Large Granular Lymphocyte Leukemia Research Articles

Survival Analysis of Patients with T-Cell Lymphoma or T-Cell Large Granular Leukemia and Concomitant Plasma Cell Dyscrasias

Introduction:Sporadic cases of patients with a combined T-cell malignancy and plasma cell dyscrasia (PCD) have been reported in the literature. While the most commonly observed association is with T-cell large granular lymphocytic leukemia (T-LGLL) and PCD, there are case reports of other T-cell malignancies and PCD, such as peripheral T-cell lymphoma (PTCL) and angioimmunoblastic T-cell lymphoma (AITL) with multiple myeloma (MM). Nearly one-third of MM patients develop clonal T-cell populations that share a similar immunophenotype to T-LGLL, suggesting likely under diagnosis of concomitant T-cell malignancies in patients with PCD. However, with the limited data available regarding the overlap between PCD and T-cell malignancy, the significance, associated pathogenesis, and impact on survival outcomes is unknown. The purpose of this study is to describe the outcomes of patients with overlapping T-cell malignancies and PCD in order to determine the survival outcomes and ultimately make management/diagnostic recommendations.Methods:In this IRB approved study, we retrospectively evaluated patients with concomitant T-cell malignancy and PCD at Ohio State University from 2010-2020. Patients were identified using a database search for T-cell malignancies as well as PCD. All patients that were included met the 2016 World Health Organization diagnostic criteria for their respective T-cell malignancy and PCD. Progression-free survival (PFS) was measured as time from the start of treatment until first progression, death, or last follow-up according to the Kaplan-Meier method with median survival times and 95% confidence intervals reported.Results:A total of 21 patients, with a median follow-up time of 22 months (range 1-153), were included in this analysis. Baseline demographics are in table 1. The most common T-cell malignancy was T-LGLL (11/21; 52%) and the most common PCD were MGUS (8/21; 38%) and MM (8/21; 38%). Ten (48%) patients presented with a T-cell malignancy as their primary malignancy, 9 (43%) presented with a PCD as their primary malignancy, 1 (5%) patient was diagnosed with both at the same time, and for 1 (5%) patient it is unknown. Within the cohort, 62% (13/21) of patients received primary treatment for their T-cell malignancy and 38% (8/21) of patients received primary treatment for their PCD. Of the 7 patients that had their PCD clone 4 were treated concomitantly and 3 were treated for only their T-cell malignancy. Overall, 9/21 (42.9%) of patients had progression of their T-cell malignancy. A summary of outcomes is provided in Table 2. 54.6% of patients with T-LGLL and 60% of patients with AITL/PTCL experienced progressive T-cell disease and no patients with CTCL had progressive T-cell disease. The median overall survival (OS) across all patients was 4.1 years. Median OS was not reached for patients with T-LGLL, 1.7 years for AITL/PTCL, and 12.4 years for CTCL. PFS was 11 months for patients with T-LGLL, 1 year for AITL/PTCL, and 12.37 years for CTCL. Survival probability is shown in Figure 1. The rates of progression, OS, and PFS were consistent with previously published data for patients with these T-cell malignancies.Conclusions:Herein, we characterize a cohort of patients with concomitant T-cell malignancies and PCD with an emphasis on survival outcomes. Our data suggests that there is no PFS or OS difference for patients with T-cell malignancies and concomitant PCD when treated with standard T-cell directed therapy. There is the potential that treating a patient's T-cell malignancy may lead to resolution of their PCD clone, even without therapy directed at the PCD. While our data is limited by small sample size, this report represents the largest data set available in this rarely described patient population. Larger retrospective cohort studies are needed to further characterize this population and validate these findings. For patients with T-cell malignancies as the primary diagnosis with concomitant PCD, treatment with standard T-cell directed therapies is recommended with continued follow-up and monitoring of the concomitant PCD. [Display omitted] DisclosuresBumma: Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees. Porcu: Viracta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Daiichi: Honoraria, Research Funding; Kiowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spectrum: Consultancy; DrenBio: Consultancy. Brammer: Seattle Genetics: Speakers Bureau; Celgene: Research Funding; Kymera Therapeutics: Consultancy.

Cyclophosphamide Induces Durable Molecular and Clinical Responses in Patients with Relapsed T-Large Granular Lymphocytic Leukemia (T-LGLL)

T-cell large granular lymphocytic leukemia (T-LGLL) is a clonal proliferation of cytotoxic T-lymphocytes that can result in severe cytopenias. The management of T-LGLL is immune-suppressive therapy, with methotrexate (MTX), cyclophosphamide (Cy), and cyclosporine (CsA) serving as primary frontline agents. While MTX has been the main front line agent to treat T-LGLL, overall response rates (ORR) are less than 40%, with complete response (CR) rates of only 5%. Data from the ECOG5998 (E5998) prospective trial and French cohort studies suggest an improved response with Cy in the 2 nd line setting. Anecdotal evidence suggests that Cy may eradicate the T-LGLL clone, producing complete molecular remission (CMR), which has not been observed with MTX or CsA. The degree to which a CMR can be attained and the lengths of such remissions with Cy remains unknown, particularly in the relapsed setting. We evaluated patients treated with Cy, to assess the duration of response and degree of CMR.We retrospectively evaluated patients treated for T-LGLL with oral Cy. Diagnosis of was based on 2016 World Health Organization Criteria. Patients needed a CD3+ CD8+ population on flow cytometry ≥500 cells/mm 3 and a positive monoclonal T-cell receptor (TCR) by PCR or restriction of TCR-Vbeta on flow cytometry. TCR-Vbeta rearrangement was deemed positive if one or more clone was detected in ≥10% of events. Disease response was defined by the E5998 study criteria. CMR was defined as CR by E5998 criteria and clearance of the TCR PCR gene rearrangement or TCR-VBeta flow cytometry. Time to response (TTR) was measured as time from start of Cy until partial response (PR) or CR, with patients who failed to respond being censored at the end of Cy treatment. Leukemia-free survival (LFS) in patients responding to Cy was measured as time from start of Cy until progression. Patients without progression were censored at last follow up. TTR and LFS were compared across variables using Kaplan-Meier curves with median survival and 95% confidence intervals.A total of 25 patients, with a mean duration of Cy treatment of 8 months, and median follow up time of 19 months, were included in this analysis. Patients were started on 50 mg daily for 2 weeks and then increased to 100 mg if tolerated. Three patients (12%) were treated with Cy as 1 st line, 14 (56%) as 2 nd line, 5 (20%) as 3 rd line, and 3 (12%) as 4 th line. Of the 3 patients that received Cy as 1 st line, none had a response. All refractory patients received MTX prior to Cy. Of the 22 refractory patients, 14 (64%) had a response (6 CR, 8 PR), 7 patients had no response, and 1 could not be determined due to development of multiple myeloma. Of the 6 patients that attained a CR, 50% had a CMR. The median TTR (CR or PR) was 6 months (95% CI: 4-7) while median time to PR was 9 months and median time to CR was 7 months. The median time to CMR was 11 months. In patients that achieved a response, median follow up time was 27 months, with a median LFS of 24 months. Median LFS for those who attained a PR was 20 months, while the median LFS for those who attained a CR was not reached as none progressed (Figure). The median follow-up for patients with a CMR was 17 months with LFS having not been reached due to no progression. There was no significant impact of age, gender, or presence of rheumatoid arthritis (RA) on LFS. Males (48%) had a shorter TTR compared to females (52%) (5 vs 7 months; p=0.05). Patients with RA (28%) also trended towards a shorter TTR (p=0.07).Herein, we demonstrate that patients treated for relapsed T-LGLL with Cy can attain durable remissions, with a prolonged response. Of particular interest is that no patients who attained a CR have relapsed, showing that durable remission is achievable, while no patients that received Cy as 1 st line had a response. Further, in patients that achieved a CR, 50% achieved a CMR which has not been previously demonstrated in the relapsed setting. While limited in cohort size, and additional follow up needed, these data suggest that Cy can produce long term remissions in patients with relapsed T-LGLL and induce CMR. While we demonstrate that CMR is attainable in the relapsed setting, the impact of this on long term disease control compared to clinical CR is unclear. Therefore, we recommend that CMR be used as an endpoint in future studies, particularly prospective trials, to evaluate response to treatment. These data clearly demonstrate that Cy is effective in the setting of relapsed T-LGLL and can induce long term disease control. [Display omitted] DisclosuresBrammer: Seattle Genetics: Speakers Bureau; Kymera Therapeutics: Consultancy; Celgene: Research Funding.

