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Tγδ large granular lymphocyte leukemia (LGLL) is a rare variant of T-cell LGLL (T-LGLL) that has been less investigated as compared with the more frequent Tαβ LGLL, particularly in terms of frequency of STAT3 and STAT5b mutations. In this study, we characterized the clinical and biological features of 137 patients affected by Tγδ LGLL; data were retrospectively collected from 1997 to 2020 at 8 referral centers. Neutropenia and anemia were the most relevant clinical features, being present in 54.2% and 49.6% of cases, respectively, including severe neutropenia and anemia in ∼20% of cases each. Among the various treatments, cyclosporine A was shown to provide the best response rates. DNA samples of 97 and 94 cases were available for STAT3 and STAT5b mutation analysis, with 38.1% and 4.2% of cases being mutated, respectively. Clinical and biological features of our series of Tγδ cases were also compared with a recently published Tαβ cohort including 129 cases. Though no differences in STAT3 and STAT5b mutational frequency were found, Tγδ cases more frequently presented with neutropenia (P = .0161), anemia (P < .0001), severe anemia (P = .0065), and thrombocytopenia (P = .0187). Moreover, Vδ2− cases displayed higher frequency of symptomatic disease. Overall, Tγδ cases displayed reduced survival with respect to Tαβ cases (P = .0017). Although there was no difference in STAT3 mutation frequency, our results showed that Tγδ LGLL represents a subset of T-LGLL characterized by more frequent symptoms and reduced survival as compared with Tαβ LGLL.

Chronic natural killer large granular lymphocyte (NK-LGL) leukemia, also referred to as chronic lymphoproliferative disorder of NK cells, is a rare disorder defined by prolonged expansion of clonal NK cells. Similar prevalence of STAT3 mutations in chronic T-LGL and NK-LGL leukemia is suggestive of common pathogenesis. We undertook whole-genome sequencing to identify mutations unique to NK-LGL leukemia. The results were analyzed to develop a resequencing panel that was applied to 58 patients. Phosphatidylinositol 3-kinase pathway gene mutations (PIK3CD/PIK3AP1) and TNFAIP3 mutations were seen in 5% and 10% of patients, respectively. TET2 was exceptional in that mutations were present in 16 (28%) of 58 patient samples, with evidence that TET2 mutations can be dominant and exclusive to the NK compartment. Reduced-representation bisulfite sequencing revealed that methylation patterns were significantly altered in TET2 mutant samples. The promoter of TET2 and that of PTPRD, a negative regulator of STAT3, were found to be methylated in additional cohort samples, largely confined to the TET2 mutant group. Mutations in STAT3 were observed in 19 (33%) of 58 patient samples, 7 of which had concurrent TET2 mutations. Thrombocytopenia and resistance to immunosuppressive agents were uniquely observed in those patients with only TET2 mutation (Games-Howell post hoc test, P = .0074; Fisher’s exact test, P = .00466). Patients with STAT3 mutation, inclusive of those with TET2 comutation, had lower hematocrit, hemoglobin, and absolute neutrophil count compared with STAT3 wild-type patients (Welch’s t test, P ≤ .015). We present the discovery of TET2 mutations in chronic NK-LGL leukemia and evidence that it identifies a unique molecular subtype.

Large granular lymphocyte (LGL) leukemia is a rare hematological disorder with expansion of the T‐cell or natural killer (NK) cell lineage. Signal transducer and activator of transcription 3 (STAT3) exhibits somatic activating mutations in 30%‐40% of LGL leukemia cases. Transcriptional targets of STAT3 include inflammatory cytokines, thus previous studies have measured cytokine levels of LGL leukemia patients compared to normal donors. Sphingolipid metabolism is a growing area of cancer research, with efforts focused on drug discovery. To date, no studies have examined serum sphingolipids in LGL leukemia patients, and only one study compared a subset of cytokines between the T‐LGL and NK‐LGL subtypes. Therefore, here, we included both LGL leukemia subtypes with the goals of (a) measuring serum sphingolipids for the first time, (b) measuring cytokines to find distinctions between the subtypes, and (c) establishing relationships with STAT3 mutations and clinical data. The serum analyses identified cytokines (EGF, IP‐10, G‐CSF) and sphingolipids (SMC22, SMC24, SMC20, LysoSM) significantly different in the LGL leukemia group compared to normal donors. In a mixed STAT3 mutation group, D661Y samples exhibited the highest mean corpuscular volume (MCV) values. We explored this further by expanding the cohort to include larger groups of single STAT3 mutations. Male D661Y STAT3 samples had lower Hgb and higher MCV compared to wild type (WT) or Y640F counterparts. This is the first report examining large groups of individual STAT3 mutations. Overall, our results revealed novel serum biomarkers and evidence that D661Y mutation may show different clinical manifestation compared to WT or Y640F STAT3., Analysis of LGL leukemia serum identified cytokines (EGF, IP‐10, G‐CSF) and sphingolipids (SMC22, SMC24, SMC20, LysoSM) that were significantly different compared to normal donors. Comparing three groups of STAT3 mutation status (WT, Y640F, D661Y) in LGL leukemia patients showed the D661Y mutation group had higher mean corpuscular volume and lower hemoglobin compared to the other groups.

Background: T-cell large granular lymphocytic leukemia (T-LGLL), a rare lymphoproliferative disorder of mature T cells, is characterized by the accumulation of activated effector T cells leading to a clonally restricted T-cell receptor (TCR) repertoire. Chronic antigen stimulation together with activating somatic STAT3 mutations have been proposed to lead to clonal expansion of leukemic cells. However, no holistic research has been done to show how leukemic and non-leukemic cells liaise to sustain abnormal immune reactivity in T-LGLL. Methods: We investigated the transcriptome and TCR repertoire in T-LGLL using: 1) single-cell RNA and TCR (scRNA+TCRαβ) sequencing from CD45+ sorted blood cells (T-LGLL n=11, healthy n=6), 2) TCRβ sequencing from blood mononuclear cells (T-LGLL n=48, healthy n=823), 3) bulk RNA sequencing (T-LGLL n=15, healthy n=5), 4) plasma cytokine profiling (T-LGLL n=9, healthy n=9), and 5) flow cytometry validations (T-LGLL n=6, healthy n=6) (Figure) Results: ScRNA+TCRαβ-seq data revealed that in healthy controls, hyperexpanded CD8+ T-cell clones (at least 10 cells with identical TCRs) preferentially had an effector memory phenotype, whereas in T-LGLL, the hyperexpanded clonotypes represented a more cytotoxic (increased expression of GZMB, PRF1, KLRB1) and exhausted (LAG3 and TIGIT) phenotype. Using flow cytometry, we confirmed that upon anti-CD3/CD28/CD49 antibody stimulation, T-LGLL clones (CD8+CD57+) expressed higher levels of cytotoxic proteins (GZMA /GZMB , PRF1) but were deficient in degranulation responses and cytokine secretion as measured by expression of CD107a/b and TNFα/IFNγ, respectively. Focused re-clustering of the extracted T-LGLL clones from the scRNA+TCRαβ-seq data revealed considerable heterogeneity among the T-LGLL clones and partly separated the mutated (mt) STAT3 and wild type (wt) STAT3 clones. STAT3wt clones upregulated T-cell activation and TCR signaling pathways, with a higher cytotoxicity and lower exhaustion score as compared to STAT3mt clones. This was validated with bulk RNA-seq data. To understand the antigen specificities of the T-LGLL clones, we combined previously profiled T-LGLL TCRs with our data to form the largest described dataset of 200 T-LGLL clones from 170 patients. Notably, T-LGLL clones were found to be private to each patient. Furthermore, the analysis by GLIPH2 algorithm grouping TCRs did not reveal detectable structural similarities, suggesting the absence of a unifying antigen in T-LGLL. However, in 67% of T-LGLL patients, the TCRs of leukemic clones shared amino acid level similarities with the rest of the non-leukemic TCR repertoire suggesting that the clonal and non-clonal immune repertoires are connected via common target antigens. To analyze the non-clonal immune repertoire in T-LGLL in detail, we compared our data to other published scRNAseq data from solid tumors (n=4) and hematologic cancers (n=8) and healthy controls (n=6). The analysis revealed that in T-LGLL also the non-leukemic CD8+ and CD4+ T cells were more mature, cytotoxic, and clonally restricted. When compared to healthy controls and other cancer patients, in non-leukemic T-LGLL the most upregulated pathway was IFNγ response. Finally, most of the upregulated cytokines in T-LGLL (e.g., CCL2/3/7, CXCL10/11, IL15RA) were secreted predominantly by monocytes and dendritic cells, which also had upregulated HLA class II expression and enhanced scavenging potential in T-LGLL patients. Ligand-receptor analysis with CellPhoneDB revealed that the number of predicted cell-cell interactions was significantly higher in T-LGLL as compared to reactive T-cell clones in healthy controls. The most co-stimulatory interactions (e.g., CD2-CD58, TNFSF14-TNFRSF14) occurred between the IFNγ secreting T-LGLL clones and the pro-inflammatory cytokine secreting monocytes. Conclusions: Our study shows a synergistic interplay between the leukemic and non-leukemic immune cell repertoires in T-LGLL, where an aberrant antigen-driven immune response including hyperexpanded CD8+ T-LGLL cells, non-leukemic CD8+ cells, CD4+ cells, and monocytes contribute to the persistence of the T-LGLL clones. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clones in patients with T-LGLL. Figure 1 Figure 1. Disclosures Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Maciejewski: Alexion: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy. Mustjoki: Novartis: Research Funding; BMS: Research Funding; Janpix: Research Funding; Pfizer: Research Funding.

