Large Granular Lymphocyte Leukemia Research Articles

Clustering Adult ACUTE Lymphoblastic Leukemia (ALL) Philadelphia Negative (Ph-) By Whole Exome Sequencing (WES) Analysis

Introduction: Adult ALL represents a biologically and clinically heterogeneous group. Incidence and cure rates differ among children and adults. In adults, ALL is less common and generally carries a worse prognosis with shorter long-term survival probability. Although the remarkable progress made in the treatment of ALL in children and, with less efficacy, in adults, several ALL subtypes continue to have a poor prognosis.Aims: focus our attention on adult Ph-negative ALL pts using whole exome experiments to discover novel insights into the mechanisms involved in leukemogenesis and to develop genetic models that accurately define novel ALL subtypes based on the genomic profile of individual patients (pts).Patients and Methods: we performed the WES analysis of 72 samples of B-cell precursor ALL acute lymphoblastic leukemia (B-ALL) cases using the Illumina Hiseq2000 platform. All were adult patients (18-79 years) and were negative for Philadelphia chromosome (BCR-ABL) translocation and negative for the recurrent known molecular rearrangements (E2A-PBX, TEL, AML1-MLL-AF4). Peripheral blood and/or bone marrow samples were collected from adult B-ALL at the time of diagnosis and/or at the time of relapse. Matched samples of primary tumour (peripheral blood or bone marrow) and germline DNA from buccal swab or peripheral blood at the remission time were analyzed.MuTect and GATK tools to call mutations (Single Nucleotide Variants=SNVs and/or INDELs) were used and we selected variants with a minor allele frequency (MAF) lower than 0.05 and filtered using dbSNP142.Results: The WES analysis of the 41 Ph negative cases identified 735 point mutations and 25 mutations that occur in splicing sites in 651 genes. The average number of somatic coding mutations was 17 per case (range 1-47). 38 genes were recurrently mutated with 11 genes mutated in at least 3 cases: PAX5, PRDM12, JAK2, TTN, TP53, PTPN11, PKHD1L1, CUL3, PIEZO2, TACC2, RBBP6. The first two genes present more point mutations, in 5 pts and in 4 pts respectively. Some mutations in genes like PAX5, JAK2, TP53, PTPN11 were deeply described in acute lymphoblastic leukemic; PKHD1L1 was described mutated in one case of T-cell large granular lymphocyte leukemia; PRDM12 disruption was described in an aggressive CML case;TACC2 expression in infant ALL was described as predictor of outcome andtranscription factor and RBBP6 expression was differentially expressed in leukemic cells that overexpressed Gfi-1B gene. The alterations in the remaining genes were not previously described in ALL and/or leukemia.Using KEGG database we mapped the 651 mutated genes to detect the mostly represented pathways. The following resulted significantly enriched (p=0.0004 to p=0.006): Jak-STAT signaling pathway (11 genes), Cell Cycle (13), Dilated Cardiomyophaty (9), Hypertrophic cardiomyophaty (8), Axon Guidance (9), Calcium Signaling pathways (10), Huntington's disease (10), Wnt signaling pathway (9), Metabolic pathways (30), Pacreat Secretion (7). Preliminar analysis lead considering both SNVs and INDELs, detected totally 956 gene variations. Again the pathways mainly significantly (p=8.05e-05 to p=0.0076) affected are the Jak-STAT signaling pathway (14) and the Cell Cycle (13). Also Huntington's disease (14), Dilated Cardiomyophaty (10), Hypertrophic cardiomyophaty (9), Wnt signaling pathway (12), Metabolic pathways (40 genes), Calcium Signaling pathways (12), Metabolic pathways (40), TGF-Beta signaling pathway (8), Pacreat Secretion (8) were alterated.Prediction of protein interactions, using STRING database, generated a network with the genes mutated in more than 5 patients. Then, the nodes were clustered with K-means identifying 4 groups that contain several of our analysis variations (Fig.1).Conclusions: Point mutations are the prevalent mechanism identified in our pts cohort (75.5%). INDELs are less represented (21.5%). Altogether the identified mutations may help cluster Ph- ALL pts. Analysis of SNVs confirmed mutations in important genes known to be involved in leukemogenesis. Relevant alterations affect crucial pathways as cell cycle and Jak-STAT signaling which may be effectively targeted by currently available JAK inhibitors. Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:AMGEN: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; ROCHE: Consultancy; MSD: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau.

Management of T-Cell Large Granular Lymphocyte Leukemia with Methotrexate or Cyclophosphamide

Background. T-Cell Large Granular Lymphocyte Leukemia (T-LGL Leukemia) is an extremely rare lymphoproliferative disorder which can originate from either mature T cells (CD3+) or natural killer cells (CD3-). T-LGL leukemia is a slowly progressing disease, rarely presenting with an aggressive course and in general patients have prolonged survival. Treatment is commonly monotherapy with methotrexate (with or without prednisone), cyclosporine, or cyclophosphamide. Herein we review our experience with the management of T-LGL in the last 15 years.Patients and Methods. We conducted a retrospective cohort study of all patients diagnosed with T-LGL at our center between 2000 and 2015. Patients were included if they had a positive study for T-Cell Receptor gene rearrangement. Response was evaluated using the criteria of Loughran et al (Leukemia 2015). Data was analyzed using descriptive statistics.Results. We included 23 patients, (16 male). All of the patients were caucasian with the exception of two. The average age at diagnosis is 65.1 years. Patients were treated for symptomatic cytopenias or splenomegaly with transfusions and/or chemotherapy. Six patients did not need any treatment. Fourteen of the 23 patients were treated with oral methotrexate monotherapy. The methotrexate dose ranged from 5mg/week to 15mg/week orally. Seven patients had a complete response, 2 had a partial response, 3 patients failed treatment, 1 patient was not evaluable, and 1 was stable. Four patients were treated with methotrexate and prednisone. Two patients obtained a complete response, with 1 partial response, and 1 patient was not evaluable due to the short duration of the prescription. Nine patients were treated with oral cyclophosphamide at doses of 50mg or 100mg po daily. Four patients achieved a complete response, 2 patients achieved a partial response, while the other 3 patients did not respond. Only 2 patients were treated with oral cyclosporine, at 400mg/day and at 500mg/day. One patient failed treatment while the other is still on therapy. Two patients died while on the study at 22 and 42 months, both deaths were due to other illnesses not associated with T-LGL Leukemia. The median time since diagnosis for the remaining patients is 41 months.Conclusion. T-LGL is a rare lymphoproliferative disorder. Most patients responded to simple oral therapy with methotrexate or cyclophosphamide and patients generally have prolonged survivals. DisclosuresLazo-Langner:Bayer: Honoraria; Pfizer: Honoraria. Kovacs:Daiichi Sankyo Pharma: Research Funding; LEO Pharma: Honoraria; Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