Chronic Lymphocytic Leukemia, T-Cell Large Granular Lymphocytic Leukemia in a Pediatric Patient without Underlying Condition

BackgroundLarge granular lymphocytic (LGL) leukemia is a rare hematological malignancy in children. The two types of LGL leukemia that have been described are T-cell and Natural Killer cell leukemia. It is most commonly diagnosed in older adults, average age of 60-year-old. About 20 cases of LGL leukemia have been reported in children and young adults. All the patients in the reported cases had immune dysregulation conditions, such as chronic graft versus host disease, common variable immunodeficiency disorder, Crohn's disease and autoimmune hemolytic anemia. Here we report a case of T-cell LGL leukemia in a 11-year-old boy without underlying condition who presented with chronic neutropenia associated with gingival hypertrophy, recurrent skin abscesses, aphthous ulcers, clubbing of nails and low bone density.MethodsMulti-institution collaboration and literature review.Case Description11-year-old male with two years history of episodic gum bleeding with gingival hypertrophy, skin abscesses, aphthous ulcers, chronic neutropenia and lymphocytosis presented to our clinic for further evaluation. Initial workup demonstrated moderate to severe neutropenia (absolute neutrophil count between 400/ul to 800/ul) with low segmented neutrophils of 2-4% and high lymphocytes of more than 80%, but normal white blood cell count, hemoglobin for age and platelet count. Peripheral blood smear showed several variant lymphocytes with cytoplasmic blebs and no immature cells present. Expansion of T-cell large granular lymphocytes were detected in peripheral blood by flow cytometry. Due to new symptom of lower back pain, a lumbar Magnetic Resonance Imaging was performed. Results showed low bone density with mild compression deformity of L1 and abnormal heterogeneous marrow signal with heterogeneous contrast enhancement. The abnormal bone marrow signal promoted the investigation of bone marrow aspiration and biopsy. Flow cytometry detected forty-five percent of lymphocytes with immuno-phenotype of CD3+, CD8+, CD57+, CD16+, CD7+ and CD5-. The morphology of minimal cytoplasm and mature chromatin along with immunophenotype were consistent with clonal T-cell large granular lymphocytic proliferation/leukemia. Further cytogenetic tests showed TCR gamma and beta genes rearrangement, STAT3 N647I mutation with normal male karyotype. A peripheral blood congenital neutropenia panel, which included a total of 18 genes, found a heterozygous mutation c 279 G>A in the Gata2 gene; a variant of uncertain significance. Next generation sequencing showed somatic mutations of TRGV10, TRGV8 TRGJ1, TNFAIP3 and STAT3. However, there was no germline mutations detected in sample from skin biopsy. Comprehensive evaluation by immunology, rheumatology and gastroenterology failed to detect any underlying conditions.ConclusionDue to rarity of LGL leukemia in pediatrics, standard of care guidelines are currently unavailable. Extrapolated from limited literature, two management options are considered: watch and wait approach versus early initiation of immunosuppressant chemotherapy. Improved diagnostics can aide management strategies in this patient population. DisclosuresNo relevant conflicts of interest to declare.

Efficient Targeting of CD38 in Mature T-Cell Neoplasms with Daratumumab and Allogeneic NK Cells