Introduction: Large Granular Lymphocyte (LGL) leukemia is a rare lymphoproliferative disorder usually manifested in elderly individuals, with mean age of onset at 65. However, LGL leukemia cases in pediatric and young adult (age 50) cohort. Methods: We retrospectively collected clinical data from 23 young adult (age 50) LGL leukemia patients. All participants signed informed consent to provide biospecimens and clinical records to the LGL Leukemia Registry. Whole exome (PBMC and saliva) and RNA sequencing (PBMC) was conducted on 115 patients. Sanger sequencing was utilized to determine STAT3 mutational status in the remaining 102 patients. Results: STAT3 is the most frequently mutated gene in LGL leukemia, and the mutation is associated with lower absolute neutrophil counts (ANC). We observed no differences in the frequency of STAT3 somatic mutations in young adult, adult, and older adult LGL leukemia (p=0.143). However, we detected a significant positive linear relationship between age of the patient and ANC values (p=0.002) for STAT3 mutant patients (Figure 1A). STAT3 wild-type (WT) patients did not display this relationship (p=0.756). No age associated trends were detected for hemoglobin values, although we detected decreasing red blood cell (RBC) values with increasing age for both STAT3 mutated and WT patients (p= 0.031, 0.010, respectively). No difference in sex (male vs female) balance was found within the young adult cohort (p=0.190). As rheumatoid arthritis (RA) is observed in ~30% of LGL leukemia patients, we asked if this prevalence also holds in the younger cohort. RA was observed in similar proportion in all young, adult, and older adult groups (p=1). To compare the severity of disease manifestation between the age groups, we first asked how many patients ever initiated LGL-related treatments in each age group. We found that patients under 50 are more likely to be on LGL related treatments compared to patients over the age of 50 (p=0.016) (Figure 1B). Next, we examined length of survival between age groups. Despite increased initiation of LGL related treatments, no statistically significant difference in overall survival (OS) was observed between patients under 50 and patients over the age of 50 (p=0.33) (Figure 1C). Therefore, we asked if the treatment response in younger patients is better compared to older patients. We observed no statistically significant differences in the routine LGL treatment responses between patients under 50 and patients over the age of 50 (p=0.732). Interestingly, epigenetic and chromatin modifier mutations are equally common in patients 50 years old and under relative to older adult patients (p= 0.271) (Figure 1D). Transcriptomic analysis of the PBMC data revealed enrichment of TNFα signaling in patients older than 50, while IFNα and IFNγ signaling was enriched in patients 50 years old and younger. Conclusions: We report the clinical and molecular analysis of LGL leukemia in pediatric/young adult and adult patients compared to the older adult patients that comprise the majority of LGL cases. We show that while STAT3 mutation frequency is similar across all age groups, younger patients with STAT3 mutation show lower ANC values compared to older patients. Importantly, we show that younger patients exhibit a more severe disease, as indicated by increased initiation of LGL treatment. However, overall survival in younger groups compared to older patients is not impacted. Finally, we show that younger patients display similar proportions of epigenetic and chromatin modifying mutations that are a prominent feature of older patients, and feature enrichment of IFNα and IFNγ signaling distinct from older patients. Figure 1 Figure 1. Disclosures Feith: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees. Loughran: Dren Bio: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Background: The PTCL are a heterogeneous group of non-Hodgkin lymphomas originating from mature T-lymphocytes. They are aggressive diseases, often resistant to conventional chemotherapy. Despite the fact that a number of new agents have been approved, treatment paradigms tailored to the biology of the disease have yet to emerge. Tolinapant (ASTX660) is a potent antagonist of both cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), and is presently in phase I/II trials in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor (TNF) receptor superfamily mediated apoptosis (Ward GA, et al. Mol Cancer Ther. 2018), are potent anti-tumor immune enhancers and induce markers of immunogenic cell death such as damage associated molecular patterns (DAMPs; Ye W, et al, Oncoimmunology, 2020). Objectives: We explored the sensitivity of a range of T-cell lymphoma (TCL) cell lines to tolinapant. We establish the synergy coefficient between tolinapant and the HDAC inhibitor, romidepsin, and interrogated the molecular basis of their synergistic interaction. Methods: A panel of human T-cell lymphoma cell lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10ng/ml of TNF alpha. For combination studies, with tolinapant and romidepsin, each drug was tested at the IC10 and IC40 concentrations in the presence or absence of TNF alpha. Synergy scores using the Excess over Bliss (EOB) model were calculated using SynergyFinder (Aleksandr Ianevski et al; Nucleic Acids Research, 2020). Additionally, the effects of tolinapant and romidepsin on the IAPs and caspases were analyzed by western blots. TNFR1 receptor expression and induction of DAMPs were also analyzed by flow cytometry. Results: TCL Lines demonstrated varying sensitivities to tolinapant in the presence or absence of TNF alpha. The most sensitive cell lines, ALK+ ALCL and SUP-M2, had IC50 concentrations ranging from 200nM ± 100nM to 20nM ± 1nM in the absence or presence of TNF alpha, respectively, at 24, 48 and 72hrs, while a resistant CTCL cell line HH had an IC50 concentration of over 20mM, even in the presence of TNF alpha. Interestingly, using western blot analysis, we found that the presence of TNF alpha increased the levels of cIAP1 in the tolinapant sensitive SUP-M2 cell line, but not in the resistant HH cell line. However, there was a concentration dependent decrease in cIAP1 but not in XIAP in both cell lines treated with tolinapant. Flow cytometry analysis demonstrated that tolinapant increases the expression of TNFR1 and DAMPs in a dose dependent manner on the sensitive SUP-M2, but not in the resistant HH cells. In combination experiments, using the EOB model, tolinapant plus romidepsin was found to be synergistic in the absence of TNF alpha, at 36hrs, in both the sensitive cell line SUP-M2 and the resistant cell line HH. In the presence of TNF alpha, synergism was seen only in the sensitive cell line SUP-M2 and antagonistic in the HH cell line (Fig. 3). In the tolinapant plus romidepsin treated samples, cIAP1 levels decreased in the SUP-M2 cell line, in the absence of TNF alpha, however, addition of TNF alpha did not alter the levels of cIAP1 in the SUP-M2 cells. The cIAP1 levels decreased in the HH cells treated with the combination, in both the presence or absence of TNF alpha (Figure). Our findings indicate that the synergy of the tolinapant plus romidepsin is not dependent on the presence of TNF alpha. Conclusion: Tolinapant has demonstrated potent cytotoxic effects against a broad range of TCL lines both as a monotherapy and in combination with the HDAC Inhibitor, romidepsin. In in vitro studies, T cell lymphoma cell lines demonstrated varying sensitivity to tolinapant with certain cell lines being more resistant, even in the presence of TNF alpha. Interestingly, the addition of romidepsin appeared to overcome the intrinsic resistance to tolinapant in the absence of TNF alpha. These data provide the rationale to continue to explore the combination of tolinapant and romidepsin in vivo and to investigate additional combinations with T-cell specific agents (e.g. pralatrexate, belinostat, azacitidine and decitabine). Figure 1 Figure 1. Disclosures Smyth: Astex Pharmaceuticals: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Marchi: Kyowa Kirin: Honoraria; Myeloid Therapeutics: Honoraria; Astex: Research Funding; BMS: Research Funding; Merck: Research Funding; Kymera Therapeutics: Other: Scientific Advisor.

Acute myeloid leukemia (AML) is the most common adult leukemia. Only a fraction of AML patients will survive with existing chemotherapy regimens. Hence, there is an urgent and unmet need to identify novel targets and develop better therapeutics in AML. In the past decade, the field of sphingolipid metabolism has emerged into the forefront of cancer biology due to its importance in cancer cell proliferation and survival. In particular, acid ceramidase (AC) has emerged as a promising therapeutic target due to its role in neutralizing the pro-death effects of ceramide. Areas covered: This review highlights key information about AML biology as well as current knowledge on dysregulated sphingolipid metabolism in cancer and AML. We describe AC function and dysregulation in cancer, followed by a review of studies that report elevated AC in AML and compounds known to inhibit the enzyme. Expert opinion: AML has a great need for new drug targets and better therapeutic agents. The finding of elevated AC in AML supports the concept that this enzyme represents a novel and realistic therapeutic target for this common leukemia. More effort is needed towards developing better AC inhibitors for clinical use and combination treatment with existing AML therapies.