Persistent Cytotoxic T Lymphocyte Expansions after Allogeneic Hematopoietic Stem Cell Transplantation: Kinetics, Clinical Impact and Absence of STAT3 Mutations

Thymic-independent peripheral expansion of CD8+ cells derived from the graft in the initial stage of post-HSCT immune recovery is a well-known physiological event. Nevertheless, the description of symptomatic LGL leukemias and aggressive malignant cases in this setting may generate uncertainty, mostly in those cases in which the cytotoxic T lymphocyte expansion CTLe persists beyond the early transplantation period. We aimed to assess the nature of CTLe in adults during the post-alloHSCT period in a series of 154 patients with a long term surveillance. We studied the longitudinal kinetics of those expansions, their relation to clinical events, and their phenotypic and molecular features, including recently reported CTL leukemia-STAT3 mutations. In our study, trying to adhere to the WHO annotation of T-LGL, we considered two definitions for a CTL expansion: an absolute increase (≥ 2000 x109/L), and a relative expansion (a CD8/CD4 ratio ≥ 1.5), persisting more than six months in both cases. Persistent relative CTLe cases are frequent (49%) and related with timoglobulin prophylaxis (p≤0.001), acute graft versus host disease (GVHD, p=0.02), reduced intensity conditioning (p=0.04) and fungal and viral infections in the early post-HSCT. No differences in the number of serious infectious events from day 180 was found. Absolute CTLe are scarce (9%), related with chronic GVHD and absence of relapses. TCR rearrangement was reported as clonal and oligoclonal in the majority of patients with CTLe. We studied in a cross sectional manner with an extended immunophenotypic panel 17 patients: 5 patients with an absolute CTLe and 12 cases with a relative CTLe. A similar cytotoxic T αβ-effector phenotype was observed in all cases, with slight differences in the expression of CD25, CD16 and 1a. One patient with a relative CTLe expressed CD56 intensely: his ratio normalized at day 730 and no immune-related events were recorded. DNA stored during the post-alloHSCT setting was available from 68/75 relative CTLe patients (14/14 absolute CTLe cases). All of them went through molecular TCR rearrangement and STAT3 exon 21 mutations determination. In the relative CTLe cohort, TCR rearrangement was described as clonal, oligoclonal or polyclonal in 77%, 16% and 7%, respectively. Regarding absolute CTLe patients, TCR rearrangement was described as clonal in all the patients (n=14) of this subset. To increase the sensibility of the Sanger PCR, it was performed on DNA from CD3+ sorted cells in 54 out of 68 cases. No STAT3 mutation could be found in the CD3+ sorted fraction of relative or absolute defined CTLe. Not using an absolute threshold would establish a diagnosis of a persistent CTL expansion in 49% of our cohort of allo-transplanted patients. Additional diagnostic tools, as an effector phenotype, the presence of a NK marker or a monoclonal TCR rearrangement would not reduce significantly that percentage: CD57 was invariably expressed in CTLe cases, and 80% of our patients with expansions showed a TCR monoclonal pattern. STAT3 mutations resulting in persistent proliferation of CTL clones are a frequent event in large granular lymphocytic leukemia, and those clones have also been described in autoimmunity-driven disorders as acquired aplastic anemia and hypocellular myelodysplastic syndromes. We establish in this study the absence of exon 21 STAT3 mutations in the persistent CTL expansions found in a large series of patients with a long-term post-alloHSCT surveillance. The absence of STAT3 mutations and the CD8/CD4 declining longitudinal kinetics in the late period, supports its benign nature, expressed clinically by the null detrimental impact of these expansions on post-transplant outcome and/or serious infectious events. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic Relevance of Integrated Genetic Profiling in Adult T-Cell Leukemia/Lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a distinct subtype of peripheral T-cell neoplasms associated with human T-cell leukemia virus type-1 retrovirus. ATL includes a heterogeneous group of patients in terms of pathological and clinical features as well as prognosis, suggesting the presence of underlying molecular pathogenesis that could explain such heterogeneity among patients. Recently, we performed an integrated molecular analysis of a large number of ATL cases and delineated a comprehensive registry of gene mutations and other genetic/epigenetic lesions in ATL. In this study, we investigated possible correlations between these genetic/epigenetic lesions and clinical/pathological phenotypes in a large set of ATL patients, with a special focus on the impact of mutations and copy number alterations (CNAs) on clinical outcome.We analyzed a total of 361 ATL samples, including acute (n = 192), lymphoma (n = 66), chronic (n = 89), and smoldering (n = 14) subtypes, for recurrent mutations and CNAs. Each subtype had characteristic genetic/epigenetic features, suggesting a distinct molecular pathogenesis therein. Aggressive (acute and lymphoma) subtypes were characterized by a higher number of mutations and CNAs including focal amplifications/deletions, hyperploid status, and CIMP phenotype, compared with indolent (chronic and smoldering) tumors. Two mutations (TP53 and IRF4) and eight focal deletions involving 1p13 (CD58), 6p21 (HLA-B), 9p21 (CDKN2A), 10p11 (CCDC7), 13q32 (GPR183), 16q23 (WWOX), 17p13 (TP53), and 19q13 (CEBPA), were more common in aggressive ATL than in indolent ATL. In contrast, showing a similar mutational distribution to those found in large granular lymphocytic leukemia, STAT3 mutations were characteristic of the indolent diseases. Gene set enrichment analysis of RNA-seq data showed a significant enrichment of MYC pathway and genes regulating cell cycle and DNA repair in upregulated genes in aggressive ATL.Next, we assessed the impact of mutations and CNVs on prognosis among 215 ATL cases, for which survival data were available. In the entire cohort, mutation in CCR4 and IRF4, focal amplification in 9p24 (CD274) and 14q32 (BCL11B), and focal deletion in 9p21 (CDKN2A) were found to be significant predictors of poor overall survival, after adjustment for disease subtype and age. Multivariate analysis revealed that disease subtype (aggressive vs. indolent) was the most significant predictor of clinical outcome in ATL. Subsequent multivariate analysis according to disease subtype showed that within the patients with aggressive ATL, older age (≥ 70 years), CCR4 mutations, and 9p24 amplification were independently associated with an adverse outcome. Based on the number of the risk factors they owned, patients with aggressive ATL were classified into three categories showing marked difference in 3-year overall survival (OS) (P < 0.001): those with no risk factors (OS, 32%), with one risk factor (18%), and with two or more (0%). Among the patients with indolent ATL, we found IRF4 and TP53 mutations, 9p24 amplification, and deletions in 9p21 and 10p11 were independently associated with reduced survival. Interestingly, these alterations, except for 9p24 amplification, were also identified as genes more frequent in aggressive ATL. More importantly, based on these risk factors, the patients with indolent ATL can be classified into two categories showing very different prognostic profiles: patients with no risk factors (OS, 89%) and those with one or more risk factors (21%) (P < 0.001, HR = 16.8, 95% CI:5.4-52.5), suggesting that patients with indolent ATL having a genetic feature of the aggressive subtypes might genetically and biologically represent a distinct subset, which should be better managed as having an aggressive disease. Among these poor prognostic factors, 9p24 amplification and CCR4 mutation are especially interesting, because these lesions might be plausible targets of available agents, including anti-PD1/PD-L1 and anti-CCR4 antibodies.In conclusion, based on the comprehensive genetic profiling, we demonstrated that the known subtypes of ATL can be further classified into genetically and biologically distinct subsets of tumors characterized by discrete sets of genetic lesions and substantially different prognosis. Our results suggest that molecular profiling can improve the prediction of prognosis in ATL patients and better guide therapy. DisclosuresTobinai:Gilead Sciences: Research Funding. Miyazaki:Shin-bio: Honoraria; Chugai: Honoraria, Research Funding; Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