Introduction: Mature T-cell neoplasms (MTCN) are a heterogeneous and aggressive group of lymphoid neoplasms with very limited options for precision immunotherapy. Targeted immunotherapy with antibodies directed against surface markers on tumor cells has emerged as an effective treatment for B-cell neoplasms, but the development of immunotherapy strategies for MTCN has been much slower. CD38 is expressed at low levels in a subset of normal resting T-cells. Data on the frequency and level of CD38 expression is MTCN are lacking. Our goal was to study the expression and stability of CD38 on a spectrum of neoplastic T-cell populations and assess the potential anti-tumor effect of anti-CD38 monoclonal antibodies in combination with allogeneic natural killer (NK) cells in MTCN.Methods and results: We searched the Thomas Jefferson University Hospital (TJUH) pathology records for all cases of MTCN for which immune-phenotypical characterization of the neoplastic T-cell population by multi-color flow cytometry (FC) was available. CD38 expression was evaluated in cases where an abnormal T-cell population (defined as loss of one on more pan T-cell markers and/or a skewed CD4 to CD8 ratio). A total of 103 unique patients with MTCN were identified. Of these, 51 had at least one biopsy whereby tumor cells had an abnormal immunophenotype which could then be assessed for CD38 expression. CD38 was expressed to some extent in all but 2 cases with expression levels on peripheral T-cell lymphoma-not other specified (PTCL-NOS)(% Mean±SEM = 80.84±10.26, N=11), angioimmunoblastic T-cell lymphoma (AITL) (% Mean±SEM = 80.56±7.34, N=6), nodal PTCL with T follicular-helper (T FH) phenotype (% Mean±SEM = 55.00±12.72, N=7), anaplastic large cell lymphoma (ALCL) (% Mean±SEM = 77.38±10.75, N=3), large granular lymphocytic leukemia (LGLL) (% Mean±SEM = 80.27±7.49, N=4), T-cell prolymphocytic leukemia (T-PLL) (% Mean±SEM = 88.26±4.20, N=7), cutaneous T-cell lymphoma (CTCL) (% Mean±SEM = 49.52±14.77, N=7), adult T‐cell leukemia/lymphoma (ATLL) (% Mean±SEM = 76.68±10.45, N=3), hepatosplenic T-cell lymphoma/monomorphic epitheliotropic intestinal T-cell lymphoma (HSTCL/MEITL) (% Mean±SEM = 75.00±11.37, N=3). The medians and ranges of the MFI of CD38 on CD38+ tumor cells were the following: PTCL-NOS 29.46 (3.1-115.62), AITL 13.64 (2.67-29.41), other PTCL-T FH 5.87 (0-48.67), ALCL 11.13 (3.6-11.28), LGL (13.52 (9.72-18.25), T-PLL 6.49 (3.09-18), CTCL 8.36 (0-116.75), ATLL 27.17 (3.54-60.5), HSTCL/MEITL 15.96 (6.96-167.85). We also measured surface expression of CD38 on the patient-derived MTCN cell lines HuT-78, HuT-102, Jurkat, H9, HH, and MOTN1, all of which expressed CD38, to determine which of these cell lines could be used for in vitro experiments. We next evaluated if the CD38 molecule is an effective target for antibody-mediated therapy in MTCN, by testing the ability of daratumumab (dara) to enhance antibody-dependent cellular cytotoxicity (ADCC) elicited by NK cells. For this, we purified normal NK-cells from TJUH Blood Bank leukoreduction filters and cultured with recombinant IL-15 for 48 hours prior to all experiment. T-cell lines and primary MTCN cells were treated with increasing concentrations (0.1 µg/mL - 2 µg/mL) of dara or isotype control. NK-cells were added at Effector:Target ratio of 5:1 and incubated for 4 hours at 37⁰ Celsius. Cytotoxicity was measured by LDH release assay. Dara induced significant cell lysis starting at doses as low as 0.1μg/mL in both T-cell lines and primary MTCN cells, reaching maximum cytotoxicity at 0.5-2μg/mL (mean±SEM cytotoxicity in isotype vs dara treated cells= 50.0±5.05% vs 97.5±2.5%, N=4, p-value=0.0002). The degree of ADCC induction also correlated with interferon-gamma (IFN-g) release by NK cells in vitro for both T-cell lines and primary MTCN cells.Conclusions: The majority of MTCN analyzed (N=49, 96%) showed any degree of CD38 expression by FC with a wide variation of intensity, including within the same subtype. Allogeneic NK cells efficiently elicited dara-mediated ADCC of tumor cells from all MTCN subtypes and produced abundant IFN-g. These data highlight the potential of targeting CD38 in MTCN with anti-CD38 antibodies and allogeneic NK cells. The strong CD38 expression observed in most tumor cells from ultra-rare and very aggressive subtypes of MTCL opens the door to much needed new treatment strategies. DisclosuresBrammer: Celgene: Research Funding; Kymera Therapeutics: Consultancy; Seattle Genetics: Speakers Bureau. Chakravarti: Kiadis Pharma: Patents & Royalties. Porcu: Viracta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Daiichi: Honoraria, Research Funding; Kiowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spectrum: Consultancy; DrenBio: Consultancy.

Single Capture High Throughput Sequencing Assay for Combined V(D)J Clonality Analysis and Oncogene Mutations in the Diagnosis of T and B Lymphoid Malignancies

IntroductionThe diagnosis of B and T cell malignancies relies on the demonstration of B-cell (BCR) or T-cell (TCR) antigen receptor clonality. This can be studied through the analysis of V(D)J rearrangements of BCR and TCR genes by PCR (van Dongen Leukemia 2003) or, more recently, by high-throughput sequencing (HTS). Amplification of a clonal population with a “primers approach” could fail in case of hybridization problems due to too fragmented DNA, somatic mutations or polymorphic variations. Here we evaluated the performance of a HTS capture system for the analysis of B and T-cell clonality in clinical samples from mature T or B malignancies. We further combined this technology to concomitant sequencing of oncogenes of interest.Patients and MethodsDNA was extracted from 58 tumoral samples from fresh/frozen (FF) cells or tissues or formalin-fixed paraffin-embedded tissue (FFPE) (n=19). These samples comprised various T-cell [i.e. 1T-cell prolymphocytic leukemia, 1 T large granular lymphocytic leukemia, 2 Sézary syndrome, 4 peripheral T-cell lymphoma not otherwise specified, 14 angioimmunoblastic T-cell lymphoma] or B-cell [i.e. 14 chronic lymphocytic leukemia, 1 mantle cell lymphoma, 5 diffuse large B-cell lymphoma, 1 grey-zone lymphoma, 13 Hodgkin lymphoma, 1 Poppema, 2 Waldenström and 1 multiple myeloma] malignancies. The Biomed-2 PCR technique was used as standard for assessing the performance of TRG, IGH and IGK clonality analysis. An extensive panel of capture probes was designed (SureSelect XT HS2 DNA system, Agilent Technologies) that covered the variable (V), + diversity (D) and junction (J) segments of the IGH, IGK, TRG, TRB loci and diagnostic/theranostic genes of interest i.e. B2M, BTK, CARD11, CD28, DNMT3A, IDH2, JAK3, PLCG1, PLCG2, ROHA, SOCS1, STAT3, STAT5B, STAT6, TET2, TNFAIP3, TP53. Paired-End sequencing was performed on a MiSeq system (Illumina) in 300, 500 and 600 cycles. Analysis of clonality profiles was performed using Vidjil software and SeqOne.ResultsHTS runs resulted in a median total read count of 1,6M (0.7-2.9) per sample. V(D)J rearrangements were identified with a median of 1503 reads (189-6824) per sample. Five samples were excluded because less than 300 rearranged reads were obtained. The number of rearranged reads and of clonotypes identified are influenced by the number of sequencing cycles (300<500 or 600) but not by the quality of DNA (FFPE vs FF).Analyses of tumoral samples with HTS versus PCR were compared. For the IGH locus (n=47), comparable PCR/HTS clonal (n=22) and polyclonal (PCL, n=20) profiles were identified. One discordant case showed a clonal PCR profile and a PCL HTS profile but the IGK was clonal. For the IGK locus (n=23), 10 clonal and 12 PCL cases were similar with both techniques. One case appeared discordant with a PCL PCR profile but a clonal HTS profile. For the TRG locus (n=31), PCR and HTS profiles were similar in 14 clonal, 5 oligoclonal and 9 PCL cases respectively. Three cases were discordant with oligoclonal PCR profiles but a clonal HTS profile.Overall in the 38 cases of B-cell malignancies, 27 and 11 cases had a concordant B-cell clonal or PCL profile with PCR and HTS. Among PCL cases, only one was discordant with a clonal HTS profile. This case and 3 other PCL cases were Hodgkin lymphomas which all disclosed another mutation (i.e. TP53, TNFAIP3, SOCS1). Of the 20 cases of T-cell malignancies, 14 displayed a clonal TRG profile with PCR and HTS. Among them, 13 showed oncogene mutations that confirmed the oncogenic nature of the clonal proliferation. Among 6 patients with a non-clonal PCR TRG profile, two cases of AITL and T-LGL had a discordant clonal TRG HTS profile and both also had specific mutations (SOCS1, RHOA and STAT3 respectively). Two other AITL samples showed a T-PCL profile with PCR and HTS but also had a mutation/CNV (RHOA, SOCS1).ConclusionA very good performance of B and T cell clonality assessment was obtained here with capture-HTS compared to Biomed-2 PCR. The combined identification of mutation/CNV allowed to confirm the malignant character in cases of clonal or PCL lymphoproliferations, while concomitantly specifying the type of lymphoproliferative disorder. The combined capture-HTS of B and T repertoires and oncogenes of diagnostic or theranostic interest thus appears as an efficient, accurate and useful approach for the diagnosis of mature B and T lymphoid malignancies in clinical practice. DisclosuresNo relevant conflicts of interest to declare.

