ALBERT

Description: Introduction: Romiplostim is a thrombopoietin mimetic protein that increases platelet production. Romiplostim has already been approved in numerous countries for treatment of immune thrombocytopenia. We previously reported a clinical trial to identify the dosage of romiplostim in aplastic anemia (AA) patients with thrombocytopenia refractory to immunosuppressive therapy (IST). Platelet, erythroid, and neutrophil responses were achieved at high rates with the initial dose at 10 μg/kg in the previous studies (Lee JW et al, ASH2016, 2017). Based on these findings, we conducted a Phase 2/3 clinical study in Japan and Korea for the purpose of evaluating the efficacy and safety of romiplostim in patients with AA who were ineligible or refractory to IST. This abstract shows the efficacy and safety results as of cut-off date (17 Nov 2017), which will be updated with 1-year follow-up result on the ASH2018 annual meeting. Methods: This study was a multi-center, open-label, intra-individual dose adjustment study in adult AA patients in Japan and Korea (NCT02773290). Patients with AA who were ineligible or refractory to IST and having thrombocytopenia with platelet count equal to or less than 30×109/L were enrolled in this study. The dosage of romiplostim was set at the initial level of 10 μg/kg and fixed for the first 4 weeks. The dose was adjusted from 5 to 20 μg/kg according to dose adjustment procedure. Patients who did not achieve a platelet response after the treatment with 20 μg/kg during 8 consecutive weeks were withdrawn from the study. The primary endpoint was the proportion of patients achieving a hematological response (any of the platelet, erythroid, and neutrophil response) at Week 27. Each response was defined as Table1. The secondary endpoints included the proportion of patients achieving hematological response at Week 53; and the time from the first romiplostim administration to hematological response; and the proportion of patients with transfusion independence or decreased platelet transfusion requirement among patients receiving platelet transfusion within 8 weeks prior to the first romiplostim administration. The bone marrow and cytogenetic analyses were performed prior to enrollment and every 6 months after treatment. Results: Of 46 patients with screening, a total of 31 patients (24 Japanese patients, and 7 Korean patients) were enrolled in this study. The median age was 46.0 years old (range: 20-78 years old). All patients had received at least 1 AA treatment, most of which were antithymocyte globulin (71.0%) and cyclosporin (96.8%). As of cut-off date (17 Nov 2017), 28 patients completed assessment of Week 27, and 18 patients completed assessment of Week 53. Three patients discontinued before Week 27, and 1 patient discontinued after Week 27 but before Week 53. In total (31 patients), 26 patients (83.9% [95% CI; 66.3%, 94.5%]) achieved any hematological response at Week 27. Eight patients (25.8%) achieved tri-lineage hematological response at Week 27. The median days to reach any hematological response were 37.0 [95% CI; 36.0, 44.0] days. Of the patients who depended on platelet transfusion before romiplostim administration (15 patients), 12 patients (80.0%) achieved transfusion independence or showed a reduction of transfusion requirements until Week 53. The frequently reported adverse events (AEs) were nasopharyngitis (38.7%) followed by upper respiratory tract infection (22.6%); pyrexia (19.4%); headache (16.1%); and diarrhoea (12.9%). The frequently reported drug-related AEs were headache (12.9%) followed by muscle spasms (9.7%); and alanine aminotransferase increased, fibrin D dimer increased, malaise, and pain in extremity (each 6.5%). In bone marrow test, 2 patients showed abnormality in karyotypes after romiplostim dosing. Monosomy 7 was shown at Week16 in 1 patient who had been receiving granulocyte-colony stimulating factor prior to the start of romiplostim. This patient did not show the transformation into acute myeloblastic leukemia and/or myelodysplastic syndrome. The other patient showed the gains of chromosomes 3, 4, 14, 16, 17, 19 and 21 at Week 27, but did not show any abnormality at Week 53. None of patient discontinued the study because of AE or karyotype abnormality. Conclusion: These results demonstrate that romiplostim is quite effective and well-tolerated in adult patients with AA ineligible or refractory to IST. Disclosures Tomiyama: Sysmex Corporation: Consultancy; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lee:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Miyazaki:Kyowa Hakko Kirin Co., Ltd.: Honoraria, Research Funding; Novartis Pharma Co., Ltd.: Honoraria. Usuki:Novartis: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Janssen Pharmaceutical K.K: Research Funding; Pfizer Japan: Research Funding, Speakers Bureau; Boehringer-Ingelheim Japan: Research Funding; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; Celgene Corporation: Research Funding, Speakers Bureau; SymBio Pharmaceuticals Limited.: Research Funding; Shire Japan: Research Funding; Sanofi K.K.: Research Funding; GlaxoSmithKline K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Nippon Shinyaku: Speakers Bureau; Mochida Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau. Kizaki:Nippon Shinyaku,: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis: Speakers Bureau. Sawa:Celgene Corporation: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis International AG: Honoraria; CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Mundipharma K.K.: Honoraria. Yonemura:Alexion Pharma: Honoraria, Research Funding. Keta:Kyowa Hakko Kirin Co., Ltd.: Employment. Matsuda:Novartis Pharma K. K.: Honoraria; GlaxoSmithKline 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. Nakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

Description: Background Several molecular markers, such as FLT3-internal tandem duplication (ITD), NPM1, CEBPA are well known to correlate with mortality in patients with acute myeloid leukemia (AML). Recently, a number of gene mutations have been implicated in the pathogenesis of AML, including mutations of DNMT3A, IDH1/2, TET2 and EZH2 in addition to RAS, KIT and FLT3. However, DNMT3A, IDH1/2, and TET2 are rare in pediatric patients with AML, thus accurate risk evaluation remained challenging even after incorporating these molecular markers. On the other hand, overexpression of the EVI1 gene is reported to be associated with adverse outcome in pediatric AML. Moreover, we have previously reported that measuring of PRDM16 gene expression was a powerful tool to predict the prognosis of pediatric AML. PRDM16 gene is highly homologous to the MDS1/EVI1 gene, which is an alternatively spliced transcript of the EVI1 gene. In this study, we investigated EVI1 gene expression to verify the prognosis of EVI1 gene expression and the relationship between EVI1 and PRDM16 gene expression. Methods Between 2006 and 2010, 485 de novo pediatric AML patients participated in the Japanese AML-05 study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). Among them, 116 patients were excluded from the study because of misdiagnosis and unavailability of their RNA samples. Therefore, 369 patients were analyzed. Quantitative RT-PCR analysis was performed in these patients using the 7900HT Fast Real Time PCR System with TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay. In addition to EVI1 and PRDM16, ABL1 was also evaluated as a control gene. We investigated the correlations between these gene expressions and other genetic alterations, and clarified the prognostic impact of EVI1 and association between EVI1 and PRDM16 genes. Results A total of 58 of 369 patients (15.7%) showed high expression of EVI1 gene. Overexpression of EVI1 gene was strongly associated with dismal prognosis; low risk (LR; 1 of 123 patients, or 0.8%); intermediate risk (IR; 38 of 147 patients, or 25.9%); high risk (HR; 6 of 50 patients, or 12%); and non-complete remission (Non-CR; 13 of 49 patients, or 26.5%), (P 〈 0.001). Overexpression of EVI1 correlated with the following characteristics: younger age at diagnosis; M4, M5, and M7 subtype; higher coincidence of MLL-rearrangement; and lower coincidence of t(8;21), and inv(16). EVI1 overexpression was very frequent among patients with de novo pediatric AML and IR/non-CR groups. Furthermore, more than half of patients in M6 (5 of 8 patients, or 62.5%) were EVI1 high expression. Interestingly, no patients with EVI1 high expression in M7 had a fusion of CBFA2T3-GLIS2. Patients with EVI1 overexpression also more frequently harbored a complex karyotype and monosomy 7. The frequencies of patients with high or low EVI1 expression differed widely with respect to each genetic alteration, as follows: t(8;21), 1% vs 99%, P 〈 0.001; inv(16), 0% vs 100%, P 〈 0.001; NUP98-JARID1A, 83% vs 17%, P 〈 0.001; OTT-MAL, 100% vs 0%, P = 0.02; and KIT, 5% vs 95%, P = 0.003. The overall survival (OS) and event-free survival (EFS) among patients with EVI1 high expressions were significantly lower than that among patients without such gene aberrant expression (3-year OS 54% vs. 77%, P=0.008G3-year EFS: 34% vs 58%, P