FTY720 (Fingolimod) Targets the Sphingolipid Pathway and Induces Autophagy-Related Apoptosis in Human Natural Killer Large Granular Lymphocyte Leukemia

Introduction: Natural killer large granular lymphocytic leukemia (NK-LGL) and chronic lymphoproliferative disorder of NK cells (CLPD-NK) are rare disorders of cytotoxic CD3-/CD56+ natural killer cells. Aggressive NK-LGL leukemia patients present with a malignant clinical course and a fatal outcome with median survival time of two months from diagnosis. Aggressive NK-LGL is refractory to conventional chemotherapy and pathogenetic mechanisms remain undefined.The 'sphingolipid rheostat' has been identified as a key player in determining cell fate. The balance between pro-apoptotic ceramide and pro-survival sphingosine-1-phosphate (S1P) in this rheostat is determined by the expression of acid ceramidase (AC) which converts ceramide to sphingosine, and sphingosine kinase-1 and -2 (SPHK1 and SPHK2), which convert sphingosine to S1P. The S1P pathway is involved in cancer pathogenesis, cell survival, and resistance to chemotherapy and radiation, therefore it is considered a potential target for anti-cancer therapy. S1P signals both extracellularly through a family of five related GPCRs; the S1P receptors (S1PR1-5) and intracellularly through various incompletely defined mechanisms. FTY720 is a sphingolipid modulator that targets the S1P system and has potent immunosuppressive and anti-cancer properties. We have previously shown that FTY720 shows therapeutic efficacy in a rat model of NK-LGL leukemia, but its mechanism-of-action in inducing cell death is incompletely defined. The aim of this study is to explore these molecular mechanisms and further characterize the actions of FTY720 in NK-LGL leukemia.Results: We identified that the key enzymes in S1P biosynthesis, SPHK1 and AC, and the S1P receptor S1PR5 were all expressed in LGL cell lines and primary PBMCs from NK-LGL patients. Increased mRNA levels of AC, SPHK1 and S1PR5 were detected in NK-LGL samples (n=8) compared to PBMCs from healthy donors (n=8) (p < 0.05). These results were verified by immunoblot in NK-LGL samples (n=6) versus PBMCs from healthy donors (n=3) (p < 0.05). In vitro treatment of human and rat LGL cell lines (NKL, RNK16 and TL-1) resulted in time- and dose-dependent decreases in cell proliferation and increased apoptosis (IC50 5.0 - 12.5 μM). Interestingly, treatment of the LGL cell lines with the phosphorylated form of FTY720, FTY720-P, which acts antagonistically at the S1PRs, resulted in minimal effects on proliferation and apoptosis. This suggests that FTY720-induced cell death is independent of its effects on S1PRs. However, treatment of LGL cell lines with FTY720 did target multiple sphingolipid pathway members. We observed dose-dependent decreases in AC and S1PR5 protein expression. Interestingly, the combination of FTY720 with LCL204, an AC inhibitor, resulted in additive effects on cell proliferation and apoptosis in the NKL cells.Further characterization of cell death mechanisms demonstrated involvement of autophagy-related apoptosis. We observed increased PARP and caspase-3 cleavage after FTY720 treatment, which occurred after increased expression of LC3A/B and the conversion of LC3-I to LC3-II, suggesting autophagy is being induced prior to apoptosis induction. We identified the mTOR pathway as a target of FTY720. The mTOR pathway negatively regulates autophagy and its inhibition can induce autophagy. FTY720 treatment suppressed mTOR signaling in LGL cell lines as demonstrated by decreased phosphorylation of mTOR, p70S6K and S6 Kinase. These effects preceded the autophagy-related changes in LC3A/B. Furthermore, PARP and caspase-3 cleavage could be blocked by pre-treatment with 3-methyladenine and BafilomycinA1, both inhibitors of autophagy. Interestingly, the effects of FTY720 in LGL cell lines could also be blocked by pre-treatment with autophagy inhibitors, further suggesting that autophagy is required for the FTY720-induced effects. Importantly, these key findings were also reproduced in PBMCs from NK-LGL patients (n=3).Conclusions: Taken together, our findings demonstrate that FTY720 targets multiple sphingolipid pathway members in NK-LGL leukemia and that FTY720-induced cell death is autophagy-dependent. The down-stream targeting of mTOR by FTY720, combined with mTOR's prominent role in inhibiting autophagy, suggests that co-treatment with specific mTOR inhibitors in combination with FTY720 may be a promising and novel therapeutic strategy in LGL leukemia. DisclosuresNo relevant conflicts of interest to declare.