A Longitudinal Evaluation of Cytopenia Severity in Patients with Clonal Cytopenia of Undetermined Significance (CCUS) and Correlation with the Presence of Sub-Clinical Dysplasia

IntroductionClonal cytopenia of undetermined significance (CCUS) represents a clonal group of cells with the presence of a hematologic malignancy-associated somatic mutation without meeting the morphologic criteria for MDS. CCUS patients have a variable risk of progression to MDS. The WHO requires at least 10% of a cell lineage have dysplastic morphology for the diagnosis of MDS, but CCUS patients have been described with no evidence of dysplasia (CCUS-ND) or with low level dysplasia (CCUS-D). We previously identified CCUS-D as biologically distinct with higher risk MDS mutations. We hypothesized that CCUS-D would be independently associated with the risk of progressive cytopenia over time and thus may have clinical relevance in the setting of CCUS.MethodsCases of CCUS were identified on review of bone marrow biopsies completed for the diagnosis of cytopenia from 8/2016 to 9/2018 with results of a custom 31-gene NGS hotspot panel to detect mutations in the following genes: ASXL1, BRAF, CALR, CBL, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA2, IDH1, IDH2, JAK2, JAK3, KIT, KRAS, MPL, MYD88, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, WT1, and ZRSR2. The classification of CCUS-D or CCUS-ND required two or more board-certified hematopathologists to score the degree of morphologic dysplasia on bone marrow aspirates with at least one of the following morphologic criteria: >2%-<10% erythroid precursors with nuclear irregularities, >50% erythroid cells with megaloblastoid changes and/or basophilic stippling, >5%-<10% hypogranular and/or hypolobated granulocytes, or ≥10% megakaryocytes that were small and hypolobated or showed other nuclear abnormalities such as grape-like nuclei. We recorded baseline characteristics including cytogenetics, results of next-generation sequencing, and lab findings and then followed patients with annual blood counts.Statistical differences of baseline characteristics were evaluated by T-test for continuous variables, and Chi-square / Fisher's exact test were used for association between categorical variables. The repeated measures over time for various outcomes (WBC, ANC, hemoglobin, platelet) were visualized using scatter plot supervised with lowess smoother. The effects of dysplasia on various longitudinal outcomes were estimated using linear mixed-effect models.ResultsWe identified 54 CCUS patients on review of 200 marrows completed for undiagnosed cytopenia. Twenty-six patients were identified with CCUS-D, and 28 patients with CCUS-ND. Baseline clinical characteristics including age, gender, race, smoking status, or presence of infection at time of initial diagnosis were similar. Hematology laboratory parameters including White Blood Cell Count (WBC), Absolute Neutrophil Count (ANC), Hemoglobin (Hgb), and Platelet Count (Plts) did not differ. (Table 1). Patients with CCUS-D had an average of 1.62 mutated genes detected per patient with the most common being TET2 (26.19%), SRSF2 (14.29%), and DNMT3A (14.29%). Patients with CCUS-ND had an average of 1.41 mutations per patient with the most common being TET2 (31.57%) and DNMT3A (31.57%).Patients were followed for a median of 32.2 months and a range from 1.0 to 48.8 months. On long term follow up, WBC, ANC, Hgb and plts trended lower in patients with CCUS-D compared to CCUS-ND, but the difference was marginally statistically significant (p = 0.09, 0.16, 0.13, 0.64 respectively). We also evaluated the rate of change in hematology parameters and found no significant difference in change rate of WBC, ANC, and hgb (p = 0.25, 0.13, and 0.28, respectively). There was a significant difference in the change rate of platelet counts between the two groups (p = 0.026). In particular, patients with CCUS-D had decreasing platelet counts at a rate of -0.04119 10E9/L/day and patients with CCUS-ND had increasing platelet counts at a rate of 0.01075 10E9/L/day.Two patients in the CCUS-D cohort progressed to MDS at an average of 938.5 days. One patient with CCUS-ND progressed to Large Granular Lymphocytic Leukemia at 805 days.ConclusionsLow level (< 10%) dysplasia in cell lineages in CCUS is associated with a higher rate of decline in platelet counts but was not independently associated with increased risk of progressive neutropenia, leukopenia, or anemia. Longer follow up may be required to further understand the clinical significance of low level dysplasia in CCUS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of Large Granular Lymphocyte Leukemia on Blood DNA Methylation and Epigenetic Clock Modeling in Fischer 344 Rats.

Age-dependent differences in methylation at specific cytosine-guanine (CpG) sites have been used in "epigenetic clock" formulas to predict age. Deviations of epigenetic age from chronological age are informative of health status and are associated with adverse health outcomes, including mortality. In most cases, epigenetic clocks are performed on methylation from DNA extracted from circulating blood cells. However, the effect of neoplastic cells in the circulation on estimation and interpretation of epigenetic clocks is not well understood. Here, we explored this using Fischer 344 (F344) rats, a strain that often develops large granular lymphocyte leukemia (LGLL). We found clear histological markers of LGLL pathology in the spleens and livers of 27 out of 61 rats aged 17-27 months. We assessed DNA methylation by reduced representation bisulfite sequencing with coverage of 3 million cytosine residues. Although LGLL broadly increased DNA methylation variability, it did not change epigenetic aging. Despite this, the inclusion of rats with LGLL in clock training sets significantly altered predictor selection probability at 83 of 121 commonly utilized CpG sites. Furthermore, models trained on rat samples that included individuals with LGLL had greater absolute age error than those trained exclusively rats free of LGLL (39% increase; p < .0001). We conclude that the epigenetic signals for aging and LGLL are distinct, such that LGLL assessment is not necessary for valid measures of epigenetic age in F344 rats. The precision and architecture of constructed epigenetic clock formulas, however, can be influenced by the presence of neoplastic hematopoietic cells in training set populations.

