ALBERT

Description: Abstract 2668 Large granular lymphocyte leukemia (LGL-L) is a proliferative disorder of cytotoxic T cells or NK cells frequently complicated with cytopenia and autoimmune phenomena. In the current WHO classification, T-cell large granular lymphocyte leukemia (T LGL-L), and chronic lymphoproliferative disorders of NK cells (CLPD-NK) are included in this category. Aggressive NK cell leukemia (ANKL) and Epstein-Barr virus (EBV)-positive T-cell lymphoproliferative disease of childhood (EBV-T LPD) are also categorized as entities in the classification based on the clinical characteristics and EBV-positivity, and their morphologies closely resemble LGL. There has been controversy concerning the nature of these diseases, whether they are reactive processes or neoplasms. Recently, recurrent somatic mutations in the src homology (SH) 2 domain of the signal transducer and activator of transcription 3 (STAT3) gene have been found in T LGL-L and CLPD-NK, leading to constitutive activation of STAT3 and dysregulation of genes downstream of STAT3. Since LGL-L consists of various disorders and is suggested to differ based on racial backgrounds, these findings prompted us to investigate mutations in STAT3 in a Japanese cohort of LGL-L. The study included a total of 36 patients (pts) with LGL. Genes of exons 19 to 24 in STAT3 were amplified by PCR and sequenced directly using genomic DNA isolated from peripheral blood mononuclear cells. Since Y604F and D661Y mutations in STAT3 gene were representative and frequently recognized, allele specific PCR (AS-PCR) assays for these mutations were also performed. Pts consisted of 18 with T LGL-L (14 with αβ T cell receptor (TCR) type and 4 with γΔ TCR type), 11 with CLPD-NK, 5 with ANKL, and 2 with EBV-T LPD; one pt with αβ TCR type and 1 with γΔ TCR type, as well as 1 with reactive NK lymphocytosis used as control. By direct sequencing, two mutations, Y640F and D661Y, were identified. Y640F was recognized in 1 pt with αβ T LGL-L and D661Y in 3 pts with CLPD-NK. By AS-PCR, 10 additional pts were found to be positive for mutations of either Y604F or D661Y. A pt with T LGL-L was positive for both mutations by AS-PCR, although no mutations were detected by direct sequencing. A pt with CLPD-NK was positive for Y640F by AS-PCR in addition to D661Y recognized by direct sequencing. All 4 pts positive for the mutations by direct sequencing were confirmed to be positive by AS-PCR. In total, 7 pts with αβ TCR type T LGL-L and three T LGL-L pts with γΔ TCR type were positive for mutations. All these mutations were heterozygous. Mutations in SH2 domains of the STAT3 gene were not found in ANKL or EBV T-LPD pts by either direct sequencing or AS-PCR. Samples from fifty healthy controls were examined by AS-PCR and all were negative for Y604F or D661Y mutations. Reactive NKL lymphocytosis and three cell lines, Jurkat, NKL and NK92, were also negative for the mutations. The frequencies of STAT3 mutations in T LGL-L and CLPD-NK were 55.6% and 27.3%, respectively (P = 0.25). Among the pts with T LGL-L and CLPD-NK, the frequency of pure red cell aplasia (PRCA) was significantly higher in pts with the mutations (8/13) than in those without the mutations (3/16) (P = 0.03). In three T LGL-L pts positive for the mutations examined on subsequent occasions, the mutation became undetectable after cyclosporine A treatment in one pt, and was persistently found at stable amounts in two other pts by quantitative PCR. Our results indicate that mutations in the SH2 domain of the STAT3 gene frequently occur in T LGL-L and CLPD-NK in a Japanese cohort and these mutations are closely associated with PRCA and treatment requirements. STAT3 mutation thus likely contributes to the pathogenesis of T LGL-L and CLPD-NK, while EBV-associated LGL diseases, such as ANKL or EBV T-LPD, might be driven by mechanisms other than STAT3-associated pathways. Disclosures: No relevant conflicts of interest to declare.