Description: Introduction: SKY59 is an anti-C5 antibody applying SMART* [Fukuzawa et al., SciRep 2017] to allow for infrequent SC dosing. A three-part adaptive clinical trial was conducted in healthy volunteers (part1 -previously reported, Röth et al., Blood 2017 130:4750) and PNH patients who are treatment naive (part 2) or previously treated with eculizumab (ecu) (part 3) to establish dose, safety and efficacy of SKY59. Methods: Part 2 was an intra-patient dose escalation study. Patients received IV doses of 375mg, 500mg, and 1000mg of SKY59 on days 1, 8 and 22, respectively, followed by weekly doses of SKY59 of 170mg SC starting on day 36. In part 3 PNH patients who had been on ecu for at least 3 months received an IV loading dose of 1000mg SKY59 on day 1, 2 weeks after their last ecu dose, and were randomized to receive 170mg SC QW, 340mg SC Q2W or 680mg SC Q4W of SKY59 starting on day 8. After 5 months all patients entered an open-label extension remaining on their previous dose regimen with SKY59. Terminal complement activity was quantified using an ex vivo liposome immunoassay (LIA). The study was approved by Ethics Committees and Health Authorities and conducted according to the principles of the declaration of Helsinki. Results: 17 PNH patients were included (Table 1), 10 in part 2 and 7 in part 3 at the time of writing this abstract. PK: After SC administration, bioavailability is estimated at 100%. For a patient of 75 kg, the terminal half-life was estimated around 25 days. PD: Following IV dosing, complete complement inhibition (defined as LIA values 〈 10 U/mL, the LLOQ of the assay), was achieved at end of infusion in all patients and maintained for all SC dose regimens. Median baseline (BL) total C5 concentration was 107 μg/mL (range: 66.9 - 130 μg/mL) in part 1, 140 μg/mL (73.6 - 184 μg/mL) in part 2, 295 μg/mL (205 - 354 μg/mL) in part 3. Following treatment with SKY59, patients in Part 2 showed a slight increase in total C5 at week 6 to 215 μg/mL (109 - 331 μg/mL). Patients in part 3 had a significant decrease in total C5 at week 6 to 228 μg/mL (184 - 305 μg/mL), likely reflecting the antigen disposing activity of SKY59. Treatment-naive patients (including one Arg885His C5 SNP patient [Nishimura et al.; N Engl J Med. 2014]) with PNH had a rapid median reduction in LDH (-79% from BL; median 1.20 x ULN, range 0.8 to 1.7 x ULN) at 6 weeks. LDH of Patients in part 3 did not show a significant change at 6 weeks or later compared to BL except for one C5 SNP patient who normalized LDH. 6/6 of naive (non-transfusion dependent) and 1/5 switch (non-transfusion dependent) patients had an increase in hemoglobin of at least 10g/L from BL to week 20, one patient in Part 2 and one patient in Part 3 normalized their hemoglobin. Overall transfusion requirements were not changed. QoL: From BL to week 10 treatment naive patients experienced a median change of +11 points (range -1 to 28) in the FACIT fatigue score. Switch patients showed no significant short-term change from BL to week 12 on the same questionnaire. Safety: Treatment with SKY59 was well tolerated, particularly, no injection site AEs and a low incidence of headache was observed. There were 2 SAEs (break-through hemolysis due to infection and atrial fibrillation) not related to study drug. No AEs resulted in withdrawal from the study or death. In 2/7 switch patients mild-moderate non-serious, likely drug-target-drug complex (DTDC) mediated reactions with clinical manifestations similar to serum sickness were observed in the initial post-switch period (day 9 and 10 respectively). These manifestations were treated with topical steroids and resolved by day 21 with no interruption in study treatment. In patients switching from ecu to SKY59, the formation of DTDC composed of SKY59, C5, and ecu are expected due to the different binding epitopes of the two antibodies. Conclusion: SKY59 administered SC in a low volume is very well tolerated, has a good benefit/risk ratio and is efficacious in treatment naive and ecu-treated patients with PNH. Transient induction of DTDC was not associated with undue toxicity. Furthermore, SKY59 has the potential to provide treatment for patients unresponsive to ecu due to SNP and significantly reduces