Acid Ceramidase Inhibition Impairs Tumor Progression in a Rat Model of LGL Leukemia

Natural killer cell large granular lymphocytic (NK-LGL) leukemia is a disorder characterized by the expansion of clonal CD3-, CD56+ cells in the peripheral blood and marrow. NK-LGL leukemia could manifest in both aggressive and chronic forms. Currently, no curative treatment is available for patients with NK-LGL leukemia. The pathogenetic mechanisms of NK-LGL leukemia are also poorly understood, which highlights the need to increase our understanding of the disorder in order to develop novel therapeutic targets. Sphingolipid dysregulation has been shown to promote tumor growth and survival in several types of cancers, including LGL leukemia. In cancer cells, the accumulation of pro-survival sphingosine-1-phosphate (S1P) throws the S1P/ceramide rheostat off balance, which leads to tumor growth and activated signaling pathways. Our laboratory previously discovered that the reversal of sphingolipid dysregulation in NK-LGL leukemia, either through targeting sphingolipid enzymes or by the addition of antiproliferative ceramide, leads to programmed cell death in leukemic cells.Acid ceramidase, a cysteine hydrolase, catalyzes the breakdown of ceramide into sphingosine and fatty acid. We found that cells from chronic NK-LGL patients have lower pro-death ceramide levels and higher S1P levels when compared to normal NK donors. Furthermore, the mRNA expression and activity of acid ceramidase (AC) were elevated in leukemic NK cells compared to normal NK cells. We further demonstrate the importance of AC in NK-LGL leukemic cells through AC knockdown in human (NKL) and rat (RNK-16) NK-LGL leukemic cell lines. AC knockdown decreased cell viability and increased ceramide levels, significantly ceramide species C16 and C24 (p<0.05), showing that AC is essential for the growth of leukemic NK cells. We further confirm that the accumulation of both C16 and C24 ceramide mediated the decreased viability of NKL and RNK-16 cells by treating the cells with C16 and C24 ceramide (6.25µM, 12.5µM and 25µM) for 24 hours.As our data showed that AC mediates the survival of NK-LGL, we did further analysis on the effects of AC knockdown on survival signals. Protein expression of AC knockdown- NKL and RNK-16 cell lines showed that AC knockdown decreased survival signaling mediators, including Bcl-2, Mcl-1 and survivin. Conversely, pro-apoptotic puma increased with AC knockdown in both cell lines. We further extended our in vitro findings in an established in vivo NK-LGL Fischer rat model. RNK-16 cells stably expressing AC shRNA (RNK-16/shRNA) or stably expressing scrambled shRNA (RNK-16/scr) were transplanted into 6-week old male rats (7 per group). The rats engrafted with RNK-16/shRNA survived significantly longer than those engrafted with RNK-16/scr (Mantel-Cox test, p<0.05). Interestingly, RNK-16/shRNA rats did not exhibit organomegaly compared to their leukemic counterparts. Splenocytes isolated from RNK-16/shRNA engrafted animals showed 50 percent reduction in AC expression compared to splenocytes from RNK-16/scr. Lipid analysis on splenocytes isolated from both RNK-16/scr and RNK-16/shRNA showed that RNK-16/shRNA cells had significant increases in total ceramide, C16 and C24 ceramide species, and a significant decrease in S1P. Collectively, our data show that targeting AC slows the progression of NK-LGL leukemia by reversing sphingolipid dysregulation and warrants further investigation as a therapeutic target in this incurable disease. DisclosuresNo relevant conflicts of interest to declare.