Neutropenia and Large Granular Lymphocyte Leukemia: From Pathogenesis to Therapeutic Options.

Large granular lymphocyte leukemia (LGLL) is a rare lymphoproliferative disorder characterized by the clonal expansion of cytotoxic T-LGL or NK cells. Chronic isolated neutropenia represents the clinical hallmark of the disease, being present in up to 80% of cases. New advances were made in the biological characterization of neutropenia in these patients, in particular STAT3 mutations and a discrete immunophenotype are now recognized as relevant features. Nevertheless, the etiology of LGLL-related neutropenia is not completely elucidated and several mechanisms, including humoral abnormalities, bone marrow infiltration/substitution and cell-mediated cytotoxicity might cooperate to its pathogenesis. As a consequence of the multifactorial nature of LGLL-related neutropenia, a targeted therapeutic approach for neutropenic patients has not been developed yet; moreover, specific guidelines based on prospective trials are still lacking, thus making the treatment of this disorder a complex and challenging task. Immunosuppressive therapy represents the current, although poorly effective, therapeutic strategy. The recent identification of a STAT3-mediated miR-146b down-regulation in neutropenic T-LGLL patients emphasized the pathogenetic role of STAT3 activation in neutropenia development. Accordingly, JAK/STAT3 axis inhibition and miR-146b restoration might represent tempting strategies and should be prospectively evaluated for the treatment of neutropenic LGLL patients.

A phase 2 biomarker-driven study of ruxolitinib demonstrates effectiveness of JAK/STAT targeting in T-cell lymphomas

AbstractSignaling through JAK1 and/or JAK2 is common among tumor and nontumor cells within peripheral T-cell lymphoma (PTCL). No oral therapies are approved for PTCL, and better treatments for relapsed/refractory disease are urgently needed. We conducted a phase 2 study of the JAK1/2 inhibitor ruxolitinib for patients with relapsed/refractory PTCL (n = 45) or mycosis fungoides (MF) (n = 7). Patients enrolled onto 1 of 3 biomarker-defined cohorts: (1) activating JAK and/or STAT mutations, (2) ≥30% pSTAT3 expression among tumor cells by immunohistochemistry, or (3) neither or insufficient tissue to assess. Patients received ruxolitinib 20 mg PO twice daily until progression and were assessed for response after cycles 2 and 5 and every 3 cycles thereafter. The primary endpoint was clinical benefit rate (CBR), defined as the combination of complete response, partial response (PR), and stable disease lasting at least 6 months. Only 1 of 7 patients with MF had CBR (ongoing PR > 18 months). CBR among the PTCL cases (n = 45) in cohorts 1, 2, and 3 were 53%, 45%, and 13% (cohorts 1 & 2 vs 3, P = .02), respectively. Eight patients had CBR > 12 months (5 ongoing), including 4 of 5 patients with T-cell large granular lymphocytic leukemia. In an exploratory analysis using multiplex immunofluorescence, expression of phosphorylated S6, a marker of PI3 kinase or mitogen-activated protein kinase activation, in <25% of tumor cells was associated with response to ruxolitinib (P = .05). Our findings indicate that ruxolitinib is active across various PTCL subtypes and support a precision therapy approach to JAK/STAT inhibition in patients with PTCL. This trial was registered at www.clincialtrials.gov as #NCT02974647.

Survival and associated comorbidities in inclusion body myositis.

ObjectiveTo evaluate survival and associated comorbidities in inclusion body myositis (IBM) in a population-based, case-control study.MethodsWe utilized the expanded Rochester Epidemiology Project medical records-linkage system, including 27 counties in Minnesota and Wisconsin, to identify patients with IBM, other inflammatory myopathies (IIM), and age/sex-matched population-controls. We compared the frequency of various comorbidities and survival among groups.ResultsWe identified 50 IBM patients, 65 IIM controls and 294 population controls. Dysphagia was most common in IBM (64%) patients. The frequency of neurodegenerative disorders (dementia/parkinsonism) and solid cancers was not different between groups. Rheumatoid arthritis was the most common rheumatic disease in all groups. A total of 36% of IBM patients had a peripheral neuropathy, 6% had Sjögren’s syndrome and 10% had a haematologic malignancy. T-cell large granular lymphocytic leukaemia was only observed in the IBM group. None of the IBM patients had hepatitis B or C, or HIV. IBM patients were 2.7 times more likely to have peripheral neuropathy, 6.2 times more likely to have Sjögren’s syndrome and 3.9 times more likely to have a haematologic malignancy than population controls. IBM was associated with increased mortality, with a 10-year survival of 36% from index, compared with 67% in IIM and 59% in population controls. Respiratory failure or pneumonia (44%) was the most common cause of death.ConclusionsIBM is associated with lower survival, and higher frequency of peripheral neuropathy, Sjögren’s syndrome and haematologic malignancies than the general population. Close monitoring of IBM-related complications is warranted.

TNFAIP3 mutation may be associated with favorable overall survival for patients with T-cell lymphoma

BackgroundT-cell lymphoma (TCL) is highly aggressive and has a poor prognosis; thus, it is worth exploring biomarkers that may predict clinical outcomes and investigate their potential role in developing targeted therapies. In this study, we characterized the mutation pattern of tumor necrosis factor-alpha-inducing protein 3 (TNFAIP3) and its role in the prognosis of TCL patients.MethodsCoding sequence (CDS) mutations in TNFAIP3 in TCL patients was explored using exome-sequencing data from 79 patients in our center (Guangdong Provincial People’s Hospital, GDPH) and 544 samples from the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Additionally, non-CDS mutations in TNFAIP3 in 41 TCL patients from our center (JNU) were investigated by polymerase chain reaction (PCR) and Sanger sequencing. Furthermore, non-CDS mutations in TNFAIP3 in 47 TCL patients from Gene Expression Omnibus (GEO) dataset were explored.ResultsIn the COSMIC database, TNFAIP3 mutations in TCL patients were located in the CDS, and the overall mutation frequency was 2.2%. However, TNFAIP3 mutations were not detected in the CDS of any of the samples in our center’s datasets. Interestingly, non-CDS TNFAIP3 mutations were found in 14.6% and 4.3% of TCL patients in the JNU and GSE15842 dataset, respectively. Importantly, there was a clear trend showing that TCL patients with a TNFAIP3 mutation were associated with a longer 5-year restricted mean survival time (RMST) and favorable OS rate compared with those without a TNFAIP3 mutation in the JNU dataset [hazard ratio (HR) = 0.29, 95% confidence interval (CI) 0.07 to 1.31, P = 0.089]. Furthermore, TNFAIP3 mutations significantly correlated with T-cell large granular lymphocytic leukemia (T-LGLL) with a favorable prognosis in the JNU dataset (P = 0.002). Notably, the different mutation patterns of TNFAIP3 when comparing our center and the COSMIC datasets might be due to different ethnic and genetic backgrounds.ConclusionsTo the best of our knowledge, we for the first time describe that TNFAIP3 mutations in non-CDS regions are associated with favorable OS for TCL patients, which might be a potential biomarker for the prognostic stratification of Chinese TCL patients.

