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: Background and Objective Primary mediastinal large B-cell lymphoma (PMBL) accounts for 2 to 4% of non-Hodgkin lymphomas and is characterized by distinct clinical, pathological and genetic features. Although the utility of DA-EPOCH-R without radiotherapy (RT) and a PET-guided RT approach were recently reported, a standard therapy has not yet been established, mostly due to the lack of data from prospective randomized studies. In addition, the prognosis for patients (pts) with relapsed PMBL is not well understood. Therefore, we conducted a multicenter, cooperative retrospective study to evaluate the clinical outcome of pts with PMBL. Patients and Methods We analyzed a total of 345 pts with newly diagnosed PMBL from 65 institutes between May 1986 and September 2012 in Japan. Pts were treated according to each institutional protocol or physicians' decisions. In pts treated with R-CHOP, the role of PET before RT was analyzed. In addition, we analyzed prognostic factors for PMBL pts and constructed a novel prognostic model using data from patients treated with R-CHOP. Results The median age was 32 (range, 17-83) years, and female pts were predominant (58%) among the patient population. Median tumor diameter was 10 cm. Stage I/II, low-risk by IPI, and PS 0/1 were also predominant (68%, 52% and 75%, respectively). The presence of pleural or pericardial effusion, elevated lactate dehydrogenase level and extra-nodal lesions were observed in 46%, 80% and 44% of pts, respectively. With a median follow-up of 48 months in surviving pts, overall survival (OS) and progression-free survival (PFS) at 4 years were 87% and 70%, respectively. The OS and PFS were improved in pts treated with rituximab(R)-containing chemotherapy (n = 267) (4-year OS: 91% vs. 77%, P 〈 0.001; 4-year PFS: 75% vs. 54%, P 〈 0.001, respectively). The OS at 4 years for patients treated with CHOP (n = 44), R-CHOP (n = 187), DA-EPOCH-R (n = 9), second- or third-generation regimens (n = 45; 28 with R and 17 without R), and chemotherapy followed by ASCT (n = 57; 43 with R and 14 without R) were 67%, 90%, 100%, 91% and 92%, respectively (P 〈 0.001). The PFS at 4 years were 40%, 71%, 100%, 83% and 76%, respectively (P 〈 0.001) (Figure 1). Consolidative RT was given to 42% of the patient population. A total of 119 of 187 pts treated with R-CHOP were assessed by PET/CT after the completion of R-CHOP, and 64 pts received consolidative RT after R-CHOP. In pts with negative PET after R-CHOP (n = 84), the OS (100% vs. 100%, P 〉 0.99) and PFS (92% vs. 72%, P = 0.36) at 4 years were similar when comparing pts treated with (n =25) or without RT (n =59). However, in pts with positive PET after R-CHOP (n = 28), both OS (100% vs. 60%, P = 0.010) and PFS (80% vs. 17%, P 〈 0.001) at 4 years were superior in pts who received RT (n = 21) than in pts who did not receive RT (n = 7). A total of 97 pts (28%) relapsed or progressed after first-line therapy. Of these, 67 (19%) and 11 pts (3%) relapsed in the mediastinum and CNS, respectively. Median time from initial diagnosis to relapse or progression was 9 months. Median OS after relapse or progression was 16 months and was higher in patients treated with stem-cell transplantation (SCT) (n = 58; 44 ASCT, 14 allogeneic SCT) than in patients who did not undergo SCT (4-year OS: 67% vs. 31%, P 〈 0.001). The IPI was predictive for OS (P = 0.002) and PFS (P 〈 0.001) in pts with PMBL treated with R-CHOP. Moreover, multivariate analysis showed that the presence of pleural or pericardial effusion was a significant and independent prognostic factor for PFS. We constructed a novel prognostic model (PMBL prognostic index; PMBIPI) and classified pts treated with R-CHOP into three different risk groups using these two factors (the presence of pleural or pericardial effusion, and IPI high/intermediate-risk or high-risk). For 93 pts (51%) classified as the low-risk group (0 factor), OS and PFS at 4 years were 97% and 89%, respectively. For 61 pts (34%) classified as the intermediate-risk group (1 factor), OS and PFS at 4 years were 85% and 59%, respectively. For 27 pts (15%) classified as the high-risk group (2 factors), OS and PFS at 4 years were 72% (P = 0.001) and 44% (P 〈 0.001), respectively (Figure 2). Conclusions The combination of R and chemotherapy improved outcomes for patients with PMBL. In addition, PET could predict the necessity for RT in pts with PMBL treated with R-CHOP. PMBIPI is a promising tool for risk-stratification of pts with PMBL. These findings require further validation in prospective studies. 