Large Granular Lymphocyte Infiltration in the Bone Marrow in Children and Young Adults May Suggest Primary Immune Deficiency

Large granular lymphocyte (LGL) leukemia is a group of rare lymphoproliferative disorders which involve inappropriate clonal expansion of either cytotoxic T-lymphocytes (CTLs) or natural killer cells (NK). The usual age at onset is between 40 to 60 years. We here describe four very young patients showing LGL infiltration in the bone marrow (BM) and all diagnosed with primary immune deficiency.Patient 1 is a 13-year old male who presented with gingivostomatitis, but no severe infections, autoimmune disease or allergies. He had persistent leukopenia (1.9x109/L), neutropenia (<0.05x109/L), thrombocytopenia (60-80x109/L) and mild anemia. The BM showed myeloid maturation arrest and infiltration of LGL cells with NK cell phenotype (CD3-, CD2+, CD16+, CD56-/+, CD57-, CD4-, CD8-) at 50% of the BM cellularity. In peripheral blood (PB) T, B and NK cell counts were normal. However, low levels of monocytes (0.09x109/L), normal levels of monocytoid (2.7x106/L) and low levels of plasmacytoid (0.1x106/L) dendritic cells (DC) were detected. While germline whole exome sequencing (WES) did not identify mutations likely to cause the disease, targeted sequencing of GATA2 mRNA showed uniallelic GATA2 expression, confirming haploinsufficiency.Patient 2 is a 15-year old female who has neonatal diabetes, autoimmune desquamative interstitial pulmonary disease, autoimmune enteropathy, exocrine pancreatic insufficiency and delayed growth. She developed transfusion-dependent, Coombs-negative anemia at age of 15. In the PB, 50% of the lymphocytes were LGLs. BM showed pure red cell aplasia plus LGL infiltration of TCRgd positive T cells (CD3+, CD2+, CD5+, CD8-/+, CD4-/+, CD57+) at 43% of BM cellularity. Clonal TCRg rearrangement was detected. Hypogammaglobulinemia presented at age of 7. T, B and NK cell counts were low normal, but DCs were non-existent. WES and functional studies revealed a germline gain-of-function mutation of STAT3 gene.Patient 3 is 42-year old female who has had severe infections (bacterial, viral and protozoan) starting at the age of 4 months. From age of 9, autoimmune manifestations occurred with immune thrombocytopenia, neutropenia and anemia, and intensively positive rheumatoid factor. Since age of 17, CD8+ LGLs were detected in PB reaching level of 50% of lymphocytes. In the BM, CD3+CD5-CD56- T -lymphocyte infiltration with clonal TCRd rearrangement was detected. In her 30ies she developed pulmonal arterial hypertension and hypogammaglobulinemia (0.8 g/L). She has low counts of B cells (0.02x109/L), and monocytoid (1.9x106/L) and plasmacytoid (0) DCs. WES identified a homozygous germline missense mutation in CECR1 gene, leading to total loss-of-function of the encoded adenosine deaminase 2 (ADA2) protein.Patient 4 presented with waxing and waning skin nodules from age of 10 years, but no severe infections or allergies. At age of 26, she was diagnosed with hemolytic anemia with spherocytosis, splenomegaly and cholelithiasis. Five years later remarkable hepatomegaly, and clinical autoimmune manifestations with SLE-type skin lesions, autoimmune hemolytic anemia and iritis were observed. Complement C3 (0.17 g/l) and C4 (<0.02 g/L) and NK cell (0.081) levels were low, but CD3, CD8 and CD4 counts and IgM and IgG levels were increased. T-cell infiltration was shown in the skin nodules and liver. BM showed mildly decreased myelo- and erythropoiesis and T cell infiltration (CD3+, CD8+, CD7+, CD45RO+, BCL2+, CD56-) up to 70% of the BM cellularity consistent with LGL infiltration. WES identified the same germline missense mutation in CECR1 gene as seen in patient 3, and a functional analysis showed no ADA2 activity.We conclude that LGL lymphoproliferation in young patients is often a sign of underlying primary immunodeficiency, highlighting the need for detailed genetic studies in these cases.Table 1PatientAge at LGLLGL in PB(109/L)LGL immune-phenotypeClonalityBM LGL infiltrationClinicalPID1130.38NKNA40%N,THepatomegalyGATA2 deficiency2151.79CTLTCRg43%AutoimmunityAHepatosplenomegalySTAT3 hyperactivation3171,49CTLTCRdNAN,T, AAutoimmunityPulmonary arterial hypertensionADA2 deficiency431NACTLTCRg weak40%AutoimmunityHepatosplenomegalyHepatopulmonal syndromeADA2 deficiencyA, anemia; N, neutropenia, CTL, cytotoxic T cell; LGL, large granular lymphocyte; NK, natural killer cell; PID, primary immune deficiency; TCR, T cell receptor; T, thrombocytopenia DisclosuresMustjoki:Signe and Ane Gyllenberg Foundation: Research Funding; Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Academy of Finland: Research Funding; the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Siitonen:Pzizer: Other: charges of EAHAD congress 2015; Novartis: Other: charges of EHA congress 2015. Saarela:Roche: Honoraria.