The Value of Flow Cytometry Clonality in Large Granular Lymphocyte Leukemia.

Simple SummaryLarge granular lymphocyte (LGL) leukemia, a lymphoproliferative disease, is characterized by an increased frequency of large-sized lymphocytes with typical expression of T-cell receptor (TCR) αβ, CD3, CD8, CD16, CD45RA, and CD57, and with the expansion of one to three subfamilies of the TCR variable β chain reflecting gene rearrangements. Molecular analysis remains the gold standard for confirmation of TCR clonality; however, flow cytometry is time and labor saving, and can be associated with simultaneous investigation of other surface markers. Moreover, Vβ usage by flow cytometry can be employed for monitoring clonal kinetics during treatment and follow-up of LGL leukemia patients.Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder of mature T or NK cells frequently associated with autoimmune disorders and other hematological conditions, such as myelodysplastic syndromes. Immunophenotype of LGL cells is similar to that of effector memory CD8+ T cells with T-cell receptor (TCR) clonality defined by molecular and/or flow cytometric analysis. Vβ usage by flow cytometry can identify clonal TCR rearrangements at the protein level, and is fast, sensitive, and almost always available in every Hematology Center. Moreover, Vβ usage can be associated with immunophenotypic characterization of LGL clone in a multiparametric staining, and clonal kinetics can be easily monitored during treatment and follow-up. Finally, Vβ usage by flow cytometry might identify LGL clones silently underlying other hematological conditions, and routine characterization of Vβ skewing might identify recurrent TCR rearrangements that might trigger aberrant immune responses during hematological or autoimmune conditions.

Large Granular Lymphocytic Leukemia: From Immunopathogenesis to Treatment of Refractory Disease.

Simple SummaryLarge Granular Lymphocytic Leukemia (LGLL) is a clonal disorder of cytotoxic T-cells. Because of the variety of clinical presentations ranging from the mere presence of lymphocytosis to cytopenias and autoimmune conditions, this rare lymphoma may require treatment to control such manifestations. Although first-line treatments are more established, refractory cases are often managed based on the experience of the attending physician. Herein, we review the pathways involved in the pathogenesis of LGLL, including refractory cases, inferring clues as to the potentially actionable targets.Large Granular Lymphocyte Leukemia (LGLL) is a rare, chronic lymphoproliferative disorder of effector cytotoxic T-cells, and less frequently, natural killer (NK) cells. The disease is characterized by an indolent and often asymptomatic course. However, in roughly 50% of cases, treatment is required due to severe transfusion-dependent anemia, severe neutropenia, or moderate neutropenia with associated recurrent infections. LGLL represents an interesting disease process at the intersection of a physiological immune response, autoimmune disorder, and malignant (clonal) proliferation, resulting from the aberrant activation of cellular pathways promoting survival, proliferation, and evasion of apoptotic signaling. LGLL treatment primarily consists of immunosuppressive agents (methotrexate, cyclosporine, and cyclophosphamide), with a cumulative response rate of about 60% based on longitudinal expertise and retrospective studies. However, refractory cases can result in clinical scenarios characterized by transfusion-dependent anemia and severe neutropenia, which warrant further exploration of other potential targeted treatment modalities. Here, we summarize the current understanding of the immune-genomic profiles of LGLL, its pathogenesis, and current treatment options, and discuss potential novel therapeutic agents, particularly for refractory disease.

TCL-232: Integrated Genomic and Transcriptomic Sequencing Reveals the Landscape of T, NK, and Gamma-Delta Large Granular Lymphocyte Leukemia

<h3>Context</h3> Large granular lymphocyte (LGL) leukemia is a rare lymphoproliferative disorder of primarily CD8+ T or natural killer (NK) cell lineage whose molecular landscape is poorly characterized. <h3>Objective</h3> In this study, we defined the landscape of non-synonymous somatic variants in LGL leukemia exomes, asked whether these mutational profiles differed across LGL leukemia subtypes, and tested whether mutational groups were associated with clinical features. <h3>Design</h3> We leveraged paired whole-exome and transcriptome sequencing in the largest LGL leukemia study to date, which included 116 patients (93 TCRαβ T-LGL, 11 NK-LGL, and 12 TCRγδ T-LGL). <h3>Patients</h3> The study was conducted under IRB-approved protocols for the LGL Leukemia Registry at the University of Virginia School of Medicine. 116 LGL patients meeting diagnosis criteria by the World Health Organization (evidence of clonality, persistence of chronic expansion >6 months) were recruited for this study. <h3>Results</h3> The mutational landscape of LGL leukemia identified ARHGAP25, CCL22, CDH8, DNMT3A, FAS, KDM6A, KMT2D, PIK3R1, STAT3, STAT5B, TET2, and TNFAIP3 as recurrently mutated putative drivers using IntOGen bioinformatic driver analysis. The majority of gene mutations spanned all LGL subtypes, with the notable exception of CCL22, which was mutated exclusively within NK-LGL (4/11; 36.4%; p=0.0008). Moreover, there was a predilection for co-occurrence of STAT3 mutations with epigenetic modifier KMT2D (p < 0.01). Hotspot mutations in PIK3R1 and FAS were detected in both T-LGL subtypes, hinting at domain-specific mechanisms. STAT3 mutant patients exhibited increased non-silent mutation burden and a mutational signature associated with increased cell division. Gene expression analysis revealed enrichment of interferon-alpha signaling for STAT3 mutant patients, while increased PI3K-Akt signaling was observed for STAT3 WT patients. Finally, STAT3 mutations were associated with reduced neutrophil counts for only the D661Y and N647I variants but not Y640F, whereas all three of these mutation groups were associated with anemia. <h3>Conclusions</h3> These findings highlight the clinical and molecular heterogeneity of this rare disorder. LGL leukemia research is supported by NCI R01CA178393, P30CA044579, T32LM012416, T32GM007267, F30CA225046. Additional support is provided by Bess Family Charitable Fund, LGL Leukemia Foundation, Dr. Charles and Katharine Hutton Tweedy, William J. Branch, Dr. Szabolcs Szentpetery, and a generous anonymous donor.