the treatment burden associated with chronic IV administration. Updated data will be presented at the meeting. * Sequential Monoclonal Antibody Recycling Technology Disclosures Röth: Bioverativ: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; Roche: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding. Peffault De Latour:Novartis: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding. Usuki:Takeda Pharmaceutical: Speakers Bureau; Boehringer-Ingelheim Japan: Research Funding; Novartis: Speakers Bureau; Pfizer Japan: Research Funding, Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; Shire Japan: Research Funding; SymBio Pharmaceuticals Limited.: Research Funding; GlaxoSmithKline K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Chugai Pharmaceutical: Speakers Bureau; Celgene Corporation: Research Funding, Speakers Bureau; Sanofi K.K.: Research Funding; Janssen Pharmaceutical K.K: Research Funding; Nippon Shinyaku: Speakers Bureau; Mochida Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau. Winter:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Hsu:Roche: Employment. Dieckmann:Roche: Employment. Anzures-Cabrera:Roche: Employment. Jordan:Roche: Employment. Shinomiya:Chugai: Employment. Klughammer:F. Hoffmann-La Roche Ag: Employment. Bucher:Roche: Employment. Jahreis:Genentech: Employment, Equity Ownership. Nishimura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Chugai Pharmaceuticals: Consultancy, Research Funding.

Description: 【 Background and purpose 】Introduction of novel drugs such as immunomodulatory drugs (IMIDs), and proteasome inhibitors has significantly prolonged survival of multiple myeloma (MM). However, MM harboring high-risk cytogenic changes such as del 17 and t(4;14) is still significantly associated with short survival. Immunotherapy including monoclonal antibodies against MM cells or chimeric antigen receptor expressing T cell (CAR-T) therapy has also showed excellent response to high-risk MM, although the cost of these treatments is expensive. Some of conventional anti-cancer drugs, such as anthracyclines, have been reported to cause induction of immunomodulatory effects by cell surface expression of calreticulin (CRT) followed by release of high mobility group box 1 (HMGB 1) and ATP from cancer cells. Dendritic cells (DCs) recognize CRT, HMGB1 and ATP through CD91, TLR4, P2X7 receptors respectively, and take up cancer cells. This series of events is called immunogenic cell death (ICD). However, there is substantially no information on ICD in MM. The purpose of this study is to investigate whether anti-MM drugs can induce ICD. 【 Methods 】Myeloma cell line harboring high-risk cytogenetic changes, MUM24, KMS34, and KMS21 were treated with anti-myeloma drugs at the IC50, including dexamethasone(320µM), melphalan(2µM), lenalidomide(3.5µM), bortezomib(3nM), carfilzomib(4nM), and panobinostat(6nM). Expression of cell surface CRT and HMGB1 release, which are indicators of ICD, were detected using flow cytometry 48hr after drug treatment and western blotting 24hr after treatment, respectively. We also checked CRT expression on CD138+ cells derived from bone marrow samples of myeloma patients (n=3). Furthermore, the phagocytosis of drug-treated MM cells by DCs generated from healthy human peripheral blood mononuclear cells was evaluated by flow cytometry. 【 Results / Discussion 】Bortezomib and carfilzomib significantly induced expression of CRT and release of HMGB1 in MM cell lines and bone marrow CD138+ cells obtained from MM patients compared with other anti-MM drugs. Especially carfilzomib induced CRT expression at lower concentration. It was also observed that MUM24 cells treated with bortezomib or carfilzomib were effectively taken up by DCs compared to cells treated with other drugs. 【 Conclusion 】Our results suggest that proteasome inhibitors could not only directly kill MM cells but also induce anti-myeloma immune response via ICD. In particular, these immune effects are expected to improve the prognosis of patients in continuous therapy setting after induction therapies by evoking immune memories against residual MM cells, which might contribute to decrease recurrence or formation of extramedullary disease. Moreover, synergistic effects with other cancer immunotherapy such as immune checkpoint inhibitors and CAR-T therapy can be expected. Figure. Figure. Disclosures Hattori: IDAC inc.: Research Funding; Takeda: Research Funding. Matsushita:Amgen: Research Funding.