Introduction: Large Granular Lymphocyte (LGL) leukemia is a rare lymphoproliferative disorder characterized by clonal expansion of either CD3+ cytotoxic T cells expressing T cell receptor (TCR) alpha beta or CD3- natural killer (NK) cells. A less frequent CD3+ T cell subtype expresses TCR gamma delta (GD). Here, we present the molecular landscape of known LGL types (T, NK, GD) from analysis of the largest patient cohort assembled to date. An integrative analysis of genomic datasets from all LGL subtypes is necessary to more precisely define the shared and unique etiology of this rare disorder. Methods: We collected paired saliva and PBMC samples and related clinical information from 116 LGL leukemia patients after informed consent. The assembled cohort consisted of 93 T-LGL, 11 NK-LGL, and 12 GD T-LGL patients. Genomic analyses were performed on the leukemic (PBMC) and germline (saliva) samples after whole exome sequencing (WES) and transcriptome sequencing (RNAseq, PBMC only). Results: Somatic mutations were detected in the previously described potential drivers STAT3 (n=56), TNFAIP3 (n=9), and PIK3R1 (n=4). We also identified somatic mutations in CDH8 (n=3) and CCL22 (n=4), which we postulate as putative drivers based on mutational clustering. CDH8 was mutated in all three LGL subtypes, but CCL22 somatic mutations were only observed in NK-LGL patients. We observed that STAT3 and CCL22 together account for 64% (7/11) of NK-LGL cases. STAT3 is the most recurrently mutated gene in LGL leukemia, yet concurrent molecular and clinical features are incompletely defined. Interestingly, patients with STAT3 mutations have a higher mutational burden (P=0.0006) compared to those with wild-type (WT) STAT3. This effect is independent of the age of the patient, which correlates with the mutational burden (R=0.26, P=0.0039) and agrees with the finding that the dominant mutational signatures in this cohort exhibit clock-like properties. We also observed that patients with STAT3 mutations are enriched (P=0.0273) for additional mutations in chromatin modifier enzymes such as KMT2D, TET2, DNMT3A, and SETD1B (Figure 1). We found that ~10% of the samples exhibit broad somatic copy-number aberrations, and a patient with somatic mutations in STAT3 and KMT2D displayed high-level microsatellite instability. STAT3 mutations were also significantly associated with increased expression of genes involved in apoptosis, complement activation, and interferon cytokine signaling compared to STAT3 WT (FDR < 0.05). Early-onset LGL patients with age 51 years or less (n=28), as defined by the bottom quartile of the cohort, displayed no differential enrichment of the somatic driver genes. Interestingly, the age of the patient was significantly associated with absolute neutrophil counts (ANC) (P = 0.0068), with younger patients exhibiting lower neutrophil counts, even after adjusting for the presence of STAT3 mutation, as it is associated with lower ANC. As neutropenia is a hallmark feature of LGL leukemia and often a trigger for initiating therapy, the association of young age with lower neutrophil counts and lower somatic mutational burden suggests other mechanisms may be involved. Focusing on the germline variants, we found that 17 of the patients (14.6%) had at least one pathogenic or likely pathogenic germline variant with known oncogenic association as annotated using CharGer. 5 patients had pathogenic mutations in known tumor suppressors including FANCC (n=1), BRCA1 (n=1), PALB2 (n=1), MUTYH (n=1), and SDHA (n=1), while 1 patient had pathogenic mutations in ALK, a known oncogene. Conclusions: We report on the genomic analyses done on whole exome and RNA-seq data from the largest cohort assembled for LGL leukemia to date. We show that the presence of STAT3 mutation is significantly associated with an increase in mutation burden and additional somatic mutations in chromatin modifiers, hinting at potential pathogenic mechanisms within STAT3 mutated patients. By combining LGL subtypes in our analysis, we were able to identify CDH8 as a putative driver that is present in T, NK, and GD subtypes. Additionally, we report CCL22 mutations specific to NK-LGL leukemia, however did not detect any subtype specific mutations in GD T-LGL. We found that about 15% of the patients carry at least one pathogenic germline variant with known oncogenic associations. These findings highlight emerging etiologic insights into this rare disorder. Disclosures Feith: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees. Loughran:Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees.

Donor-derived CD4(+) T lymphocytes are the major effector cells directly involved in the development of graft-versus-host disease (GVHD). As a negative regulator of immune cell differentiation and development, microRNA-150 (miR-150) induces immunological tolerance in CD4(+) T cells after transplantation. However, the specific mechanisms have not been fully elucidated. In this study, we demonstrated that miR-150 is capable of not only inhibiting proliferation and activation of CD4(+) T cells but also promoting apoptosis. Mechanistically, miR-150 targets v-akt murine thymoma viral oncogene homolog 3 (AKT3), and subsequently downregulates B-cell lymphoma 2 (Bcl-2) interacting mediator of cell death (BIM). We have also demonstrated that re-expression of AKT3 reversed miR-150-mediated inhibition of CD4(+) T lymphocyte development. Therefore, we conclude that miR-150 negatively regulates CD4(+) T cell function by inhibiting the AKT3/BIM signaling pathway. These findings also suggest that manipulating the levels of miRNA-150 could be a valuable strategy in prevention and/or treatment of acute graft-versus-host disease.

Key Clinical Message In this report, a case of relapsed Ph+ ALL was remedied by reinduction, and consolidation regimen of TKI and Flu+ Ara‐C+ IDA (FLAI) combination, followed by haploidentical SCT. Results suggest that FLAI together with TKI and subsequently with haploidentical SCT could be applied for relapsed Ph+ ALL.

Background: The pathogenesis of chronic lymphoproliferative disorder of NK-cells (CLPD-NK) is poorly understood. Mutations in the JAK/STAT pathway (especially STAT3) are found in 30% of patients, but the genetic or exogenous drivers responsible for other cases are unknown. Here we comprehensively define the genetic drivers of CLPD-NK by integrated genome and transcriptome sequencing (WGS/WTS) of a large cohort of cases and complementary functional analyses. Methods: We studied 63 CLPD-NK patients (M/F: 42/21, median age: 71 years [35-89 y]) by WGS, WTS and flow cytometry. A validation cohort of 67 patients (M/F: 43/24, median age: 64 years [7-91]) was analyzed by targeted sequencing. To study the role of CCL22 in CLPD-NK pathogenesis, we examined internalization of the CCL22 receptor, CCR4, and cell chemotaxis in response to exogenous wild type (wtCCL22) or mutant CCL22 (mtCCL22: L45R, P79R, IL87_88NF) in CCR4-expressing Ba/F3 cells (Ba/F3-CCR4). To examine potential autocrine/paracrine activity, we exchanged supernatants of Ba/F3-CCR4-wtCCL22 and -mtCCL22 cells and examined CCR4 expression. To examine the in vivo effects of the mutations on proliferation and phenotype, GFP-tagged empty vector, wtCCL22, or mtCCL22-transduced NK-92 cells were engrafted into IL15-transgenic NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ (NSG) mice. Human NK-92 cells isolated from spleens of moribund mice were analyzed by WTS and immunophenotyping. Results: WGS of 63 CLPD-NK identified STAT3 mutations in 18 (29%) cases, with mutually exclusive CCL22 mutations (mtCCL22) in 14 (22%) patients. WGS of 4500 hematological malignancies showed that mtCCL22 were only found in CLPD-NK. Recurrent co-mutations in both groups were found in ATM (n=3), FAS (n=2) and TET2 (n=5). Of the remaining patients, 23/31 had one or more mutation including epigenetic regulators (n=12), signaling components (n=7) or TP53 (n=4). Our findings of CCL22 mutations were confirmed in an independent validation cohort with STAT3 mutations in 19/67 (28%) and mtCCL22 in 13/67 (19%). CCL22 mutations were clustered at the conserved leucine 45 and proline 79 residues (Fig. 1A). Sequencing of purified CD3+ T and CD56+ NK cells showed that the mtCCL22 were somatic mutations acquired by the CD56+ NK population. mtCCL22 defined a subgroup of CLPD-NK, with high NCAM1 (CD56) positivity by flow cytometry (p 1.3, adj P CCL22 induces chemotaxis of CCR4-expressing Th2 T cells, and also drives CCR4 internalization mediated by G-protein coupled receptor signaling via β-arrestin as a negative feedback mechanism. All three tested mtCCL22 resulted in significant and prolonged attenuation of CCR4 internalization compared to wtCCL22 (Fig 1C). Exchanging the supernatant of mtCCL22-treated Ba/F3-CCR4 cells for that from wtCCL22-treated cells decreased CCR4 expression, and vice versa, suggesting that mtCCL22 regulates CCR4 surface expression and signaling through an autocrine or paracrine mechanism (Fig 1D). Furthermore, mtCCL22 increased chemotaxis of CCR4-Ba/F3 cells in a transwell cell migration assay compared to wtCCL22 (Fig 1E). Expression of mtCCL22 enhanced engraftment of NK-92 cells which exhibited higher XCR1,GZMK and surface CD56 expression compared to controls, as well as deregulation of type I interferon response pathways indicating reactive cytokine production similar to that observed in CLPD-NK patients. Conclusions: mtCCL22 were detected in 19-22% of CLPD-NK, were mutually exclusive with STAT3 mutations, and define a distinct subgroup of CLPD-NK with genetic features that recapitulate CD56bright cytokine-producing NK cells. We show that mtCCL22 enhanced cellular chemotaxis by preventing CCR4 internalization and promoted expansion of CD56+ NK cells in vivo. We recommend to study mtCCL22 in cases suspected of CLPD-NK, in order to distinguish clonal NK neoplasms from polyclonal reactive expansion. Disclosures Feith: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees. Loughran:Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Mullighan:Pfizer: Honoraria, Research Funding, Speakers Bureau; Illumina: Consultancy, Honoraria, Speakers Bureau; AbbVie, Inc.: Research Funding.