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: Abstract 1549 Poster Board I-572 Classical Hodgkin lymphoma (CHL) is characterized by Hodgkin and Reed Sternberg (H-RS) cells, which are B-cell origin in many cases. Recently, we highlighted an adverse prognostic significance of cytotoxic molecule (CM) expression among CHL patients (Asano N, et al. J Clin Oncol. 2006). However, the clinical characteristics of CM-positive CHL still remain controversial. We here document the clinicopathologic profiles of 35 patients with CM-positive CHL, consisting of 23 men and 12 women with a median age of 50 years (range, 16 - 84 years). All patients had lymphoadenopathy, and 14 cases showed mediastinal involvement at presentation. Physical findings included hepatomegaly and splenomegaly in six and five patients, respectively. Four patients had a bulky mass, and nine showed stage IV disease. As for laboratory data, four patients had elevated white blood cell counts (greater than 15.0 × 103/mm3). Anemia (hemoglobin level; less than 10.5 g/dl) was also present in six patients. The pathological diagnoses were nodular sclerosis type (NS) in 22 cases and mixed cellularity (MC) in 12 cases. The H-RS cells of CM-positive CHL had a prototypic immunophenotype of CD15+ CD30+ and Fascin+. All of the cases completely lacked CD20 positivity on H-RS cells, while the expression of CD3e and CD45RO was found in 2 and 1, respectively. The H-RS cells were further positive for EBV RNA transcripts in 14 of 32 (44%) cases studied by in situ hybridization method. All except three cases were negative for Pax5. A clonal TCR-g chain gene arrangement was undetected in any of the cases with successful amplification of control GAPDH DNA by PCR analysis. No cases show B-cell clonality. 27 of 35 patients received systemic multi-agent chemotherapy consisting of first-line treatment regimens as follows: doxorubicin, bleomycin, vinblastine, and dacarbacin (ABVD) (22 patients); cyclophosphamide, doxorubicin, vincristin, and prednisone (CHOP) (2 patients); two with C-MOPP; and one BEACOPP regimens. Three patients died within 6 months, before completing induction treatment because of disease progression. Overall, 22 patients responded to first-line treatment: 13 with complete response and 9 with partial response. 13 of them had relapses, and 8 died with a clinical course ranging from 7 to 142 months. Effective therapeutic approaches should be explored for CM-positive CHL patients, who resist standard treatment for CHL. Comparing CM-positive nodal peripheral T-cell lymphomas of not otherwise specified type (PTCL-N)(n=55) with CM-positive CHL(n=35), no significant differences were detected in clinical parameters except for the frequency of elevated lactate dehydrogenase level in CM-positive PTCL-N. Immunophenotypically, CM-positive CHL cases showed significantly higher rates for CD15, CD30, and Fascin expression, while CM-positive PTCL-N cases showed significantly higher CD3e, CD8, CD45RO, and CXCR3 positivity. The survival curve of CM-positive CHL showed poorer prognosis than that of CM-negative CHL (P = .0002) and better than that of CM-positive PTCL-N (P = .002) (Figure). These findings suggest that CM-positive CHL is characterized by an unfavorable clinical feature, although their histologic and phenotypic features are more suggestive of CHL. Moreover, one case in CM-positive CHL had presented the skin lesion as a CD30-positive lymphoproliferative disorder or an ALK-negative anaplastic large cell lymphoma (ALCL) at the time of recurrence. These findings suggest that CM-positive CHL represent a distinct variant form based on clinicopathologic and phenotypic traits beyond the framework of CHL. Significant overlaps in biologic and morphologic features has been identified in CHL and non-Hodgkin lymphoma. CM-positive CHL cases may be ‘intermediate lymphoma‘ between CHL and PTCL or ALK-negative ALCL. Further studies, including array CGH profiling analysis are needed to clarify the origin of CM-positive CHL. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Description: Background. Idiopathic pure red cell aplasia (PRCA) and secondary PRCA not responding to the treatment of the underlying diseases are generally thought be immune-mediated and treated by immunosuppressive therapy. We previously conducted the PRCA2004/2006 study and reported that poor response to induction therapy and relapse of anemia were associated with death. Principal causes of death were infections and organ failure. Based on the literatures, idiopathic PRCA may represents the prodrome to myelodysplastic syndromes. Theoretically, there are two potential mechanisms of unresponsiveness to immunosuppression; the clonal hematopoiesis by the stem/progenitor cells that have undergone somatic mutations during disease progression of PRCA and the clonal changes of auto-aggressive lymphocytes reacting against erythroid progenitors. Objectives. In this study, we investigated the somatic mutations of myeloid malignancy-associated genes in acquired PRCA in order to determine how often clonal hematopoiesis is detected in this disorder. Materials and Methods. This study included 23 patients with chronic acquired PRCA (12 idiopathic, 7 thymoma-, 2 LGL leukemia- and 2 systemic lupus erythematosus-associated PRCA) with a median age of 62 (range: 40-62). Disease status was varying. After obtaining informed consent, heparinized blood was drawn and mononuclear cells were separated by density gradient centrifugation. Extracted genomic DNA samples were subjected to targeted sequencing for 54 myeloid malignancy-associated genes using a TruSight Myeloid Sequencing Panel kit according to the manufacturer's instruction (Illumina). Criteria for the significant somatic mutations of myeloid malignancy-associated genes in the present study were as follows: potential functional consequences such as missense, nonsense or frameshift mutations; exclusion of previously reported SNPs; being recurrently detected in two sequencing runs; variant allele frequency (VAF) exceeding 0.02 and less than 0.40. The institutional review board approved the experimental protocol. Results. We detected some mutations of the targeted genes in 20 out of 23 patients, and the somatic mutations defined by the criteria mentioned above were found in 10 patients including 6 idiopathic, 3 thymoma-associated and one LGL leukemia-associated PRCA (Fig. 1). These 10 patients had 38 distinct mutations in 20 genes. Variant allele frequencies were 0.02 to 0.37 (median, 0.04; average, 0.06, Fig. 2). Four patients had more than one mutated genes and multiple genes were mutated in some patients (Fig. 1). The most frequently mutated gene was CUX1 that was found in four patients, and STAG2, DNMT3A, KDM6A, SMC3A, ASXL1, TET2 and TP53 were mutated in more than one patient. Discussion/Conclusion. This study demonstrated that myeloid malignancy-associated genes were somatically mutated in 43% of acquired chronic PRCA patients. This figure appears to exceed the prevalence rate of clonal hematopoiesis of indeterminate potential (CHIP) in the general population with the age of 60s. These mutations were presumably carried by monocytes, because DNA samples were prepared from PBMCs in this study cohort. Profiles of mutated genes in PRCA appear to be different from those of aplastic anemia that were previously reported by other groups. It is yet to be known whether this could result from the different nature of both diseases, or the difference in the experimental protocols. Our findings strongly encourage conducting a prospective study to confirm our observation and clarify the diagnostic and predictive values of somatic mutations of myeloid malignancy-associated genes in acquired PRCA. This project is ongoing in collaboration with the prospective cohort study PRCA2016 being conducted in Japan. Disclosures Nakao: Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Matsuda:GlaxoSmithKline K.K.: Honoraria; Novartis Pharma K. K.: Honoraria; Chugai Pharmaceutical Co, Ltd.: Honoraria; Kyowa Hakko Kirin Co, Ltd.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria; Celgene Corporation: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Sanofi K.K.: Honoraria; Beckman Coulter K.K.: Honoraria. Mitani:Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau; Bristol-Myesr Squibb: Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Chugai: Research Funding; Astellas: Research Funding; Sumitomo Dainippon: Research Funding; Novartis: Research Funding; Toyama Chemical: Research Funding.

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.

