 1.8 ± 0.9 G/L.MCV ranged from 64.3‐111.1 fL, with a mean of 90.6 ± 6.1 and a median of 90.9; the Control median was 90.2 (74.1‐104.7 fL) with a mean of 90.5 ± 6.4 (p = NS); 91.0% of samples were normo‐, 5.8% macro‐ and 3.2% microcytic (p = NS vs Control). The mean Reactive MCV was 88.7 ± 4.7, 88.8 ± 6.3 for Polyclonal and 91.5 ± 6.4 for Monoclonal pts (p &lt; 0.001), with microcytosis in 1.1%, 5.7% and 3.5% of pts, respectively (p = 0.044). Within B Cell lineage Monoclonal pts, the mean MCV was 92.3 ± 5.5 fL in CLL (p = 0.04 vs Control), 89.1 ± 7.9 in the indolent lymphomas (MZL, FL, WM; p = NS vs Control) and 84.5 ± 9.2 in MCL (p = 0.02 vs Control) (p &lt; 0.001), with microcytosis in 0.5%, 7.8% and 50% of pts, respectively (p &lt; 0.001).A posteriori longitudinal analysis of the MCV in microcytic pts during and after chemotherapy documented a correlation between the decrease in lymphocytosis and an increase in MCV, with improvement/resolution of microcytosis.Summary/Conclusion:We observed that reactive polyclonal lymphocytosis in non‐hematologic patients and in malignant hematology pts associated with identical average MCVs, overlapping with the mean MCV of a control population without lymphocytosis. Similarly, monoclonal lymphocytosis in the context of the indolent B cell lymphomas (MZL, FL and WM) did not significantly affect normocytosis. On the other hand, CLL tended to associate with significantly higher MCVs than controls or reactive polyclonal lymphocytoses.In contrast, a monoclonal lymphocytosis in the context of a leukemic phase of MCL associated significantly with a microcytic phenotype ‐ remarkably, we found that microcytosis was 100x more frequent in MCL (50% of cases) than CLL (0.5%).The effective treatment of the underlying LPD, and the resolution of clonal lymphocytosis, associated with a normalization of the MCV.

Proportion of Cytotoxic Peripheral Blood Natural Killer Cells and T-Cell Large Granular Lymphocytes in Recurrent Miscarriage and Repeated Implantation Failure: Case-Control Study and Meta-analysis.

We aimed to compare the proportion of peripheral blood natural killer (NK) cells (CD3-CD56+) and T-cell large granular lymphocytes (CD8+CD57+) during preconception in a homogenous group of women with unexplained well-defined recurrent miscarriage (RM) and repeated implantation failure (RIF) vs healthy controls in relation to pregnancy outcomes. This case-control study followed by a literature review and meta-analysis was conducted in three university hospitals. Patients and controls were consecutively recruited from December 2015 to October 2017. In total, 115 women were included in the study: 54 with RM, 41 with RIF and 20 healthy controls with ≥ 2 term births. Percentages of CD3-CD56+ and CD8+CD57+ cells and sub-populations of CD3-CD56+ cells did not differ between cases and controls. The results for women with subsequent miscarriage did not differ from those with live births. The meta-analysis of the literature showed higher NK-cell proportions in RM [mean difference 3.47 (95% CI 2.94-4.00); p < 0.001] and RIF [mean difference 1.64 (95% CI 0.82-2.45); p < 0.001] than controls. However, the heterogeneity between the different studies was high. The proportion of peripheral blood CD3-CD56+ and CD8+CD57+ cells in the preconception period does not reflect the risk of implantation failure or miscarriage and should not be recommended indicators for the management of RM and RIF. Further prospective large studies are needed to develop a reliable peripheral blood marker of immune deregulation.

Subclonal STAT3 mutations solidify clonal dominance

AbstractT large granular lymphocyte leukemia (T-LGLL) is a clonal lymphoproliferative disorder that can arise in the context of pathologic or physiologic cytotoxic T-cell (CTL) responses. STAT3 mutations are often absent in typical T-LGLL, suggesting that in a significant fraction of patients, antigen-driven expansion alone can maintain LGL clone persistence. We set out to determine the relationship between activating STAT3 hits and CTL clonal selection at presentation and in response to therapy. Thus, a group of patients with T-LGLL were serially subjected to deep next-generation sequencing (NGS) of the T-cell receptor (TCR) Vβ complementarity-determining region 3 (CDR3) and STAT3 to recapitulate clonal hierarchy and dynamics. The results of this complex analysis demonstrate that STAT3 mutations produce either a sweeping or linear subclone within a monoclonal CTL population either early or during the course of disease. Therapy can extinguish a LGL clone, silence it, or adapt mechanisms to escape elimination. LGL clones can persist on elimination of STAT3 subclones, and alternate STAT3-negative CTL clones can replace therapy-sensitive CTL clones. LGL clones can evolve and are fueled by a nonextinguished antigenic drive. STAT3 mutations can accelerate this process or render CTL clones semiautonomous and not reliant on physiologic stimulation.