Calcitriol, the active form of vitamin D, has been well documented to act directly on immune cells and malignant cells. Activated T cells are one of the best characterized targets of calcitriol, with effects including decreasing inflammatory cytokine output and promoting anti-inflammatory cytokine production. However, the effects of calcitriol on natural killer (NK) cells are less clear. Reports suggest that only immature NK cell populations are affected by calcitriol treatment resulting in impaired cytotoxic function and cytokine production, while mature NK cells may have little or no response. NK cell large granular lymphocyte leukemia (NK-LGLL) is a rare leukemia with CD3-CD16+CD56+NK cell clonal expansion. The current standard treatments are immunosuppressant therapies, which are not curative. The Janus kinase (JAK) – signal transducer and activator of transcription (STAT) pathway is hyperactivated in LGLL and is one pathway of interest in new drug target investigations. We previously demonstrated the ability of calcitriol to decrease STAT1 tyrosine 701 (p-STAT1) and STAT3 tyrosine 705 (p-STAT3) phosphorylation as well as inflammatory cytokine output of T cell large granular lymphocyte leukemia cells, but did not determine the effects of calcitriol on NK-LGLL. Therefore, in the present study, we investigated whether NKL cells, a model of NK-LGLL, and NK-LGLL patient peripheral blood mononuclear cells (PBMCs) are susceptible to treatment with calcitriol or seocalcitol (EB1089), a potent analog of calcitriol. NKL cells are dependent on interleukin (IL)-2 for survival and we show here for the first time that treatment with IL-2 induced tyrosine phosphorylation of STATs 1 through 6. Both calcitriol and EB1089 caused significant upregulation of the vitamin D receptor (VDR). IL-2 induction of p-STAT1 and p-STAT3 phosphorylation was significantly decreased after calcitriol or EB1089 treatment. Additionally, IL-10, interferon (IFN)-γ, and FMS-like tyrosine kinase 3 ligand (Flt-3L) extracellular output was significantly decreased at 100 nM EB1089 and intracellular IL-10 was decreased with either calcitriol or EB1089 treatment. We treated NK-LGLL patient PBMCs with calcitriol or EB1089 and found decreased p-STAT1 and p-STAT3 while VDR increased, which matched the NKL cell line data. We then measured 75 serum cytokines in NK-LGLL patients (n = 8) vs. age- and sex-matched normal healthy donors (n = 8), which is the first serum cytokine study for this LGLL subtype. We identified 15 cytokines, including IL-10 and Flt-3L, which were significantly different between normal donors and NK-LGLL patients. Overall, our results suggest that activating the vitamin D pathway could be a mechanism to decrease STAT1 and 3 activation and inflammatory cytokine output in NK-LGLL patients.

Large granular lymphocyte (LGL) leukemia results from clonal expansion of CD3(+) cytotoxic T lymphocytes or CD3(−) natural killer (NK) cells. Chronic antigen stimulation is postulated to promote long-term survival of LGL leukemia cells through constitutive activation of multiple survival pathways, resulting in global dysregulation of apoptosis and resistance to activation-induced cell death. We reported previously that nuclear factor κB (NF-κB) is a central regulator of the survival network for leukemic LGL. However, the mechanisms that trigger constitutive activation of NF-κB in LGL leukemia remain undefined. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis in tumor cells but can also activate NF-κB through interaction with TRAIL receptors 1, 2, and 4 (also known as DR4, DR5, and DcR2, respectively). The role of TRAIL has not been studied in LGL leukemia. In this study, we hypothesized that TRAIL interaction with DcR2 contributes to NF-κB activation in LGL leukemia. We observed upregulated TRAIL messenger RNA and protein expression in LGL leukemia cells with elevated levels of soluble TRAIL protein in LGL leukemia patient sera. We also found that DcR2 is the predominant TRAIL receptor in LGL leukemia cells. We demonstrated that TRAIL-induced activation of DcR2 led to increased NF-κB activation in leukemic LGL. Conversely, interruption of TRAIL-DcR2 signaling led to decreased NF-κB activation. Finally, a potential therapeutic application of proteasome inhibitors (bortezomib and ixazomib), which are known to inhibit NF-κB, was identified through their ability to decrease proliferation and increase apoptosis in LGL leukemia cell lines and primary patient cells.

T cell large granular lymphocyte leukemia (T-LGLL) is a rare incurable disease that is characterized by defective apoptosis of cytotoxic CD8+ T cells. Chronic activation of the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway is a hallmark of T-LGLL. One manifestation is the constitutive phosphorylation of tyrosine 701 of STAT1 (p-STAT1). T-LGLL patients also exhibit elevated serum levels of the STAT1 activator, interferon-γ (IFN-γ), thus contributing to an inflammatory environment. In normal cells, IFN-γ production is tightly controlled through induction of IFN-γ negative regulators. However, in T-LGLL, IFN-γ signaling lacks this negative feedback mechanism as evidenced by excessive IFN-γ production and decreased levels of suppressors of cytokine signaling 1 (SOCS1), a negative regulator of IFN-γ. Here we characterize the IFN-γ-STAT1 pathway in TL-1 cells, a cell line model of T-LGLL. TL-1 cells exhibited lower IFN-γ receptor protein and mRNA expression compared to an IFN-γ responsive cell line. Furthermore, IFN-γ treatment did not induce JAK2 or STAT1 activation or transcription of IFN-γ-inducible gene targets. However, IFN-β induced p-STAT1 and subsequent STAT1 gene transcription, demonstrating a specific IFN-γ signaling defect in TL-1 cells. We utilized siRNA targeting of STAT1, STAT3, and STAT5b to probe their role in IL-2-mediated IFN-γ regulation. These studies identified STAT5b as a positive regulator of IFN-γ production. We also characterized the relationship between STAT1, STAT3, and STAT5b proteins. Surprisingly, p-STAT1 was positively correlated with STAT3 levels while STAT5b suppressed the activation of both STAT1 and STAT3. Taken together, these results suggest that the dysregulation of the IFN-γ-STAT1 signaling pathway in TL-1 cells likely results from low levels of the IFN-γ receptor. The resulting inability to induce negative feedback regulators explains the observed elevated IL-2 driven IFN-γ production. Future work will elucidate the best way to target this pathway, with the ultimate goal to find a better therapeutic for T-LGLL.

Felty syndrome is a rare disease defined by neutropenia, splenomegaly, and rheumatoid arthritis. Sometimes the differential diagnosis between Felty syndrome and large granular lymphocyte leukemia is problematic. Recently, somatic STAT3 and STAT5B mutations were discovered in 30-40% of patients with large granular lymphocyte leukemia. Herein, we aimed to study whether these mutations can also be detected in Felty syndrome, which would imply the existence of a common pathogenic mechanism between these two disease entities. We collected samples and clinical information from 14 Felty syndrome patients who were monitored at the rheumatology outpatient clinic for Felty syndrome. Somatic STAT3 mutations were discovered in 43% (6/14) of Felty syndrome patients with deep amplicon sequencing targeting all STAT3 exons. Mutations were located in the SH2 domain of STAT3, which is a known mutational hotspot. No STAT5B mutations were found. In blood smears, overrepresentation of large granular lymphocytes was observed, and in the majority of cases the CD8(+) T-cell receptor repertoire was skewed when analyzed by flow cytometry. In bone marrow biopsies, an increased amount of phospho-STAT3 positive cells was discovered. Plasma cytokine profiling showed that ten of the 92 assayed cytokines were elevated both in Felty syndrome and large granular lymphocyte leukemia, and three of these cytokines were also increased in patients with uncomplicated rheumatoid arthritis. In conclusion, somatic STAT3 mutations and STAT3 activation are as frequent in Felty syndrome as they are in large granular lymphocyte leukemia. Considering that the symptoms and treatment modalities are also similar, a unified reclassification of these two syndromes is warranted.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a valuable therapeutic strategy for a wide variety of diseases. Acute graft-versus-host disease (aGVHD) is a major complication in up to 75% of allo-HSCT. The absence of a reliable predicative marker for aGVHD onset prevents preemptive treatment and impedes widespread and successful application of this therapy. In this study we found that after allo-HSCT, the levels of miR-181a were reduced significantly prior to the onset of aGVHD. More importantly, the degree of its reduction correlated with the severity of aGVHD. Mechanistically, miR-181a affects the function of T lymphocytes by down-regulating IFN-γ in a dose-dependent manner. Meanwhile, we confirmed that miR-181a can effectively preserve the anti-leukemic effect in vitro. Using a murine allo-HSCT model, we demonstrated that murine miR-181b, the human miR-181a homolog, served as an effective predictor of aGVHD. Moreover, expression of this microRNA ameliorated the severity of aGVHD. Collectively, these results show that the level of miR-181a may serve as a reliable marker for the diagnosis and prognosis the onset of aGVHD. Am. J. Hematol. 90:998-1007, 2015. © 2015 Wiley Periodicals, Inc.

Introduction: Large granular lymphocyte (LGL) leukemia is a rare lymphoproliferative disease of cytotoxic T lymphocytes. It almost always is a chronic, indolent disease; however, rarely it can present with an aggressive clinical course. The lack of a definitive cure for this disease warrants a review of current therapeutic options and potential future treatments.Areas covered: In this paper, we provide an overview of the clinical background of LGL leukemia and discuss its epidemiology, clinical features and diagnosis. We then explore the underlying pathophysiological changes that contribute to disease development and progression. Finally, we discuss current therapeutic strategies and our recommended therapeutic regimen.Expert opinion: We discuss our recommend therapeutic strategy and the underlying difficulty in developing a targeted approach for this disease. We discuss how the future opportunities of treatment for LGL leukemia could be explored and highlight how personalized medicine could be applied an...