Description: Age-related Epstein-Barr virus–associated B-cell lymphoproliferative disorder (aEBVLPD) is a disease group characterized by EBV-associated large B-cell lymphoma in elderly without predisposing immunodeficiency. In nearly one- third of cases, aEBVLPD occurs as a polymorphous subtype with reactive cell-rich components, bearing a morphologic similarity to classic Hodgkin lymphoma (cHL). The aim of this study was to clarify clinicopathologic differences between the polymorphic subtype of aEBVLPD (n = 34) and EBV+ cHL (n = 108) in patients aged 50 years or older. Results showed that aEBVLPD was more closely associated with aggressive clinical parameters than cHL, with a higher age at onset (71 vs 63 years); lower male predominance (male-female ratio, 1.4 vs 3.3); and a higher rate of involvement of the skin (18% vs 2%), gastrointestinal tract (15% vs 4%), and lung (12% vs 2%). aEBVLPD was histopathologically characterized by a higher ratio of geographic necrosis, greater increase (〉 30%) in cytotoxic T cells among background lymphocytes, higher positivity for CD20 and EBNA2, and absence of CD15 expression. As predicted by the clinical profile, aEBVLPD had a significantly poorer prognosis than EBV+ cHL (P 〈 .001). The polymorphous subtype of aEBVLPD constitutes an aggressive group with an immune response distinct from EBV+ cHL, and requires the development of innovative therapeutic strategies.

Description: Introduction Aggressive natural killer cell leukemia (ANKL) is a rare leukemic form of mature natural killer cell neoplasms that is closely associated with Epstein-Barr virus. ANKL presents a fulminant clinical course, resulting in a poor prognosis with a median overall survival of approximately 2 months. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only curative treatment, but the long-term outcomes after allo-HSCT remain unclear. Methods Using the national Japanese transplant registry database, the outcomes of 59 ANKL patients who underwent first allo-HSCT between 1997 and 2016 were analyzed. Correlation between groups was examined by the c2 test, Mann-Whitney U test, and Kruskal-Wallis test. Patient survival data were analyzed using the Kaplan-Meier method and compared by the log-lank test. The cumulative incidence of relapse and non-relapse mortality were calculated considering competing risks. Results The median patient age was 37 years (range, 9 to 66), and males accounted for 68%. The median time from diagnosis to allo-HSCT was 3.7 months (range, 1.1 to 13.9). Twenty-nine patients received stem cells from cord blood (CB), 18 received them from peripheral blood (PB), and 12 received them from bone marrow (BM). Two patients had a prior history of autologous HSCT. Twenty-one patients (36%) had a complete response (CR) and 7 (12%) had a partial response (PR), but 31 (52%) were not responding at the time of allo-HSCT. Forty-four patients received myeloablative conditioning and 15 received non-myeloablative conditioning. Thirty-two patients received tacrolimus-based GVHD prophylaxis, including 1 with additional post-transplant cyclophosphamide as part of haploidentical HSCT, whereas 26 received cyclosporin-based GVHD prophylaxis. The median follow-up of survivors was 62 months (range, 0.9 to 193). The median OS and relapse-free survival were 3.9 months and 2.6 months, respectively. The probability of OS and relapse-free survival, and cumulative incidence of relapse and non-relapse mortality 1 year after HSCT were 33.9%, 32.4%, 55.5%, and 12.1%, respectively. The probability of OS was significantly higher for patients with CR or PR at allo-HSCT than for those without a response (40.6% vs 16.1% at 5 years; P = 0.046). Among the 24 patients with primary induction failure (PIF) at allo-HSCT, 15 achieved CR after allo-HSCT. The prognosis of these 15 patients was almost equivalent to that of those who received allo-HSCT in CR or PR, as shown in the Figure (P = 0.95). Regarding the stem cell source, the probability of OS was significantly higher