Description: Hepatitis B virus (HBV) surface antigen (HBsAg)-positive patients treated with anti-B-cell lymphoma therapy are at high risk of HBV reactivation; indeed, the incidence is as high as 60-80% without antiviral prophylaxis. Hepatitis caused by HBV reactivation usually leads to discontinuation of chemotherapy, which in itself may be fatal. Therefore, several guidelines recommend antiviral prophylaxis for HBsAg-positive patients treated with chemotherapy. However, there is limited evidence regarding the clinical impact of antiviral prophylaxis on HBV reactivation and subsequent hepatitis and long-term outcome, particularly in patients treated with rituximab (R)-containing regimens. To answer these questions, we conducted a nationwide multicenter retrospective analysis for HBsAg-positive patients with diffuse large B-cell lymphoma (DLBCL) treated with R-chemotherapy. Data were collected from HBsAg-positive patients with DLBCL who received R-CHOP or R-THP-COP regimens as an initial chemotherapy (regardless of steroid use) at 29 participating institutions between January 2004 and December 2014. Key exclusion criteria were as follows: seropositive for HCV or HIV; alanine transaminase (ALT) levels ≥ 100 U/L at the time of diagnosis; and a history of decompensated cirrhosis or hepatocellular carcinoma. Medical data were also obtained for HBsAg-negative DLBCL patients (controls), regardless of anti-HBc or anti-HBs seropositivity; these patients were diagnosed within 2 month (before and after 1 month) of the diagnosis date of each HBsAg-positive patient. Hepatitis was defined as an absolute serum ALT level of ≥ 100 U/L. HBV reactivation-related hepatitis was defined as hepatitis accompanied by an absolute serum HBV DNA level of ≥ 3.3 Log IU/mL or an absolute increase of ≥ 2 Log compared with the baseline value. Statistical analysis was performed at the Japanese Data Center for Hematopoietic Cell Transplantation using Stata software Version 13.1 and EZR 1.35. The study was registered at UMIN (ID: 000025574). A total of 394 patients were eligible: 116 (29.4%) were HBsAg-positive and 278 (70.6%) were HBsAg-negative. The baseline characteristics of the two cohorts were similar; exceptions was HBV status (Table). R-CHOP was the most common regimen (n=337, 85.5%), followed by R-THP-COP (n=57, 14.5%). For HBsAg-positive patients with detectable and quantifiable HBV DNA (n=65, 56.0%), the median baseline HBV DNA level was 2.9 Log IU/mL (interquartile range, 2.0-3.7). HBsAg-positive patients were divided into three groups based on prophylactic nucleoside analogue (NA) therapy: no prophylactic therapy (non-NA, n=9), prophylactic therapy with lamivudine (LAM, n=20), and prophylactic therapy with entecavir (ETV, n=87). Of the HBsAg-negative patients, 64 (23.0%) were seropositive for anti-HBc or anti-HBs. The median follow-up time for HBsAg-positive and HBsAg-negative patients was 4.3 years and 4.5 years, respectively. The 4-year cumulative incidence of hepatitis in HBsAg-positive and HBsAg-negative patients was 21.1% (95% confidence interval (CI): 14.1-28.9%) and 14.6% (95% CI: 10.7-19.2%), respectively (p=0.081). Importantly, the 4-year cumulative incidence of HBV reactivation-related hepatitis was higher in HBsAg-positive patients than in HBsAg-negative patients (8.0% (95% CI: 3.9-14.0%) vs. 0.4% (95% CI: 0.0-2.0%, respectively; p