Previously, we had shown that although only 8% of patients with large granular lymphocytic leukemia (LGLL) were infected with human T cell lymphoma/leukemia virus (HTLV)-2, almost half had antibodies to HTLV Gag and Env peptides. Herein, we investigated whether this could be due to cross-reactive antibodies to two homologous peptides in the Gag protein of the endogenous retrovirus HTLV-related endogenous sequence-1 (HRES-1). In addition, we had previously shown that patients with HTLV neurodegenerative diseases had increased seroreactivity to homologous HERV-K10 endogenous retrovirus peptides. Hence, in this study we also examined whether these patients had increased seroreactivity to the aforementioned HRES-1 Gag peptides. Sera from 100 volunteer blood donors (VBD), 53 patients with LGLL, 74 subjects with HTLV-1 or 2 infection (58 nonmyelopathy and 16 myelopathy), and 83 patients with multiple sclerosis (MS) were evaluated. The HTLV-positive myelopathy (HAM) patients had a statistically increased prevalence of antibodies to both HRES-1 Gag peptides (81%) vs. the VBD (0%), LGLL patients (13%), and MS patients (1%), and the HTLV-positive nonmyelopathy subjects (21%). The data suggest that cross-reactivity to HRES-1 peptides could be involved in the pathogenesis of HAM. The difference between the VBD and LGLL patients was also statistically significant, also suggesting a possible association in a minority of patients.

Large granular lymphocyte (LGL) leukemia is a chronic clonal lymphoproliferative disorder. Here, a T-LGL leukemia patient developed NK-LGL leukemia with residual leukemic T-LGL. TCRVβ usage and CDR3 sequence drifts were observed with disease progression. A STAT3 S614R mutation was identified in NK but not T-cells in the mixed leukemic stage. Multiple, non-dominant T-cell clones with distinct STAT3 mutations were present throughout. Our results suggest that T and NK-LGL leukemia may share common pathogenesis mechanisms and that STAT3 mutation alone is insufficient to bring about clonal expansion. Mutational and immunological monitoring may provide diagnostic and therapeutic significance in LGL leukemia., Highlights • Coexistence of NK and T cell clones in LGL leukemia. • Demonstration for the first time of a shift from T-LGL to NK type of LGL leukemia. • Emergence of a dominant STAT3-mutated clone in NK cells during disease progression. • Presence of additional STAT3-mutated clones that fail to become dominant over time.

This section reviews the diagnostic criteria and pathogenesis of large granular lymphocyte (LGL) leukemia. There is a particular focus on the overlap of LGL leukemia and rheumatoid arthritis (Felty's syndrome). Current understanding of the mechanisms of neutropenia in these disorders is discussed. Finally, treatment indications and therapeutic recommendations are outlined.

A facultative heterochromatin mark, histone H3 lysine 9 dimethylation (H3K9me2), which is mediated by histone methyltransferases G9a/GLP (EHMT2/1), undergoes dramatic rearrangements during myeloid cell differentiation as observed by chromatin imaging. To determine whether these structural transitions also involve genomic repositioning of H3K9me2, we used ChIP-sequencing to map genome-wide topography of H3K9me2 in normal human granulocytes, normal CD34+ hematopoietic progenitors, primary myeloblasts from acute myeloid leukemia (AML) patients, and a model leukemia cell line K562. We observe that H3K9me2 naturally repositions from the previously designated “repressed” chromatin state in hematopoietic progenitors to predominant association with heterochromatin regions in granulocytes. In contrast, AML cells accumulate H3K9me2 on previously undefined large (> 100 Kb) genomic blocks that are enriched with AML-specific single nucleotide variants, sites of chromosomal translocations, and genes downregulated in AML. Specifically, the AML-specific H3K9me2 blocks are enriched with genes regulated by the proto-oncogene ERG that promotes stem cell characteristics. The AML-enriched H3K9me2 blocks (in contrast to the heterochromatin-associated H3K9me2 blocks enriched in granulocytes) are reduced by pharmacological inhibition of the histone methyltransferase G9a/GLP in K562 cells concomitantly with transcriptional activation of ERG and ETS1 oncogenes. Our data suggest that G9a/GLP mediate formation of transient H3K9me2 blocks that are preserved in AML myeloblasts and may lead to an increased rate of AML-specific mutagenesis and chromosomal translocations.

Large granular lymphocyte (LGL) leukemia has been recognized by the World Health Organization classifications amongst mature T-cell and natural killer (NK) cell neoplasms. There are 3 categories: chronic T-cell leukemia and NK-cell lymphocytosis, which are similarly indolent diseases characterized by cytopenias and autoimmune conditions as opposed to aggressive NK-cell LGL leukemia. Clonal LGL expansion arise from chronic antigenic stimulation, which promotes dysregulation of apoptosis, mainly due to constitutive activation of survival pathways including Jak/Stat, MapK, phosphatidylinositol 3-kinase–Akt, Ras–Raf-1, MEK1/extracellular signal-regulated kinase, sphingolipid, and nuclear factor-κB. Socs3 downregulation may also contribute to Stat3 activation. Interleukin 15 plays a key role in activation of leukemic LGL. Several somatic mutations including Stat3, Stat5b, and tumor necrosis factor alpha-induced protein 3 have been demonstrated recently in LGL leukemia. Because these mutations are present in less than half of the patients, they cannot completely explain LGL leukemogenesis. A better mechanistic understanding of leukemic LGL survival will allow future consideration of a more targeted therapeutic approach than the current practice of immunosuppressive therapy.

T-cell large granular lymphocytic leukemia and chronic lymphoproliferative disorder of natural killer cells are intriguing entities between benign and malignant lymphoproliferation. The molecular pathogenesis has partly been uncovered by the recent discovery of somatic activating STAT3 and STAT5b mutations. Here we show that 43% (75/174) of patients with T-cell large granular lymphocytic leukemia and 18% (7/39) with chronic lymphoproliferative disorder of natural killer cells harbor STAT3 mutations when analyzed by quantitative deep amplicon sequencing. Surprisingly, 17% of the STAT3-mutated patients carried multiple STAT3 mutations, which were located in different lymphocyte clones. The size of the mutated clone correlated well with the degree of clonal expansion of the T-cell repertoire analyzed by T-cell receptor beta chain deep sequencing. The analysis of sequential samples suggested that current immunosuppressive therapy is not able to reduce the level of the mutated clone in most cases, thus warranting the search for novel targeted therapies. Our findings imply that the clonal landscape of large granular lymphocytic leukemia is more complex than considered before, and a substantial number of patients have multiple lymphocyte subclones harboring different STAT3 mutations, thus mimicking the situation in acute leukemia.

BACKGROUND: Mutations to epigenetic and spliceosome regulators frequently occur in acute myeloid leukemia (AML) and contribute to the underlying pathobiology of the disease. In addition, recent studies have sought to improve the anti-AML efficacy of sphingolipid-based therapeutics by identifying the underlying mechanisms responsible for dysfunctional sphingolipid metabolism. Sphingolipids are an extensive classification of lipids that play profound roles in membrane structure as well as regulation of cellular function and fate. Ceramide, as the hydrophobic moiety of sphingolipids, serves as the hypothetical center of sphingolipid metabolism. It regulates cellular stress responses and apoptosis, whereas many of its metabolites regulate opposing processes such as proliferation and survival. Growth/differentiation factor 1 (GDF1) is a TGF-beta superfamily member, of which other members have been attributed roles in stem cell biology and hematopoiesis. Mutations in GDF1 have been found to be associated with congenital cardiovascular malformations, yet little is known about the role of GDF1 outside of cardiac development. GDF1 is encoded from a bicistronic gene that also encodes for ceramide synthase 1 (CERS1), which is responsible for generating the C18:0 species of ceramide. Recently, an anti-AML role was attributed to CERS1 in FLT3-ITD-mutated AML (Dany et. al. Blood 2016). However, a potential role for GDF1 was not evaluated in this study. Intriguingly, in preliminary work using FLT3ITD transgenic mice we observed an inverse correlation between the expression of GDF1 and UGCG which encodes for the ceramide detoxifying enzyme glucosylceramide synthase. Therefore, our present study tested the hypothesis that GDF1, encoded from the bicistronic CERS1-GDF1 gene, exerts anti-AML efficacy by downregulating ceramide neutralization and promoting stem cell differentiation METHODS & RESULTS: Studies were carried out using AML cell lines as well as primary cells harvested from transgenic murine models of myelodysplastic syndrome (MDS) and AML (FLT3ITD, Nup98-HoxD13, Srsf2P95H-mutant, Tet2-deficient, Asxl1-deficient, and Tert-deficient). The expression of GDF1 was evaluated by real time qPCR in hematopoietic cells isolated from these models of MDS and AML. GDF1 expression was greatest in Srsf2P95H-mutant (MDS) samples, which corresponded to enhanced sensitivity to ceramide-based therapies. We had recently reported this MDS-specific sensitivity for nanoliposomal ceramide (Barth et. al. Blood Advances 2019), which is a ceramide-based therapy currently in a clinical trial for solid tumor malignancies (ClinicalTrials.gov identifier: NCT02834611). Next, cell lines and models were treated with recombinant GDF1. Real time qPCR revealed that GDF1 treatment downregulated the expression of the genes in the ceramide neutralization pathway including UGCG. In addition, flow cytometry was used to show that GDF1 treatment promoted hematopoietic stem cell differentiation. Lastly, C57BL/6J mice engrafted with C1498 AML cells was used to show that GDF1 enhanced the therapeutic efficacy of cytarabine and nanoliposomal ceramide. CONCLUSIONS: Overall, this study provided evidence for differential expression of GDF1 in subtypes of MDS and AML and showed that GDF1 can regulate sphingolipid metabolism by downregulating ceramide neutralization. Importantly, we have demonstrated that GDF1 exerts anti-AML efficacy in combination with either standard care therapy or ceramide-based therapy. Therefore, GDF1-elevating strategies are well-positioned as novel therapeutic approaches for the treatment of AML and related myeloid hematological disorders. This work was funded by NIH/NCI K22 CA190674 (B.M.B.) and University of New Hampshire COBRE Pilot Project Grant NIH/NIGMS P20 GM113131 (B.M.B.). The authors acknowledge US Provisional Patent 62/602,437, issued to B.M.B. and the University of New Hampshire. Disclosures Loughran: Bioniz: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees. Barth:University of New Hampshire: Patents & Royalties: US Provisional Patent 62/602,437; NIH (NCI and NIGMS): Research Funding.