for patients who received stem cells from CB than for those who received them from PB or BM (CB 37.3% vs PB 15.8% and BM 16.7% at 5 years; P = 0.04). Seventeen patients (59%) of those who received stem cells from CB were CR at HSCT, whereas only 4 patients (13%) of those who received stem cells from PB or BM were CR at HSCT. In addition, 5 (83%) of 6 patients who received stem CB transplantation in PIF achieved CR after allo-SCT, whereas 10 (56%) of 18 patients who received PB stem cell transplantation or BM transplantation in PIF achieved CR. The age and year at HSCT were not different between the groups. The time from diagnosis to allo-HSCT did not differ among stem cell sources (median CB 3.3 months, PB 3.7 months and BM 4.1 months; P = 0.31). Regarding the conditioning regimen, the probability of OS was not different between myeloablative and non-myeloablative conditioning regimens (P = 0.58). Patients who developed acute GVHD grade 1/2 had a significantly better prognosis than those with grade 3/4 or without GVHD (P 〈 0.001). In contrast, chronic GVHD development did not affect the prognosis (P = 0.60). At the last follow-up, 42 patients (71%) had died. The most common cause of death was primary disease (62%), followed by infection (14%) and organ dysfunction (7%). Conclusion Allo-HSCT can lead to long-term survival even for patients with PIF at HSCT. CB is useful as a stem cell source, providing good outcomes and timely allo-HSCT for this rapidly progressive disease. To confirm our findings and evaluate the outcome of allo-SCT in more detail, further studies including patients who did not receive allo-SCT for ANKL are warranted. Figure Disclosures Ishida: Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; Astellas Pharma: Research Funding; Eli Lily and Company: Research Funding; Celgene: Honoraria; Chugai Pharmaceutical: Consultancy, Research Funding. Izutsu:Celgene: Consultancy, Research Funding; Chugai: Honoraria, Research Funding; Daiichi Sankyo: Honoraria, Research Funding; Astra Zeneca: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Symbio: Research Funding; Ono: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Solasia: Research Funding; Zenyaku: Research Funding; Incyte: Research Funding; Novartis: Honoraria, Research Funding; Sanofi: Research Funding; HUYA Bioscience: Honoraria, Research Funding; MSD: Honoraria, Research Funding; Astellas Amgen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; ARIAD: Research Funding; Takeda: Honoraria, Research Funding; Pfizer: Research Funding; Kyowa Kirin: Honoraria; Nihon Medi-physics: Honoraria; Janssen: Honoraria; Dainihon Sumitomo: Honoraria; Bristol-Byers Squibb: Honoraria; Mundi: Honoraria; Otsuka: Honoraria; Asahi Kasei: Honoraria. Suzumiya:Celgene, Kyowa Kirin, Chugai-Roche, Eisai, Takeda, Celltrion, SymBio, Astellas, Ono, AstraZeneca, Ootsuka, Taiho, Mundi, Dainihon-Sumitomo: Research Funding. Mitsui:MSD pharmaceutical: Research Funding; Maruho pharmaceutical: Research Funding; JCR pharmaceutical: Research Funding; Teijin pharmaceutical: Research Funding; Chugai pharmaceutical: Research Funding; Daiichi Sankyo pharmaceutical: Research Funding; Astellas pharmaceutica: Research Funding; Shionogi pharmaceutical: Research Funding. Kanda:Kyowa Hakko Kirin: Honoraria; Otsuka: Honoraria; Daiichi Sankyo Company: Honoraria; MSD: Honoraria; Chugai: Honoraria; Bristol-Meyers Squib: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Astellas: Honoraria; JCR Pharmaceuticals: Honoraria; Takeda: Honoraria; NextGeM Incorporation: Patents & Royalties: 2019-011392. Atsuta:CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Kyowa Kirin Co., Ltd: Honoraria. Suzuki:Celgene: Honoraria; Novartis: Honoraria; AbbVie: Honoraria; Kyowa Hakko Kirin: Honoraria; Chugai Pharmaceutical Co.,Ltd.: Honoraria; Meiji Seika: Honoraria; Bristol-Myers Squibb: Honoraria; Merck Sharp & Dohme: Honoraria; Takeda Pharmaceutical Co., Ltd.: Honoraria; Eisai: Honoraria; ONO Pharmaceutical Co., Ltd.: Honoraria; Janssen: Honoraria.