Identification of a STAT3-miRNA Axis in T-LGL Leukemia

IntroductionT large granular lymphocytes leukemia (T-LGLL) is a rare disease characterized by the abnormal expansion of T-large granular lymphocytes (T-LGLs) in the peripheral blood. The etiology of this disease is still largely unknown. LGL proliferation is maintained through an impairment of the apoptotic machinery due to the activation of many survival signals. Among these, JAK/STAT signaling represents one of the most important deregulated pathways in T-LGLL. In particular, leukemic LGLs are equipped with STAT3 constitutively over-expressed and over-activated. Moreover, in 30-40% of patients, STAT3 has been demonstrated carrying hot-spot mutations, likely resulting in STAT3 activation. Although STAT3 is an inducer of transcription of a large number of oncogenes, its relationship with microRNAs (miRNAs) has not yet been extensively evaluated in T-LGLL patients. As a matter of fact, several miRNAs contribute to normal hematopoietic processes and many miRNAs act both as tumor suppressors and oncogenes in the pathology of hematological malignancies, including acute and chronic leukemias and lymphomas, where they contribute to lymphomagenesis acting in various cellular functions, such as the regulation of cell survival and proliferation.AimsWe investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. A high throughput quantitative and qualitative analysis of the miRNA expression profile in leukemic LGLs compared to healthy controls was performed with the aim to investigate whether STAT3 activation and/or mutation were correlated to some miRNAs in leukemic LGLs.MethodsSix patients (3 characterized by STAT3 mutations and 3 with wild type STAT3) and three healthy controls were enrolled in a pilot study. STAT3 mRNA expression and protein activation levels were analyzed by Real Time-PCR and Western Blot, respectively. The expression level of 756 mature miRNAs was assessed by using a TaqMan-based Low Density Array on purified LGLs. Experimental data were analyzed by ViiA7 RUO software and the relative miRNA expression values were calculated using U6 as endogenous control. miRNA array data underwent hierarchical cluster analysis (HCL) by using MEV. miRNAs with a 2 or 0.5 fold change and p value < 0.05 in samples as compared to controls were considered as differentially expressed. Results of this pilot study were validated on additional 12 T-LGLL and 3 healthy controls subjects.ResultsTwo clusters were identified by HCL analysis: cluster A included healthy controls and LGL patients characterized by comparably low levels of STAT3 activation (S3low) and absence of STAT3 mutations. Cluster B included four patients characterized by high levels of STAT3 activation (S3high). Remarkably, three out of four LGL patients in cluster B shared STAT3 mutation. Comparative analysis of the miRNAs expressed identified 33 miRNAs upregulated and 9 miRNAs downregulated in S3high as compared to S3low. Interestingly, the level of expression of these selected miRNAs correlated with the level of STAT3 expression/activation in LGL. Among these, three miRNAs, miR-484, miR-501 and miR-1249, have been validated and confirmed in the validation cohort.ConclusionsThese data firstly describe the miRNA pattern in T-LGLL, providing evidence that a series of miRNAs are correlated with relevant key factors in T-LGLL pathogenesis, including STAT3 activation/expression and mutations. Our results suggest the hypothesis that STAT3 could mediate its role through some defined miRNAs. DisclosuresNo relevant conflicts of interest to declare.

Recent Advances in Celiac Disease from TTG to Gluten in Pee.

Recent Advances in Celiac Disease from TTG to Gluten in Pee.

Clinicopathologic, Immunophenotypic, Cytogenetic, and Molecular Features of γδ T-Cell Large Granular Lymphocytic Leukemia: An Analysis of 14 Patients Suggests Biologic Differences With αβ T-Cell Large Granular Lymphocytic Leukemia. [corrected

T-cell large granular lymphocytic (T-LGL) leukemia is a rare disorder in which the neoplastic cells usually express the αβ T-cell receptor (TCR). To determine the significance of γδ TCR expression in this leukemia, we compared the clinicopathologic, immunophenotypic, and genetic features of patients with T-LGL leukemia expressing γδ TCR or αβ TCR. We used the World Health Organization classification criteria to confirm the diagnosis. All patients were diagnosed and treated at our institution. We identified 14 patients with γδ T-LGL leukemia, 11 men and three women; six (43%) patients had a history of rheumatoid arthritis, 10 (71%) had neutropenia, four (29%) had thrombocytopenia, and three (21%) had anemia. Eight (67%) of 12 patients had a CD4-/CD8- phenotype, and four (33%) had a CD4-/CD8+ phenotype. The median overall survival was 62 months. Patients with γδ T-LGL leukemia were more likely to have rheumatoid arthritis (P = .04), lower absolute neutrophil count (P = .04), lower platelet count (P = .004), and a higher frequency of the CD4-/CD8- phenotype (P < .0001). However, there was no significant difference in overall survival between the two groups (P = .64). Although patients with γδ and αβ T-LGL leukemia show some different clinical or phenotypic features, overall survival is similar, suggesting that γδ TCR expression does not carry prognostic significance.

Abstract A86: Harnessing T-cell homeostasis to diagnose and treat solid and liquid tumors

Abstract T-cell populations are subject to homeostatic control from cytokines and microenvironmental signaling. Disruption of homeostasis can cause changes to the dynamics of the system that have implications for the progression of cancer. Here we present two mathematical models that examine the progression of tumors in the context of T-cell homeostasis and provide therapeutic decision support in the clinic. Model 1: During a chronic disease such as cancer, T cells often become tolerant to the antigens presented by the disease. This tolerant state effectively limits the response of the immune system to the tumor. Experimental evidence has shown that depletion of T-cells can lead to a loss of T-cell tolerance. During the homeostatic phase of T-cell compartment repopulation, there is a temporary window of opportunity during which T cells lose their tolerant state, allowing them to respond to tumor antigens. In addition, clonal expansion of the tumor-specific T-cell clone may be enhanced during the regrowth phase due to increased stimulation. We use an ordinary differential equation (ODE) model to explore the effect of T-cell depletion and homeostatic repopulation on the loss of tolerance in the T-cell compartment and subsequent effectiveness of immune-mediated tumor cytotoxicity. The model predicts different outcomes for the tumor and T-cell compartment, dependent on the strength and schedule of the depletion therapy. The optimal regimen can lead to tumor control in some cases, but T-cell exhaustion is also common dynamic predicted by the model. By understanding the effects of T-cell depletion, immune depleting therapies can be optimized to enhance immune potential. Model 2: Large Granular Lymphocytic Leukemia (LGLL) is a T-cell lymphoproliferative disorder that exhibits clonal expansion of a subset of T cells. Since there are no clinical biomarkers to predict the aggressiveness of the disease, treatment decisions are often made on a watch and wait approach. Using a set of ODEs, we develop a model of LGLL that uses clinical patient data from diagnosis to predict the timeframe for progression of the disease. Our experimental results have suggested that the disease is caused by a change in sensitivity to both positive and negative regulators of T-cell homeostasis. The model incorporates these cell-specific mechanisms to investigate their effect when placed in a homeostatic setting. The level of dysregulation as measured from patient-specific data determines the rate of outgrowth of the diseased T-cell clone, and therefore serve as a useful predictive tool for managing treatment decisions in the clinic. Citation Format: Mark Robertson-Tessi, Derek Park, Kimberly Luddy, Adam Mailloux, Pearlie K. Burnette, Alexander Anderson. Harnessing T-cell homeostasis to diagnose and treat solid and liquid tumors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A86.