Myelodysplastic syndrome (MDS) is a clonal hematopoietic disorder characterized by ineffective hematopoiesis, cytopenias, and an increased risk of transformation to acute myeloid leukemia. New studies are urgently needed to understand the underlying processes that govern MDS and related bone marrow failure disorders and to develop better therapeutic modalities. Sphingolipids are an extensive classification of lipids which play prominent roles in cellular signaling in addition to being essential components of membranes. The most well studied sphingolipid is ceramide, which serves as a hypothetical center of sphingolipid metabolism. Ceramide is an important cellular signal that can lead to apoptosis. Overall ceramide biology is significant because novel metabolic routes that persist in MDS may be exploited for therapeutic development. Interestingly, the gene encoding ceramide synthase 1 is encoded from a bicistronic transcript that also encodes for GDF1. Little is known about the roles of GDF1 outside of cardiac development. However, GDF1 is in the TGF-beta superfamily of which many members have been attributed roles in stem cell biology. Therefore, a better understanding of the role of GDF1 and its regulation of ceramide metabolism and hematopoiesis may lead to better treatment approaches for MDS and other bone marrow failure syndromes. The current study tested an overarching hypothesis that GDF1 regulates SMAD and STAT signaling to promote ceramide generation and restore effective myelopoiesis in MDS. Initially, the expression of GDF1 was evaluated in hematopoietic cells isolated from transgenic murine models of MDS (Nup98-HoxD13; Srsf2P95H-mutant) as well as myeloproliferation (Flt3ITD). Interestingly, GDF1 expression was greatest in the bone marrow of MDS models. This was significant because we recently reported that nanoliposomal ceramide (Lip-C6), which delivers a bioactive ceramide analog, exerts unique therapeutic efficacy towards acute myeloid leukemia arising out of MDS (Barth et. al. Blood Advances 2019). Lip-C6 is a ceramide-based therapy that currently is in a clinical trial for solid tumor malignancies (ClinicalTrials.gov identifier: NCT02834611). Next, treatment of transgenic MDS mice with either recombinant GDF1 or Lip-C6 was shown to stimulate the expansion of erythroid progenitors. This was concomitant with a decrease in immature myeloid cells, which was revealed to be due to granulo-monocytopoietic differentiation. Mechanistic studies subsequently revealed that GDF1 increased SMAD2/3 phosphorylation while simultaneously down regulating STAT3 (Y705) phosphorylation, both in a TGF-beta receptor 1-dependent fashion. This dual effect was unique to GDF1, whereas TGF-beta or GDF3 only recapitulated individual the effects on SMAD2/3 and STAT3 signaling, respectively. Finally, STAT3 binding sites were identified in the promoter region of the ceramide detoxifying enzyme glucosylceramide synthase. This is important because glucosylceramide synthase expression was shown to be downregulated by recombinant GDF1 treatment. Therefore, GDF1-mediated downregulation of STAT3-dependent glucosylceramide synthase expression provides a mechanistic link where GDF1 can augment intracellular ceramide levels. Overall, this study enhances our understanding of a fundamental hematopoietic process where GDF1 regulates ceramide metabolism and myelopoiesis. This is impactful because the effects of GDF1, which are unique from other related factors including TGF-beta and GDF3, are due to a novel mechanism of action that both upregulates SMAD2/3 signaling while downregulating STAT3 signaling. More so, this study demonstrated that GDF1, and/or ceramide, can exert an anti-MDS therapeutic role by restoring normal aspects of myelopoiesis. This work was funded by NIH/NCI K22 CA190674 (B.M.B.) and University of New Hampshire COBRE Pilot Project Grant NIH/NIGMS P20 GM113131 (B.M.B.). The authors acknowledge US Provisional Patent 62/602,437, issued to B.M.B. and the University of New Hampshire. Disclosures Barth: University of New Hampshire: Patents & Royalties: US Provisional Patent 62/602,437; NIH (NCI and NIGMS): Research Funding. Loughran:Bioniz: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees.

Large granular lymphocyte (LGL) leukemia is an indolent lymphoproliferative malignancy which dysregulates humoral immunity and underlies the myriad autoimmune phenomena. We describe a 62-year-old woman with Felty's syndrome who developed a severe bleeding diathesis. Laboratory evaluation demonstrated acquired inhibitors to both factor VIII (FVIII) and fibrinogen, likely secondary to T-cell LGL leukemia. After a complicated course, the patient's inhibitors were extinguished with rituximab and high-dose corticosteroids. Bleeding was controlled with alternating FEIBA (factor eight inhibitor bypassing activity) and recombinant activated FVII. This report reviews the literature comparing the efficacy of various treatment modalities for both disorders. To our knowledge, this is the first reported case of a patient with LGL leukemia acquiring an inhibitor to FVIII or fibrinogen.

Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias and can cooccur in the context of aplastic anemia (AA) and myelodysplastic syndromes (MDS). We took advantage of the recent description of signal transducer and activator of transcription 3 (STAT3) mutations in LGL clonal expansions to test, using sensitive methods, for the presence of these mutations in a large cohort of 367 MDS and 140 AA cases. STAT3 clones can be found not only in known LGL concomitant cases, but in a small proportion of unsuspected ones (7% AA and 2.5% MDS). In STAT3-mutated AA patients, an interesting trend toward better responses of immunosuppressive therapy and an association with the presence of human leukocyte antigen-DR15 were found. MDSs harboring a STAT3 mutant clone showed a lower degree of bone marrow cellularity and a higher frequency of developing chromosome 7 abnormalities. STAT3-mutant LGL clones may facilitate a persistently dysregulated autoimmune activation, responsible for the primary induction of bone marrow failure in a subset of AA and MDS patients.

Large granular lymphocytic (LGL) leukemia is characterized by clonal expansion of cytotoxic T cells or natural killer cells. Recently, somatic mutations in the signal transducer and activator of transcription 3 (STAT3) gene were discovered in 28% to 40% of LGL leukemia patients. By exome and transcriptome sequencing of 2 STAT3 mutation-negative LGL leukemia patients, we identified a recurrent, somatic missense mutation (Y665F) in the Src-like homology 2 domain of the STAT5b gene. Targeted amplicon sequencing of 211 LGL leukemia patients revealed 2 additional patients with STAT5b mutations (N642H), resulting in a total frequency of 2% (4 of 211) of STAT5b mutations across all patients. The Y665F and N642H mutant constructs increased the transcriptional activity of STAT5 and tyrosine (Y694) phosphorylation, which was also observed in patient samples. The clinical course of the disease in patients with the N642H mutation was aggressive and fatal, clearly different from typical LGL leukemia with a relatively favorable outcome. This is the first time somatic STAT5 mutations are discovered in human cancer and further emphasizes the role of STAT family genes in the pathogenesis of LGL leukemia.

Evaluation of: Waldmann TA, Conlon KC, Stewart DM et al. Phase 1 trial of IL-15 trans presentation blockade using humanized Mik-β-1 mAb in patients with T-cell large granular lymphocytic leukemia. Blood 121(3), 476–484 (2013).IL-15 is a cytokine that stimulates the proliferation of NK and T cells. Previous studies have shown that IL-15 is critical to the induction of T-cell large granular lymphocyte (T-LGL) leukemia. A Phase I trial of a humanized antibody (Hu-Mikβ1) to the IL2/IL15Rβ receptor, expressed on T-LGL, is explored in this trial to evaluate the safety, pharmacokinetics, specificity and clinical efficacy of Hu-Mikβ1. The study demonstrated no toxicity and favorable saturation of IL2/IL15Rβ receptor, but no clinical efficacy in this Phase I study.