Description: Introduction The programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway is an inhibitory immune checkpoint that can suppress T-cell-mediated tumor cytotoxicity. Anti-PD-1 monoclonal antibodies have recently been recognized as promising therapy for adult patients with lymphoma, particularly in classical Hodgkin's lymphoma. However, little information is available regarding the expression patterns of PD-1 and PD-L1 in pediatric lymphoma. Therefore, this study aimed to investigate the expression patterns of PD-1 and PD-L1 in pediatric lymphoma. Methods Immunohistochemical analysis was performed on paraffin-embedded pretherapeutic tumor biopsies from 36 newly diagnosed pediatric patients (aged 0-15 years) with lymphoma or lymphoproliferative disorders treated at Kobe Children's Hospital (Kobe, Japan) from 2003 to 2018. Results Thirty-six samples comprising 11 of Burkitt lymphoma (BL), 7 of anaplastic large-cell lymphoma (ALCL), 6 of T-lymphoblastic lymphoma (T-LBL), 5 of diffuse large B-cell lymphoma (DLBCL), 3 of Hodgkin's lymphoma (HL), 2 of chronic active EBV-associated lymphoproliferative disorders (CAEBV-LPD), 1 of T cell/histiocyte rich B-cell lymphoma (T/HRBCL), and 1 of subcutaneous panniculitis-like T-cell lymphoma (SPTCL) were evaluated. PD-L1 and PD-1 staining results in each lymphoma type are explained below and in Table. Burkitt lymphoma None of the 11 samples stained for PD-L1 or PD-1 in BL cells. PD-1 was expressed in a small proportion of tumor-infiltrating lymphocytes (TIL) in 3 of the 11 samples. Anaplastic large-cell lymphoma PD-L1 was robustly expressed in ALCL cells in 5 of the 7 samples. However, PD-1 was not expressed in any ALCL cell samples but expressed in a small proportion of TIL in only 1 sample. T-lymphoblastic lymphoma None of the 6 samples stained for PD-L1 in T-LBL cells. PD-1 was stained in T-LBL cells in only 1 sample. Moreover, PD-1 was not stained in TIL in any samples. Diffuse large B-cell lymphoma None of the 3 samples with DLBCL-not otherwise specified (DLBCL-NOS) or 1 sample with DLBCL with IRF4 rearrangement expressed PD-L1 on tumor cells. Conversely, PD-L1 was overexpressed in tumor cells in 1 sample with DLBCL with an interfollicular pattern of proliferation (DLBCL-IF). However, PD-1 was not expressed in any DLBCL cell samples. PD-1 was expressed in a small proportion of TIL in 1 sample with DLBCL with IRF4 rearrangement. Hodgkin's lymphoma PD-L1 was overexpressed in HL cells in both nodular sclerosis classic HL (NScHL), whereas PD-L1 was not expressed in nodular lymphocyte predominant HL (NLPHL) cells. PD-1 was not expressed in HScHL or NLPHL cells but was expressed in a small proportion of TIL in 1 sample with NLPHL. Chronic active EBV-associated lymphoproliferative disorders PD-L1 was overexpressed in tumor cells in both samples with CAEBV-LPD. PD-1 was not expressed in tumor cells but was expressed in a small proportion of TIL in 1 sample. T cell/histiocyte-rich B-cell lymphoma PD-L1 was overexpressed on tumor cells in T/HRBCL. PD-1 was weakly expressed in a part of T/HRBCL cells but strongly expressed in TIL. Subcutaneous panniculitis-like T cell lymphoma PD-L1 was overexpressed in tumor cells in SPTCL. However, PD-1 was not expressed in SPTCL cells or TIL. Discussion In this pediatric cohort, PD-L1 was overexpressed in tumor cells in ALCL (5/7), DLBCL-IF (1/1), NScHL (2/2), CAEBV-LPD (2/2), T/HRBCL (1/1), and SPTCL (1/1), but not in BL, T-LBL, DLBCL-NOS, or NLPHL. While the PD-L1 expression in EBV-positive lymphoma cells has been reported before, this study demonstrated the PD-L1 overexpression in CAEBV-LPD. In addition, we demonstrated the PD-L1 overexpression on SPTCL and DLBCL-IF cells, whereas the PD-L1 overexpression in T/HRBCL cells was consistent with previous reports. This study demonstrated that the PD-1 expression in tumor cells was rare in pediatric lymphoma. In addition, PD-1 expressions in TIL tended to be low in pediatric lymphoma, except for NLPHL and T/HRBCL. Besides classic HL, PD-1 blockade might be a promising treatment strategy for ALCL, DLBCL-IF, CAEBV-LPD, T/HRBCL, and SPTCL in children. Indeed, anectodal reports showed promising efficacy in ALCL. Therefore, further investigations are required to assess the role of the PD-1-PD-L1 pathway in pediatric lymphoma. Disclosures No relevant conflicts of interest to declare.