Neutropenia With Respiratory Syncytial Virus Infection in Large Granular Lymphocyte (LGL) Leukemia

Neutropenia With Respiratory Syncytial Virus Infection in Large Granular Lymphocyte (LGL) Leukemia

Good syndrome presenting with CD8⁺ T-Cell large granular lymphocyte leukemia.

Good Syndrome is an adult-onset combined immunodeficiency defined by hypogammaglobulinemia, low or absent number of B cells, T cell deficiency and thymic tumor. We have characterized CD8+ T cells from a patient with Good syndrome that presented with CD8+T-cell large granular lymphocytic leukemia (LGL). Characterization of peripheral blood CD8+ T cells revealed that majority of CD8+ T cells were terminally differentiated effector memory phenotype (TEMRA; CD8+CCR7-CD45RA+), and were PD-1high (CD279), ICOSlow (CD278), and granzymehigh. Almost all CD8+ T cells were IFN-γ+. CD8 Treg (CD8+CD183+CCR7+CD45RA-) were decreased. TEMRA phenotype along with CD279high, demonstrates that these are exhausted CD8+ T cells. This phenotype along with CD278low may also explain severe T cell functional deficiency in our patient. In the present patient, T-LGL appears to be a clonal expansion of CD279+granzyme+IFN-γ+CD8+TEMRA cells. To best of our knowledge this is the first case of CD8+T-cell LGL leukemia associated with Good syndrome.

ID: 204: Interleukin-6-Induced and constitutive activation of signal transducer and activator of transcription 3 relies on protein kinase II activity

Cytokines of the Interleukin (IL)-6-family have essential functions in the control of inflammatory responses and immune defense. All members signal through the β -receptor gp130, albeit they activate different homo- and heterodimeric receptor complexes. Complex formation results in activation of the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway which is known to play an important role in diseases including autoimmunity and cancer, making a stringent regulation necessary. The Protein kinase II (CK2) is a constitutively active serine/threonine kinase that regulates proliferation through diverse interactions with many signaling pathways and that is shown to be overexpressed in various cancer types including hematopoietic tumors. We show that STAT1 and STAT3 activation by IL-6 and all other family members is abolished by CK2 inhibition. Furthermore, cytokine independent Jak/STAT activation via an activating deletion mutation in domain 2 of gp130 (gp130 Δ YY) is critically dependent on the activity of both CK2 and Jak1. An activating mutation in STAT3 that was found in large granular lymphocytic leukemia (STAT3 Y640F) leads to constitutive STAT3 phosphorylation, which could be blocked via pharmacological inhibition of CK2. We furthermore confirmed the dominant role of Jak1 and not Jak2 for cytokine induced and constitutive STAT signaling with the help of different murine embryonic fibroblasts (MEFs). In summary, we show a novel link between the Jak/STAT signaling pathway and the activity of the protein kinase CK2.

T-cell large granular lymphocytic leukemia

Decrite pour la premiere fois en 1985, la leucemie a grands lymphocytes granuleux (LGL, pour large granular lymphocytes) est un syndrome lymphoproliferatif rare caracterise par une expansion clonale de lymphocytes T CD3+ cytotoxiques ou de cellules natural killer (NK) CD3- infiltrant la moelle osseuse, la rate et le foie, et frequemment associee a des maladies auto-immunes. La classification de l’Organisation mondiale de la sante (OMS) de 1999 inclut les leucemies LGL a cellules T [...]

Impact of Molecular Parameters on Treatment outcomes in Large Granular Lymphocyte Leukemia (LGL)

Context: The ability of cytotoxic CD4+ T lymphocytes (CD4+ CTLs) to exert anti-tumor effects has been demonstrated in murine models of melanoma and an increase in the number of CD4+CTLs has previously been identified in the peripheral blood of patients with CLL. Objective: To further characterize these cells in patients with CLL, including their immune checkpoint receptor expression, and to identify their presence in the CLL lymph node tumor microenvironment. Design/Patients: The peripheral blood of 54 consecutive patients with CLL, who were not receiving treatment and who were attending the out-patient clinic at University College Hospital, was analyzed by flow cytometry. Ethical approval and consent had been obtained. Multi-color immunohistochemistry was performed on formalin-fixed, paraffin-embedded lymph node biopsies infiltrated with CLL and reactive lymph nodes. Results: CD4+ Granzyme B+ cells (CD4+CTLs) were detected in the peripheral blood of all patients with a median percentage of 8.54% (range: 0.616 e 65.1%). There was a significant increase in the percentage of CD4+GZB+ cells in those patients who were above the age of 60 years (P¼0.0455). An extended phenotype analysis of the CD4+GZB+ cells found that the transcription factors EOMES (median 78.05%, range: 51.6-95.1%) and Tbet (median: 90.5%, range: 88.3-96.1%), which have both been strongly implicated in the development of cytotoxicity in CD8+ cells, were highly expressed

Sphingosine kinase inhibitors decrease viability and induce cell death in natural killer-large granular lymphocyte leukemia

Sphingolipid metabolism has been identified as a potential therapeutic target in cancer. Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid metabolite produced by sphingosine kinases-1 and −2 (SPHK1 and SPHK2). Elevated SPHK1 has been found in numerous cancer types and been shown to contribute to survival, chemotherapeutic resistance and malignancy. However, its role in large granular Natural Killer (NK) large granular lymphocyte (LGL) leukemia has not been investigated. Here, we examine SPHK1 as a therapeutic target in LGL leukemia. We found that SPHK1 is overexpressed in peripheral blood mononuclear cells (PBMCs) from LGL leukemia patients which results in elevated S1P in the sera. The use of SPHK1 inhibitors, SKI-II or SKI-178, decreased leukemic NK cell viability and induced caspase-dependent apoptosis. SKI-II and SKI-178 restored the sphingolipid balance by increasing ceramide and decreasing S1P in leukemic NKL cells. SKI-II and SKI-178 also induced apoptosis in primary NK-LGLs from leukemia patients. Mechanistic studies in NK-LGL cell lines demonstrated that SKI-178 and SKI-II induced cell cycle arrest at G2/M. We found that SKI-178 induced phosphorylation of Bcl-2 at Ser70, and that this was dependent on CDK1. We further show that SPHK1 inhibition with SKI-178 leads to decreased JAK-STAT signaling. Our data demonstrate that SPHK1 represents a novel therapeutic target for the treatment of NK-LGL leukemia.