Evaluation of: Mishra A, Liu S, Sams GH et al. Aberrant overexpression of IL-15 initiates large granular lymphocyte leukemia through chromosomal instability and DNA hypermethylation. Cancer Cell 22(5), 645–655 (2012). There is increasing evidence identifying a link between inflammation and cancer. A potent proinflammatory cytokine, IL-15, stimulates the proliferation and maintenance of both NK and T cells, and it is therefore likely that it may play a prominent role in certain hematologic malignancies. Previous studies have demonstrated that IL-15 overexpression can initiate leukemic transformation in murine models and that both NK- and T-cell malignancies can develop; the mechanism is explored in this article. The authors illustrate that IL-15 can cause chromosomal instability and DNA hypermethylation in large granular lymphocytes. These aberrations led to an aggressive acute large granular lymphocyte leukemia. Through studying the affected pathways, the authors were able to identify potential therapeutic targets and induce remission in a murine model.

To the editor: Large granular lymphocyte (LGL) leukemia is a group of chronic lymphoproliferative disorders of cytotoxic T or natural killer (NK) cells frequently complicated with cytopenia and autoimmune phenomena.[1][1],[2][2] In the current World Health Organization (WHO) classification, T-LGL

Telomeres are specialized structures providing chromosome integrity during cellular division along with protection against premature senescence and apoptosis. Accelerated telomere attrition in patients with myelodysplastic syndrome (MDS) occurs by an undefined mechanism. Although the MDS clone originates within the myeloid compartment, T-lymphocytes display repertoire contraction and loss of naive T-cells. The replicative lifespan of T-cells is stringently regulated by telomerase activity. In MDS cases, we show that purified CD3+ T-cells have significantly shorter telomere length and reduced proliferative capacity upon stimulation compared with controls. To understand the mechanism, telomerase enzymatic activity and telomerase reverse transcriptase (hTERT), gene expression were compared in MDS cases (n=35) and healthy controls (n=42) within different T-cell compartments. Telomerase activity is greatest in naive T-cells illustrating the importance of telomere repair in homeostatic repertoire regulation. Compared with healthy controls, MDS cases had lower telomerase induction (P

Chronic lymphoproliferative disorders of natural killer cells (CLPD-NKs) and T-cell large granular lymphocytic leukemias (T-LGLs) are clonal lymphoproliferations arising from either natural killer cells or cytotoxic T lymphocytes (CTLs). We have investigated for distribution and functional significance of mutations in 50 CLPD-NKs and 120 T-LGL patients by direct sequencing, allele-specific PCR, and microarray analysis. STAT3 gene mutations are present in both T and NK diseases: approximately one-third of patients with each type of disorder convey these mutations. Mutations were found in exons 21 and 20, encoding the Src homology 2 domain. Patients with mutations are characterized by symptomatic disease (75%), history of multiple treatments, and a specific pattern of STAT3 activation and gene deregulation, including increased expression of genes activated by STAT3. Many of these features are also found in patients with wild-type STAT3, indicating that other mechanisms of STAT3 activation can be operative in these chronic lymphoproliferative disorders. Treatment with STAT3 inhibitors, both in wild-type and mutant cases, resulted in accelerated apoptosis. STAT3 mutations are frequent in large granular lymphocytes suggesting a similar molecular dysregulation in malignant chronic expansions of NK and CTL origin. STAT3 mutations may distinguish truly malignant lymphoproliferations involving T and NK cells from reactive expansions.

Large granular lymphocyte (LGL) leukemia is a clonal lymphoproliferative disorder of cytotoxic lymphocytes characterized by an expansion of CD3+ cytotoxic T lymphocytes or CD3- natural killer cells. Patients present with various cytopenias including neutropenia, anemia and thrombocytopenia. In addition, there is an association of T-cell large granular lymphocytic leukemia with rheumatoid arthritis. It is believed that LGL leukemia begins as an antigen-driven immune response with subsequent constitutive activation of cytotoxic T lymphocytes or natural killer cells through PDGF and IL-15 contributing to their survival. Consequently, this leads to a dysregulation of apoptosis and dysfunction of the activation-induced cell death pathway. Treatment of LGL leukemia is based on a low-dose immunosuppressive regimen using methotrexate or cyclophosphamide. However, no standard of therapy has been established, as large prospective trials have not been conducted. In addition, some patients are refractory to treatment. The lack of a curative therapy for LGL leukemia means that new treatment options are needed. Insight into the various dysregulated signaling pathways in LGL leukemia may provide novel therapeutic treatment modalities.

NK-cell leukemia is a clonal expansion of NK cells. The illness can occur in an aggressive or chronic form. We studied cell lines from human and rat NK-cell leukemias (aggressive NK-cell leukemia) as well as samples from patients with chronic NK-cell leukemia to investigate pathogenic mechanisms. Here we report that Mcl-1 was overexpressed in leukemic NK cells and that knockdown of Mcl-1 induced apoptosis in these leukemic cells. In vitro treatment of human and rat NK leukemia cells with FTY720 led to caspase-dependent apoptosis and decreased Mcl-1 expression in a time- and-dose-dependent manner. These biologic effects could be inhibited by blockade of reactive oxygen species generation and the lysosomal degradation pathway. Lipidomic analyses after FTY720 treatment demonstrated elevated levels of sphingosine, which mediated apoptosis of leukemic NK cells in vitro. Importantly, systemic administration of FTY720 induced complete remission in the syngeneic Fischer rat model of NK-cell leukemia. Therapeutic efficacy was associated with decreased expression of Mcl-1 in vivo. These data demonstrate that therapeutic benefit of FTY720 may result from both altered sphingolipid metabolism as well as enhanced degradation of a key component of survival signaling.

Leukemia is one of the most common and aggressive adult cancers, as well as the most prevalent childhood cancer. Leukemia is a cancer of the hematological system and can be divided into a diversity of unique malignancies based on the onset of the disease as well as the specific cell lineages involved. Cancer stem cells, including recently identified leukemia stem cells (LSCs), are hypothesized to be responsible for cancer development, relapse, and resistance to treatment, and new therapeutics targeting these cellular populations are urgently needed. Nontoxic and nonaggregating calcium phosphosilicate nanoparticles (CPSNPs) encapsulating the near-infrared fluoroprobe indocyanine green (ICG) were recently developed for diagnostic imaging and drug delivery as well as for photodynamic therapy (PDT) of solid tumors. Prior studies revealed that specific targeting of CPSNPs allowed for enhanced accumulation within breast cancer tumors, via CD71 targeting, or pancreatic cancer tumors, via gastrin receptor targeting. In the present study, ICG-loaded CPSNPs were evaluated as photosensitizers for PDT of leukemia. Using a novel bioconjugation approach to specifically target CD117 or CD96, surface features enhanced on leukemia stem cells, in vitro ICG-CPSNP PDT of a murine leukemia cell line and human leukemia samples were dramatically improved. Furthermore, the in vivo efficacy of PDT was dramatically enhanced in a murine leukemia model by utilizing CD117-targeted ICG-CPSNPs, resulting in 29% disease-free survival. Altogether, this study demonstrates that leukemia-targeted ICG-loaded CPSNPs offer the promise to effectively treat relapsing and multidrug-resistant leukemia and to improve the life of leukemia patients.

Natural killer (NK) cell lymphomas/leukemias are rare neoplasms with an aggressive clinical behavior. The majority of the cases belong to extranodal NK/T-cell lymphoma, nasal type (ENKTL) in the current WHO classification scheme. Gene-expression profiling (GEP) of 21 ENKTL and NK-cell lymphoma/leukemia patients, 17 NK- and T-cell lines and 5 indolent NK-cell large-granular-lymphocytic proliferation was performed and compared with 125 peripheral T-cell lymphoma (PTCL) patients previously studied. The molecular classifier derived for ENKTL patients was comprised of 84 transcripts with the majority of them contributed by the neoplastic NK cells. The classifier also identified a set of gamma delta-PTCLs both in the ENKTL cases as well as in cases initially classified as PTCL-not otherwise specified. These gamma delta-PTCLs expressed transcripts associated with the T-cell receptor (TCR)/CD3 complex, suggesting T cell rather than NK-cell lineage. They were very similar to NK-cell tumors by GEP, but were distinct from cytotoxic (alpha beta)-PTCL and hepatosplenic T-cell lymphoma, indicating derivation from an ontogenically and functionally distinct subset of gamma delta T cells. They showed distinct expression of V gamma 9, V delta 2 transcripts and were positive for TCR gamma, but negative for TCR beta by immuno-histochemistry. Targeted inhibition of two oncogenic pathways (AURKA and NOTCH-1) by small-molecular inhibitors induced significant growth arrest in NK-cell lines, thus providing a rationale for clinical trials of these inhibitors in NK-cell malignancies. Leukemia (2011) 25, 348-358; doi:10.1038/leu.2010.255; published online 5 November 2010

Large granular lymphocyte (LGL) leukemia results from chronic expansion of cytotoxic T cells or natural killer (NK) cells. Apoptotic resistance resulting from constitutive activation of survival signaling pathways is a fundamental pathogenic mechanism. Recent network modeling analyses identified platelet-derived growth factor (PDGF) as a key master switch in controlling these survival pathways in T-cell LGL leukemia. Here we show that an autocrine PDGF regulatory loop mediates survival of leukemic LGLs of both T- and NK-cell origin. We found high levels of circulating PDGF-BB in platelet-poor plasma samples from LGL leukemia patients. Production of PDGF-BB by leukemic LGLs was demonstrated by immunocytochemical staining. Leukemic cells expressed much higher levels of PDGFR-β transcripts than purified normal CD8+ T cells or NK cells. We observed that phosphatidylinositol-3-kinase (PI3 kinase), Src family kinase (SFK), and downstream protein kinase B (PKB)/AKT pathways were constitutively activated in both T- and NK-LGL leukemia. Pharmacologic blockade of these pathways led to apoptosis of leukemic LGLs. Neutralizing antibody to PDGF-BB inhibited PKB/AKT phosphorylation induced by LGL leukemia sera. These results suggest that targeting of PDGF-BB, a pivotal regulator for the long-term survival of leukemic LGLs, may be an important therapeutic strategy.