CLL-156: Novel CCL22 Mutations Drive Chronic Lymphoproliferative Disorder of NK Cells Through Biased GPCR Signaling

Context: Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is a subset of large granular lymphocyte (LGL) leukemia included as a provisional entity in the 2016 WHO classification of lymphoid neoplasms, which is characterized by clonal expansion of NK cells with no standardized therapy. STAT3 and TET2 mutations were previously each reported in 15%–30% of cases; however, the genomic basis of the remaining cases is still unknown. Objective: We aimed to perform a comprehensive genomic and transcriptomic analysis of CLPD-NK to elucidate the genomic landscape, investigate mechanisms of pathogenesis, and improve diagnostic classification and treatment strategies. Methods: CLPD-NK samples (n=121) were examined by whole-genome, targeted, and/or whole-transcriptome sequencing. Structural modeling, G-protein signaling, β-arrestin signaling, G-protein-coupled receptor (GPCR) for CCL22, CCR4 internalization, and chemotaxis were examined. Cell line-derived xenografts were used to examine cell proliferation and phenotype. Results: Somatic mutations in the chemokine CCL22 were found in 21.5% of CLPD-NK patients but not in 2837 cases of other hematological malignancies, including T-LGL and other NK leukemias. Clinically, there was a trend to an association with cutaneous complications and/or neurological symptoms. CCL22 mutations were enriched at the highly conserved residues Leu45, Pro46, and Pro79 and were mutually exclusive of STAT3 mutations. CCL22 mutations were associated with dysregulated gene expression, similar to normal CD56bright NK cells, with enrichment for activation of cytokine signaling pathways, representing a distinct subgroup. Most CCL22 mutations were substitutions to Arg, a basic residue, and Leu45Arg was predicted to promote electrostatic interactions with CCR4-Tyr22/Glu23 acidic residues. The mutations resulted in ligand-biased CCR4 signaling, in which activation of G-protein signaling was unchanged, but recruitment of β-arrestin, which mediates receptor internalization, was impaired, which, in turn, resulted in enhanced cell chemotaxis. CCL22 mutations drove enhanced cell proliferation in vivo in hIL-15-transgenic mice and induced an expression profile resembling that of primary samples, indicating the importance of microenvironmental crosstalk in pathogenesis. Conclusions: Somatic CCL22 mutations defined a new subgroup of CLPD-NK similar to CD56bright normal NK cells and represented a novel mechanism of leukemogenesis in which gain-of-function chemokine mutations drive tumorigenesis by biased GPCR signaling and dysregulation of microenvironmental crosstalk.

Poster: TCL-232: Integrated Genomic and Transcriptomic Sequencing Reveals the Landscape of T, NK, and Gamma-Delta Large Granular Lymphocyte Leukemia

Poster: TCL-232: Integrated Genomic and Transcriptomic Sequencing Reveals the Landscape of T, NK, and Gamma-Delta Large Granular Lymphocyte Leukemia

Clinicobiological Characteristics and Outcomes of Patients with T-Cell Large Granular Lymphocytic Leukemia and Chronic Lymphoproliferative Disorder of Natural Killer Cells from a Single Institution.

Simple SummaryT-LGLL and CLPD-NK are two indolent lymphoproliferative syndromes whose main symptoms derive from the cytopenia(s). A proportion of patients harbor STAT3 mutations that appear to play an important role in the pathogenesis of the disease. The aim of our retrospective study was to describe the main clinicobiological characteristics, response to therapy and outcome in a large cohort of T-LGLL and CLPD-NK patients diagnosed at a single institution. The impact of STAT3 mutations on the behaviour of the disease and the survival of these patients compared to the age- and sex-matched general population are also evaluated. For the first time, we show that the survival of patients with these two diseases does not differ from that of a matched general Spanish population, confirming the indolent course of these entities.T-cell large granular lymphocytic leukemia (T-LGLL) and chronic lymphoproliferative disorder of natural killer (NK) cells are two infrequent diseases characterized by clonal expansions of cytotoxic T lymphocytes and NK cells, respectively. Somatic mutations of STAT3 are involved in the pathogenesis of these entities. We describe the clinicobiological features, mutational status of STAT3/STAT5B, treatment and outcome of 131 patients. Neutropenia was the most frequent finding at diagnosis, followed by anemia. Concurrent hematological disorders were diagnosed in 37% of patients and autoimmune conditions and solid tumors in 17% and 15%, respectively. All patients who needed treatment belonged to the CD8+CD57+ group. Remarkably, patients included in the CD4+ group had a higher association with solid tumors (p = 0.037). STAT3 mutations were found in 17% of patients, mainly Y640F and D661Y mutations. Patients carrying STAT3 mutations more frequently presented with anemia, neutropenia, high LDH, high large granular lymphocyte counts and need for treatment (p = 0.0037). Methotrexate was the most frequently used agent (72% of cases). The overall response rate to all treatments was 50%. The 10-year overall survival of this series was 78%, with no differences according to the mutational status of STAT3. We compared the survival of these patients with the general Spanish population and no differences were found, confirming the indolent nature of these hematological malignancies. Our study further extends findings documented by others on the clinical behavior of the disease and the impact of STAT3, and for the first time analyzes survival compared to a matched general Spanish population.

T-cell Large Granular Lymphocytic Leukemia Presenting as Post-transplant Lymphoproliferative Disorder: A Report of Two Cases and Literature Review

T-cell Large Granular Lymphocytic Leukemia Presenting as Post-transplant Lymphoproliferative Disorder: A Report of Two Cases and Literature Review