Description: Large granular lymphocyte leukemia (LGL L) includes T-cell LGL L and chronic lymphoproliferative disorders of NK cells (CLPD-NK) with indolent clinical course. STAT signaling system has been shown to play a crucial role in LGL L. We reported somatic mutations in the STAT3 gene in LGL L, which is associated with pure red cell aplasia among an Asian cohort. Since STAT5b mutations in LGL leukemia had also been discovered, we investigated mutations in STAT5b gene in Japanese patients with LGL L. Among 28 T-LGL L and 11 CLPD-NK, N642H and Y665F activating mutations in the SH2 domain of the STAT5b were found in 3 and 1 patient (pt)s, respectively, by direct sequencing, which was confirmed with the corresponding allele-specific (AS) PCR. The frequencies of STAT5b mutations in T-LGL L and CLPD-NK were 14.3% and 0%, respectively. Median age of the 4 pts was 76.5 years and mild neutropenia was recognized in 2pts, but no one was anemic. None of them showed apparent aggressive clinical courses, in contrast to previous reported cases (Rajala HLM et al, BLOOD 2013). Interestingly, STAT5b mutations were associated with a unique phenotype of LGL L with CD4+CD56+TCR αβ type (P = 0.0001). The mutations of STAT5b and STAT3 were mutually exclusive. In contrast to STAT3 mutation-positive patients, among whom significant part of the patients possessed STAT3-mutated subclones only detected with AS-PCR, no pt was identified to be positive for STAT5b mutation only with AS-PCR. We also investigated STAT5b mutations in 29 Chinese patients cohort with T cell LGL L by AS-PCR for N642H and Y665F, and found that no patient was positive for the mutations. Our results indicate that the STAT5b gene is mutated in T-LGL L, which would affect a specific immunophenotype of LGL. STAT5b and STAT3 mutations have significant but distinct contributions to the pathobiology of LGL L and STAT5b-mutated LGL L may consist a unique subtype of LGL L. Disclosures No relevant conflicts of interest to declare.

Description: [Background] We report the results of a prospective multi-institutional clinical trial of BMT from an HLA-matched URD following RIC. [Patients and Methods] The conditioning regimen included cladribine 0.11 mg/kg on day -8 to day -3, busulfan 4 mg/kg po on day -6 and day -5, and 4 Gy TBI on day -1. GVHD prophylaxis included cyclosporine and short-term methotrexate. Patients with hematologic diseases were eligible for this study if they were either older than 50 years or had significant medical contraindications to undergo conventional transplantation. Primary endpoints were neutrophil engraftment and achievement of complete donor-type chimerism (CD3+ cells 〉90%) on day 90. Regimen-related toxicities (RRT) between day -8 and day 28 were assessed by the NCI-CTC v2.0. A total of 27 patients were registered, but one patient was removed before transplant because of severe fungal infection. [Results] The median follow-up time was 722 days (range, 324–996) among survivors. The median age of patients was 56.5 years (36–64). Nine of the 26 patients (36%) had advanced-stage diseases and 3 (11%) had failed previous high-dose autologous or allogeneic transplantation. The diagnoses included AML (n=9), MDS/MPD (n=7), NHL (n=3), ALL (n=2), CML, ATLL, PCL, biphenotypic acute leukemia, and severe aplastic anemia (n=1). The median number of infused nucleated cells was 2.2 × 108/kg. After transplant, while one patient experienced engraftment failure and subsequent sepsis, and died on day 34, the remaining 25 patients achieved neutrophil engraftment (median, 17th day). Another patient was censored from the study due to grade 4 liver dysfunction, which developed on day 19, which left 24 patients for the chimerism analysis. The percentage of donor chimerism in CD3+ cells on days 28, 56 and 90 was, respectively, 88% (21/24), 100% (24/24) and 100% (24/24). Grade 3 RRT included arrhythmia (n=1), hypoxia (n=3), hyperbilirubinemia or hypertransaminasemia (n=7), stomatitis (n=18) and diarrhea (n=4), and grade 4 RRT included hypoxia (n=1) and hyperbilirubinemia (n=1). Acute GVHD of grade II, III and IV occurred in 27%, 27% and 4%, respectively. Ten of 15 evaluable patients (67%) had extensive chronic GVHD. CMV reactivation occurred in 23 patients (89%); 4 had histologically confirmed CMV colitis, 1 had CMV pneumonitis and 1 had CMV hepatitis, while the remaining patients had asymptomatic viremia. Of the 16 patients with measurable disease at the time of BMT, 15 achieved complete remission. The 100-day and 1-year cumulative incidences of non-relapse mortality (NRM) estimated by the Kaplan-Meier method were 20% and 54%, respectively. The cause of death that contributed to NRM was infection, with grade 0–I acute GVHD in 29% and grade II–IV acute GVHD in 71%. The 100-day and 1-year cumulative incidences of relapse were 8% and 35%, respectively, and the 1-year overall and progression-free survival rates were 42% and 30%, respectively. [Conclusions] The results support the feasibility of this procedure with a high response rate, but there is still a problem with the high NRM due to uncontrollable infections primarily associated with GVHD.