Samples were obtained from 53 large granular lymphocytic leukemia (LGLL) patients and 10,000 volunteer blood donors (VBD). Sera were screened in an HTLV-1 enzyme immunoassay (EIA) and further analyzed in peptide-specific Western blots (WB). DNAs were analyzed by HTLV-1, -2, -3, and -4-specific PCR. Forty four percent of LGLL patients vs. 0.12 % of VBD had anti-HTLV antibodies via EIA (p < 0.001). WB and PCR revealed that four LGLL patients (7.5%) vs. one VBD patient (0.01%) were infected with HTLV-2 (p < 0.001), suggesting an HTLV-2 etiology in a minority of cases. No LGLL patient was positive for HTLV-1, -3, or -4, whereas only one EIA-positive VBD was positive for HTLV-1 and none for HTLV-3 or -4. The HTLV EIA-positive, PCR-negative LGLL patients' sera reacted to epitopes within HTLV p24 gag and gp21 env. Other then the PTLV/BLV viruses, human endogenous retroviral element HERV K10 was the only sequence homologous to these two HTLV peptides, raising the possibility of cross-reactivity. Although three LGLL patients (5.7%) vs. none of 110 VBD patients tested positive for antibodies to the homologous HERV K10 peptide (p = 0.03), the significance of the anti-HTLV seroreactivity observed in many LGLL patients remains unclear. Interestingly, out of 36 HTLV-1-positive control subjects, 3 (8%) (p = 0.014) were positive for antibodies to HERV K10; all three had myelopathy. Out of 64 HTLV-2-positive control subjects 16 (25%) (p =

Large granular lymphocyte (LGL) leukemia is a rare disorder of mature cytotoxic T or natural killer cells. Large granular lymphocyte leukemia is characterized by the accumulation of cytotoxic cells in blood and infiltration in the bone marrow, liver, and spleen. Herein, we review clinical features of LGL leukemia. We focus our discussion on known survival signals believed to play a role in the pathogenesis of LGL leukemia and their potential therapeutic implications.

Large granular lymphocyte (LGL) leukemia is a clonal proliferative disease of T and natural killer (NK) cells. Interleukin (IL)-15 is important for the development and progression of LGL leukemia and is a survival factor for normal NK and T memory cells. IL-15 alters expression of Bcl-2 family members, Bcl-2, Bcl-XL, Bim, Noxa, and Mcl-1; however, effects on Bid have not been shown. Using an adoptive transfer model, we show that NK cells from Bid-deficient mice survive longer than cells from wild-type control mice when transferred into IL-15-null mice. In normal human NK cells, IL-15 significantly reduces Bid accumulation. Decreases in Bid are not due to alterations in RNA accumulation but result from increased proteasomal degradation. IL-15 up-regulates the E3 ligase HDM2 and we find that HDM2 directly interacts with Bid. HDM2 suppression by short hairpin RNA increases Bid accumulation lending further support for HDM2 involvement in Bid degradation. In primary leukemic LGLs, Bid levels are low but are reversed with bortezomib treatment with subsequent increases in LGL apoptosis. Overall, these data provide a novel molecular mechanism for IL-15 control of Bid that potentially links this cytokine to leukemogenesis through targeted proteasome degradation of Bid and offers the possibility that proteasome inhibitors may aid in the treatment of LGL leukemia. [Cancer Res 2009;69(9):3986–94]

Large granular lymphocyte (LGL) leukemia is commonly associated with poor hematopoiesis. The first case of pulmonary artery hypertension (PAH) was observed in a 57-year-old woman with natural killer (NK)–LGL leukemia and transfusion-dependent anemia. Using a genetic approach, we demonstrated that killing of pulmonary endothelial cells by patient NK cells was mediated by dysregulated balance in activating and inhibitory NK-receptor signaling. Elevated pulmonary artery pressure and erythroid differentiation improved after disrupting the NK-receptor signaling pathway with 4 courses of a farnesyltransferase inhibitor, tipifarnib. Coincidental association between PAH and LGL leukemia suggest a causal relationship between the expanded lymphocyte population and these clinical manifestations. This trial is registered at www.ClinicalTrials.gov as NCI 6823.

T cell large granular lymphocyte (T-LGL) leukemia features a clonal expansion of antigen-primed, competent, cytotoxic T lymphocytes (CTL). To systematically understand signaling components that determine the survival of CTL in T-LGL leukemia, we constructed a T-LGL survival signaling network by integrating the signaling pathways involved in normal CTL activation and the known deregulations of survival signaling in leukemic T-LGL. This network was subsequently translated into a predictive, discrete, dynamic model. Our model suggests that the persistence of IL-15 and PDGF is sufficient to reproduce all known deregulations in leukemic T-LGL. This finding leads to the following predictions: ( i ) Inhibiting PDGF signaling induces apoptosis in leukemic T-LGL. ( ii ) Sphingosine kinase 1 and NFκB are essential for the long-term survival of CTL in T-LGL leukemia. ( iii ) NFκB functions downstream of PI3K and prevents apoptosis through maintaining the expression of myeloid cell leukemia sequence 1. ( iv ) T box expressed in T cells (T-bet) should be constitutively activated concurrently with NFκB activation to reproduce the leukemic T-LGL phenotype. We validated these predictions experimentally. Our study provides a model describing the signaling network involved in maintaining the long-term survival of competent CTL in humans. The model will be useful in identifying potential therapeutic targets for T-LGL leukemia and generating long-term competent CTL necessary for tumor and cancer vaccine development.

T-cell large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of CD3+CD8+ cells. Leukemic LGLs correspond to terminally differentiated effector-memory cytotoxic T lymphocytes (CTLs) that escape Fas-mediated activation-induced cell death (AICD) in vivo. The gene expression signature of peripheral blood mononuclear cells from 30 LGL leukemia patients showed profound dysregulation of expression of apoptotic genes and suggested uncoupling of activation and apoptotic pathways as a mechanism for failure of AICD in leukemic LGLs. Pathway-based microarray analysis indicated that balance of proapoptotic and antiapoptotic sphingolipid-mediated signaling was deregulated in leukemic LGLs. We further investigated sphingolipid pathways and found that acid ceramidase was constitutively overexpressed in leukemic LGLs and that its inhibition induced apoptosis of leukemic LGLs. We also showed that S1P5 is the predominant S1P receptor in leukemic LGLs, whereas S1P1 is down-regulated. FTY720, a functional antagonist of S1P-mediated signaling, induced apoptosis in leukemic LGLs and also sensitized leukemic LGLs to Fas-mediated death. Collectively, these results show a role for sphingolipid-mediated signaling as a mechanism for long-term survival of CTLs. Therapeutic targeting of this pathway, such as use of FTY720, may have efficacy in LGL leukemia.

We report on the clinico-biological characteristics of 20 cases of gammadelta T cell large granular lymphocyte (LGL) leukemia. All the data were compared to that of 196 cases with alphabeta T cell subtype, which represents the majority of T cell LGL leukemias. Clinical findings were quite similar in the two groups regarding age, sex ratio, recurrent infections, and association with auto-immune diseases especially rheumatoid arthritis. Gammadelta LGL predominantly expressed a CD3+/CD4-/CD8+/CD16+/CD57+ phenotype, in 50% of cases. Clinical outcome was favorable for these patients with overall survival of 85% at 3 years. Fifty percent of gammadelta patients required treatment and the response to therapy was estimated at 55%. gammadelta and alphabeta T cell LGL leukemia harbor a very similar clinico-biological behavior and represent part of an antigen-driven T cell lymphoproliferation.

Clonal disorders of large granular lymphocytes (LGLs) represent a spectrum of biologically distinct lymphoproliferative diseases originating either from mature T cells (CD3+) or natural killer (NK) cells (CD3-). Both subtypes, T-cell and NK-cell LGL leukemia, can manifest as indolent or aggressive disorders. The majority of patients with T-cell LGL leukemia have a clinically indolent course with a median survival time >10 years. Immunosuppressive therapy with low-dose methotrexate, cyclophosphamide, or cyclosporine A can control symptoms and cytopenias in more than 50% of patients, but this approach is not curative. Several cases of an aggressive variant (CD3+ CD56+) of T-cell LGL leukemia with a poor prognosis have also been reported. Aggressive NK-cell LGL leukemia is usually a rapidly progressive disorder associated with Epstein-Barr virus (EBV), with a higher prevalence in Asia and South America. This disease is usually refractory to conventional chemotherapy, with a median survival time of 2 months. Chronic NK-cell leukemia/lymphocytosis is a rare EBV-negative disorder with an indolent clinical course. The malignant origin of this subtype is uncertain because clonality is difficult to determine in LGLs of NK-cell origin.