Therapeutic Efficacy of Mcl-1 Antagonism in Large Granular Lymphocyte Leukemia

Introduction: Large granular lymphocyte (LGL) leukemia is a spectrum of rare clonal lymphoproliferative disorders, all of which involve expansion of large granular lymphocytes. Immunosuppressive regimens using methotrexate or cyclophosphamide are utilized as front-line treatments in LGL leukemia but are not curative nor are they effective in all patients. The aggressive LGL variants have no effective treatment and are refractory to conventional chemotherapy. The lack of effective and targeted therapies results from an incomplete picture of survival mechanisms that contribute to the pathogenesis of leukemic LGLs.We have previously reported that STAT3 is constitutively active in LGL leukemia cells and up to 40% of patients harbor activating mutations in STAT3 within the leukemic clone. We have also reported that the pro-survival Bcl-2-family protein Mcl-1 is upregulated in LGL leukemia patients. Additionally, we have identified MCL1 as a transcriptional target of STAT3 in leukemic LGLs. Recently, novel small molecule inhibitors of Mcl-1 have been identified including marinopyrrole A (maritoclax) and its derivative KS18. These inhibitors effectively induced apoptosis in Mcl-1-dependent cells and suppressed the growth of Mcl-1-dependent hematologic cancer xenografts in nude mice without apparent toxicity. Thus, this study further investigates the role of Mcl-1 in LGL leukemia and the potential therapeutic application of the Mcl-1 antagonists maritoclax and KS18.Results: Knockdown of Mcl-1 in human-derived LGL leukemia cell lines (TL1 and NKL) resulted in decreased cell viability by MTS assay and increased apoptosis by flow cytometry in Mcl-1 siRNA versus scramble siRNA-transfected cells (NKL and TL-1 cells: p < 0.001). Treatment with maritoclax or KS18 decreased cell proliferation and increased apoptosis in NKL, TL1 and RNK16 (rat-derived LGL leukemia cell line) cells (Maritoclax, EC50: 2.45, 1.43 and 1.23 μM respectively) (KS18, EC50: 1.30, 0.60 and 0.31 μM respectively). Ex vivo treatment with maritoclax and KS18 induced selective apoptosis in PBMCs from LGL leukemia patients (NK-LGL, n = 6; T-LGL, n = 12) but not normal donor PBMCs (n = 4) (Maritoclax and KS18, p < 0.0001). Importantly, there was no significant difference in treatment efficacy in patient cells with wild-type STAT3 (n = 9) versus mutant STAT3 (Y640F, n = 5; D661Y, n = 3). We then proceeded to mechanistic studies of Mcl-1 antagonism. Both maritoclax and KS18 induced selective and dose-dependent Mcl-1 degradation by western blot in NKL, TL1, RNK16 and PBMCs from LGL leukemia patients. We further delineated that this effect was proteasome dependent as it is reversible by pre-treatment with MG132, a proteasome inhibitor.Preclinical in vivo studies were then conducted in Fisher F344 rats that were transplanted with primary leukemic LGL cells and monitored for signs of leukemia through serial CBC and body weight measurements. Leukemic rats developed progressive anemia, thrombocytopenia and leukocytosis and significant weight loss. Rats were treated with vehicle or KS18 (2.5 or 5.0 mg/kg i.p. daily) upon detection of leukemic features. In non-leukemic KS18-treated rats, there were no observed toxicities and there was no difference in survival or weight gain versus vehicle-treated non-leukemic rats. However, leukemic KS18-treated rats exhibited findings consistent with tumor lysis syndrome, including sudden death associated with severely increased serum LDH and increased apoptosis in heart and spleen tissues. At necropsy, leuk/KS18 rats exhibited significantly decreased infiltration and elevated apoptosis of leukemic cells in heart and spleen tissues. Conversely, in leuk/veh rats, spleen tissue lacked normal histologic structure with red pulp replaced almost completely by leukemia cells. Ex vivo KS18 treatment also decreased the viability of PBMC and spleen cells from leukemic animals (p < 0.0001). These data indicate that Mcl-1 is a promising therapeutic target as evidenced by potent therapeutic efficacy of KS18 in LGL cell lines and primary patient material and in an aggressive rat model of LGL leukemia. Further studies are required in order to optimize drug dose, timing and concentrations of KS18 in order to prevent potential tumor lysis syndrome and optimize therapeutic benefit. DisclosuresGru:Seattle Genetics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Recurrent STAT3 Mutations in the Lymphocytes of Pure Red Cell Aplasia without T-Cell Large Granular Lymphocytic Leukemia

▪Background: Activating somatic mutations of STAT3 are one of the most frequently recognized genetic alterations in T-cell lymphoma including T-cell large granular lymphocytic leukemia (T-LGLL). T-LGLL is associated with certain proportions of patients (pts) with acquired aplastic anemia (AA) and/or myelodysplastic syndromes (MDS). Pure red cell aplasia (PRCA), especially acquired PRCA, is a syndrome defined by an anemia with marked reduction or absence of erythroid production associated with immunological mechanisms. PRCA is classified into primary and secondary types including T-LGLL. Previous reports have described that T-LGLL with STAT3 mutations was closely associated with PRCA. However, it is yet to be clear whether STAT3-mutated T cells would be involved in the pathogenesis of other cytopenias, such as AA, MDS, primary PRCA or secondary PRCA without (w/o) T-LGLL. In this study, we analyzed the profiles of STAT3 gene mutations in bone marrow failure syndrome (BMFS)s including AA/MDS and PRCA.Methods: Peripheral blood samples were collected from 94 patients with BMFSs, including AA, MDS, AA/PNH, and PRCA with or w/o T-LGLL. When possible, T-cell receptor (TCR) Vβ repertoire of the patients was determined by flow cytometry. DNA was extracted from mononuclear cells (MNC), and STAT3 Y640F and D661Y, two hot spot mutations, were detected with sensitive allele specific-PCR (AsPCR) methods. Then we performed deep amplicon sequencing analysis of STAT3 in PRCA pts. CD8+ T cells were sorted if possible, and also subjected to the analyses.Results: The recruited pts included 38 AA, 20 MDS, 7 AA/PNH, 11 PRCA with T-LGLL, and 18 PRCA w/o T-LGLL. Among PRCA pts w/o apparent T-LGLL, 10 had primary PRCA and 8 had secondary PRCA including 2 with thymoma. The clinical characteristics of PRCA are shown in Table 1. TCR Vβ repertoire analysis were performed in 8 PRCA w/o T-LGLL and 5 PRCA with T-LGLL. All pts with PRCA associated with T-LGLL showed restricted Vβ repertoire. On the other hand, only 1pt showed monoclonal Vβ pattern in PRCA w/o TLGLL. AsPCR did not detect Y604F or D661Y mutations in any of 65 pts with AA, MDS, or AA/PNH. In contrast, 2 pts with PRCA and 4 pts with PRCA-T-LGLL were positive for either of the mutations, suggesting STAT3 mutations may be unique to PRCA pts. Further analysis with deep amplicon analysis in the 29 PRCA pts revealed STAT3 mutations in 11 pts (61%) of PRCA w/o T-LGLL, which included 6 pts with primary PRCA and 5 pts with secondary PRCA associated with autoimmune diseases (n=2), 2 thymoma (n=2) and thymic cancer (n=1). The median variant allele frequency of STAT3 mutations was 2.3% (0.2-48.9). N538Y and I653V were most frequent mutations in PRCA w/o T-LGLL. Eight pts (73%) with T-LGLL-associated PRCA were also positive for STAT3 mutation. Thirty-nine % and 70% of STAT3 mutation were found in SH2 domain in PRCA w/o T-LGLL and PRCA with T-LGL, respectively. There was no difference in clinical features between pts with and w/o STAT3 mutations.Discussion: The high frequency of STAT3 mutations in PRCA pts regardless of etiology and the absence of the mutation in pts with AA/MDS suggest that STAT3 mutations may serve as a molecular marker of PRCA. The discordant results with regard to AA/MDS pts from a previous report which detected STAT3 mutations in 4.9% of AA/MDS patients by AsPCR methods (Jerez et al. BLOOD 2013) may be due to the difference in ethnic backgrounds of the pts studied. Given very low frequencies of STAT3 mutations in AA and MDS that were revealed by several previous studies, JAK/STAT signaling systems in the axis of STAT3 mutations may have unique roles in the pathogenesis of PRCA.Conclusion: STAT3 mutations are frequently detected in pts with PRCA even in pts w/o T-LGLL and the JAK/STAT pathway may therefore be therapeutic targets of PRCA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.