Description: Background: Dysregulation of T-cell mediated immunity is responsible for acquired pure red cell aplasia (PRCA). Although STAT3 mutations are frequently detected in patients with T-cell large granular lymphocytic leukemia (T-LGLL), which is often complicated by PRCA and is also reported to be associated with acquired aplastic anemia (AA) and myelodysplastic syndrome (MDS), whether STAT3-mutated T cells are involved in the pathophysiology of PRCA and other types of bone marrow failure (BMF) remains unknown. Methods: Patients with AA, AA-paroxysmal nocturnal hemoglobinuria (AA-PNH) syndrome, MDS or PRCA were enrolled in this study. We performed STAT3 mutation analyses of the peripheral blood mononuclear cells using an allele-specific PCR (AsPCR) to detect STAT3 Y640F or D661Y mutations and amplicon sequencing using primers covering entire coding region of STAT3. CD4+ T cells, CD8+ T cells or granulocytes were sorted, if possible, and also subjected to the analyses. The T-cell receptor (TCR) Vβ repertoire was analyzed using whole blood samples by flow cytometry. Results: A total of 124 patients including PRCA (n=42), AA (n=54), AA-PNH (n=7) and MDS (n=21) were enrolled. The subtypes of PRCA were as follows: idiopathic, n= 15, T-LGLL-associated, n=13; thymoma or thymic cancer-associated, n=7; autoimmune disease-associated, n=5; adverse drug reactions, n=1; and human parvovirus B19 infection complicated by T-LGLL, n=1. As an initial step in the STAT3 mutational analysis, we screened all patients with an AsPCR. Among the 42 patients with PRCA, 3 (10%) of 29 patients without T-LGLL and 6 (46%) of 13 patients with T-LGLL were positive for mutations. In contrast, none of the patients with AA, MDS or AA-PNH were positive (P value= 0.000031). Then, we examined MNC-derived DNA from 73 patients (PRCA, n=42 and AA, n=31) using amplicon sequencing. In this sequencing analysis, the median depth of coverage was 6,219x (range; 1,065-14,188). No STAT3 mutations were detected in 31 AA patients. In contrast, 15 (36%) PRCA patients possessed STAT3 mutations. The variant allele frequency of STAT3 mutations ranged from 0.0057 to 0.489. In all of the 7 patients studied, the STAT3 mutations were restricted to sorted CD8+ T cells. Three patients were negative for STAT3 mutations in MNCs but found to be positive when sorted CD8+ T cells were analyzed. The prevalence of STAT3 mutation in idiopathic, thymoma-associated, autoimmune disorder-associated and T-LGLL-associated PRCA was 33% (5/15), 29% (2/7), 20% (1/5), and 77% (10/13), respectively. In total, STAT3 mutations were detected in 8 of 29 (28%) PRCA patients without T-LGLL and 10 of 13 (77%) PRCA patients with T-LGLL. When TCRVβ repertoires of CD8+ T cells sorted from 3 STAT3 mutation(+) PRCA patients without T-LGLL were analyzed, skewed TCRVb repertoires were evident in all patients, and STAT3 mutations were detected in skewed TCRVβ fractions from 2 patients whose samples were available for cell sorting. The STAT3-mutation(+) patients were younger (median age of 63 years vs 73 years, P= 0.026) and less responsive to cyclosporine (CsA) (46% [6/13] vs 100% [8/8], P= 0.0092) in comparison to STAT3-mutation(-) patients. Of note, 4 of 8 STAT3 mutation(+) patients who had been refractory to CsA were treated with CY and all of them responded well, whereas none of 8 STAT3 mutation(-) patients required secondary treatment with CY owing to a sustained response to CsA. Discussion: This study is the first to reveal frequent STAT3 mutations in PRCA patients, even in thouse without T-LGLL, using a large cohort of patients. The presence of STAT3-mutated CD8+ T cells may be unique background of PRCA, irrespective of disease etiology. Poor response to CsA in STAT3 mutation(+) patients suggests that STAT3-mutated CD8+ T cells may be less sensitive to the inhibitory effects of CsA than non-mutated CD8+ T cells. In contrast, our analyses failed to detect STAT3 mutations in any of 52 AA patients, suggesting that STAT3 mutation(+) T cells had little impact on the development of AA in Japanese patients. Conclusion: STAT3 mutations are frequently detected in the CD8+ T cells of PRCA patients, regardless of the presence of T-LGLL. The identification of STAT3 mutations may be useful for appropriately managing patients with PRCA. Figure. Figure. Disclosures Nakao: Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.