Description: Abstract 2511 Background: Although prognosis of acute myeloid leukemia (AML) with t(8;21) is better than other types of AML, outcome of the patients has not been satisfied. Previously, aberrant antigen expression has been reported as risk factor for AML with t(8;21). However, in the reported series, number of cases was not large enough and chemotherapy regimens were variable. We investigated the association of prognosis and several biomarkers including immunophenotype, WBC count, age, and performance status for large number of AML patients with t(8;21) uniformly treated in JALSG AML97 regimen. Patients and Methods: Seven hundred eighty-nine eligible AML patients were evaluated for the multicenter JALSG AML97 study. Adult patients with de novo AML except for APL, ages 15–64 years, were registered consecutively from 103 institutions that participated in JALSG from December 1997 to July 2001. One hundred forty-four patients with AML with t(8;21) were analyzed in this study with a median 1205 days of observation term from diagnosis. Complete remission (CR), relapse-free survival (RFS), and overall survival (OS) rates were analyzed by Fisher's exact test and log-rank test. Factors that would affect clinical outcome were analyzed by multivariate Cox proportional hazard regression model. Results: AML with t(8;21) frequently expressed CD19, CD34, and CD56 compared to other subtypes of AML. CD11b was rarely expressed. Expression of CD19 favorably affected on CR rate (96% in CD19 positive and 87% in negative patients, p20×109/L, CD19 negativity, and CD56 positivity were adverse factors for RFS. CD56 expression was the only independent adverse factor for RFS by multivariate analysis (73.7% in CD56 negative and 48.2% in CD56 positive patients at 3 yrs) although its expression did not affect on OS. There was no difference of age, sex, WBC count, presence or absence of Auer rod, performance status, or CD15 expression between CD56 positive and negative cases. Expression of CD19 was more common in CD56 negative patients (50% in CD56 negative and 30.6% in CD56 positive patients, p

Description: Background Natural killer (NK) cell malignancies are rare lymphoid neoplasms characterized by aggressive clinical behavior and poor treatment outcomes. Clinically they are classified as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK cell leukemia (ANKL). Both subtypes are almost invariably associated with Epstein-Barr virus (EBV). Recently, genomic studies in NKTCL have identified recurrent somatic mutations in JAK-STAT pathway molecules STAT3 and STAT5b as well as in the RNA helicase gene DDX3X in addition to previously detected chromosomal aberrations. Here, we identified somatic mutations in 4 cases of ANKL in order to understand whether these entities share common alterations at the molecular level. To further establish common patterns of deregulated oncogenic signaling pathways operating in malignant NK cells, we performed drug sensitivity profiling using NK cell lines representing ANKL, NKTCL and other malignant NK cell proliferations. We aimed to identify sensitivities to agents that selectively target components of pathways required for survival of malignant NK cells in an unbiased manner. Methods Exome sequencing was performed on peripheral blood or bone marrow of ANKL patients using the NK cell negative fraction or other healthy tissue as control. Profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs. The NK cell lines KAI3, KHYG-1, NKL, NK-YS, NK-92, SNK-6 and YT and IL-2-stimulated and resting NK cells from healthy donors were used as sample material. All drugs were tested on a 384-well format in 5 different concentrations over a 10,000-fold concentration range for 72 h and cell viability was measured. A Drug Sensitivity Score (DSS) was calculated for each drug using normalized dose response curve values. Results The ANKL patients displayed mutations in genes reported as recurrently mutated in NKTCL, such as FAS, TP53, NRAS, STAT3 and DDX3X. Additionally, novel alterations in genes previously implicated in the pathogenesis of NKTCL were detected. These included an inactivating mutation in INPP5D (SHIP), a negative regulator of the PI3K/mTOR pathway and a missense mutation in PTPRK, a negative regulator of STAT3 activation. Interestingly, the total number of nonsilent somatic mutations in 3 out of 4 ANKL patients (97, 82 and 45) was remarkably high compared to other hematological malignancies analyzed in our variant calling pipeline. Analysis of drug sensitivities in NK cell lines showed a close correlation between all cell lines and a markedly higher correlation with those of IL-2 stimulated than resting healthy NK cells, suggesting that malignant NK cells may share a common drug response pattern. Furthermore, in an unsupervised hierarchical clustering the NK cell lines formed a distinct group from other leukemia cell lines tested (Fig. A). Among pathway-selective compounds (namely, kinase inhibitors and rapalogs), the drugs most selective for malignant NK cells fell into two major categories: PI3K/mTOR inhibitors (e.g. temsirolimus, buparlisib) and inhibitors of aurora and polo-like kinases such as rigosertib and GSK-461364 (Fig. B). JAK inhibitors (e.g. ruxolitinib, gandotinib) and CDK inhibitors (e.g. dinaciclib) showed strong efficacy in both malignant NK cells and IL-2 activated healthy NK cells. Conclusions Our exome sequencing results suggest that candidate driver alterations affecting similar signaling pathways underlie the pathogenesis of ANKL as has been reported in NKTCL. Drug sensitivity profiling highlights the PI3K/mTOR pathway as a potential major driver of malignant NK cell proliferation, whereas JAK-STAT signaling appears to be essential in both healthy and malignant NK cells. Components of these pathways harbored mutations in our small cohort of ANKL patients and have been shown to be deregulated by mutations or other mechanisms in previous studies, underlining their importance as putative drivers. The systematic large-scale characterization of drug responses also identified these pathways as potential targets for novel therapy strategies in NK cell malignancies. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Disclosures Mustjoki: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.