Abstract 606: Novel somatic mutations in the DNA-binding and coiled-coil domain of the STAT3 gene in LGL-leukemia

Abstract Introduction T-cell large granular lymphocyte (T-LGL) leukemia is a rare, clonal disease characterized by the expansion of CD8+ cytotoxic T-cells. We recently discovered that 40% of T-LGL leukemia patients have somatic mutations in the SH2-domain of the STAT3 gene (Koskela et al. NEJM 2012). As aberrant STAT3 activation can be observed in all patients with LGL-leukemia, we now aimed to discover whether patients without mutations in the STAT3 hotspot area harbor mutations in the other parts of the STAT3 gene. Methods Targeted STAT3 sequencing covering all 23 coding exons was done with in-house developed deep amplicon sequencing panel using the Illumina Miseq platform. The data was analyzed with a bioinformatics pipeline, which is based on calling of variants with specific counts/frequencies and filtering out false positives using the estimated error rate and quality data of amplicon reads. All samples with a frequency ratio ≥0.9 were considered to be true mutations after filtering of SNPs and low coverage variants. 111 LGL-leukemia patients with no known STAT3-mutations in the SH2 domain were analyzed. To explore the functional effects of mutations, expression constructs were generated with the identified variants and wild-type STAT3. The variants were expressed in HEK-293 cells carrying a STAT3-responsive SIE-reporter driven luciferase expression sequence to establish increased basal and IL6-stimulated STAT3 activity. Results With targeted amplicon sequencing, 3 patients were discovered to have STAT3 missense mutations in the DNA-binding domain. Two patients presented with the same H410R mutation with a variant allele frequency (VAF) of 49% and 8.8% respectively while another patient had a S381Y mutation (VAF 7%). The mutation H410R occurs in the DNA-binding domain in a highly conserved position, and results in conversion of histidine to arginine, which would predict for a slight increase in hydrophilicity. In addition to STAT3 DNA-binding domain mutations, one T-LGL patient had a novel F174S mutation in the coiled-coil domain of STAT3 (VAF 43%). The coiled-coil domain of STAT3 has previously been shown to be essential in SH2-domain mediated receptor binding and subsequent activation. Luciferase measurements of SIE-reporter HEK-293 cells transfected with constructs expressing either wild-type, variant F174S, H410R or Y640F STAT3 (the most common activating mutation in LGL leukemia) revealed the F174S and H410R variants to be as activating as the Y640F mutation in both unstimulated and IL-6 stimulated conditions. Conclusions T-LGL leukemia patients without STAT3 SH2-domain mutations harbor novel activating mutations in the DNA-binding and coiled-coil domain of STAT3. The frequency of mutations was 3.6% (4 of 111 patients). These findings further highlight the importance of screening the whole STAT3 gene in the diagnostic workup of LGL-leukemia and the central role of STAT3 in the pathogenesis of the disease. Citation Format: Emma I. Andersson, Hanna Rajala, Heikki Kuusanmäki, Arjan van Adrichem, Samuli Eldfors, Sonja Lagström, Thomas Olson, Michael Clemente, Pekka Ellonen, Caroline Heckman, Thomas P. Loughran, Jaroslaw P. Maciejewski, Satu Mustjoki. Novel somatic mutations in the DNA-binding and coiled-coil domain of the STAT3 gene in LGL-leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 606. doi:10.1158/1538-7445.AM2015-606

Recurrent alterations ofTNFAIP3(A20) in T-cell large granular lymphocytic leukemia

The pathogenesis of T-cell large granular lymphocytic leukemia (T-LGL) is poorly understood, as STAT3 mutations are the only known frequent genetic lesions. Here, we identified non-synonymous alterations in the TNFAIP3 tumor suppressor gene in 3 of 39 T-LGL. In two cases these were somatic mutations, in one case the somatic origin was likely. A further case harbored a SNP that is a known risk allele for autoimmune diseases and B cell lymphomas. Thus, TNFAIP3 mutations represent recurrent genetic lesions in T-LGL that affect about 8% of cases, likely contributing to deregulated NF-κB activity in this leukemia.

Spectrum of Clonal Large Granular Lymphocytes (LGLs) of αβ T Cells: T-Cell Clones of Undetermined Significance, T-Cell LGL Leukemias, and T-Cell Immunoclones.

Clones of T-cell large granular lymphocytes (LGLTs) were detected by flow cytometry. Disease associations are described. Flow cytometry on blood or marrow detected clonal LGLTs by analyzing variable regions of the T-cell receptor β chain. LGLT clones were detected in 20% (54/264) of tested patients. The clone sizes were less than 2.0 × 10(9)/L in the blood in 73% and less than 10% of marrow space in 94%. Blood counts showed cytopenias. Clinical associations included B-cell clones, myeloid neoplasms, nonneoplastic disorders of blood or marrow, transplants, systemic immune disorders, carcinomas, or hypothyroidism. Twelve patients had LGLT leukemia. Most (76%) had small LGLT clones with limited impact on the clinical management. Most of the LGLT clones detected by flow cytometry were small and did not change the clinical management. We propose the following terminology: T-cell clones of undetermined significance, LGLT leukemias, and T-cell immunoclones.