Description: Background Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/ myeloproliferative neoplasm that occurs during infancy and early childhood. The clinical course of the disease varies widely. The majority of children require allogenic hematopoietic stem cell transplantation (HSCT) for long term survival, but the disease will eventually resolve spontaneously in ~15% of patients. Previous studies have identified clinical and molecular risk factors in JMML. More recently, three groups independently discovered that genome-wide methylation profiling using 450K Illumina array revealed that the high methylation (HM) subgroup was significantly associated with poor survival compared to the low methylation (LM) subgroup (Murakami 2018 Blood, Stieglitz 2017 Nat. Commun., Lipka 2017 Nat. Commun.). 450K could be a standard assay for stratification of JMML. However, it is now unavailable because the manufacture replaced it with EPIC array. Here, we developed a next-generation sequencing-based clinical test recapitulate 450K clustering results using the digital restriction enzyme analysis of methylation (DREAM) method (Jelinek 2012 Epigenetics). Patients and Methods We studied 99 children (67 boys and 32 girls) with JMML. All the patients were included in our previous publications. First, we assessed JMML samples with DREAM. Briefly, genomic DNA was sequentially cut with two enzymes SmaI and XmaI recognizing the same sequence, CCCGGG sites in DNA. Enzyme-treated DNA was then used to generate sequencing libraries according to the Illumina protocols, and run on an Illumina Hiseq 2500. We assessed 10 JMML samples with reduced representation bisulfite sequencing (RRBS) (Meissner 2005 Nucleic Acids Res.). In brief, purified genomic DNA was digested by the methylation-insensitive restriction enzyme MspI to generate short fragments that contain CpG dinucleotides at the ends. The CpG-rich DNA fragments (40-220 bp) were size selected, subjected to bisulfite conversion, PCR amplified and end sequenced on an Illumina Genome analyzer. Results We analyzed 99 samples using the DREAM with 8.87 (4.09-16.35) million reads (median, [range]), and determined methylation level in 62,525 (52,356-75,185) CpG sites (median [range]). We observed a strong correlation between DREAM methylation ratio and 450K beta-value of overlapping CpG sites (Pearson r2 = 0.95 [0.913-0.962], median [range]). We performed unsupervised consensus clustering with DREAM methylation data of 7,704 CpG sites within ±1 kb from TSS on autosomal chromosomes detected in ≥95% of the samples with imputation of the missing data using the median of each CpG site methylation level. Clustering identified two distinct subgroups, the HM subgroup (n = 35) and the LM subgroup (n = 64), matching 95% (94 of 99) with the 450K clustering results. The HM subgroup patients showed significantly poorer 5-year OS than the LM subgroup patients (41.9% [95% confidence interval {CI}], 25.3%-57.6%) vs. 71.4% [95% CI, 56.2%-82.1%]; P = 0.00345). Discrepancies in the clustering results between DREAM and 450K were observed in only 5 patients (2 survived and 3 died); all 5 patients were reclassified as those with LM with DREAM from being HM with 450K. We also performed RRBS methylation analysis on 10 patients. Unsupervised consensus clustering using promoter-associated 4,971 CpG sites measured with RRBS identified HM (n = 5) and LM (n = 5) subgroups and completely matched with the classification made using DREAM and 450K. Then, we developed a prediction model of the methylation subgroups using a machine-learning program. We selected 85 CpG sites from 7,704 CpG sites used for unsupervised clustering of the DREAM assay that showed a distinct difference in the average methylation level (〉0.3) between the HM and LM subgroups of the learning cohort (n = 70) and developed a support vector machine (SVM) model. As a validation cohort, we analyzed the remaining 29 JMML samples with a SVM model and confirmed a high matching rate with 450K clustering results (100%, 29 of 29). Conclusions We could develop a methylation test for JMML using the DREAM assay. Both the unsupervised clustering analysis and SVM model could repeat the result of 450K-based methylation classification, i.e., the HM and LM subgroups. The relatively lower cost of the DREAM assay (US$200/sample) enabled us to incorporate methylation classification in JMML in most settings. Disclosures No relevant conflicts of interest to declare.

Description: Key Points PD-L1 expression is associated with poor prognosis in DLBCL. The double-staining technique is a useful method for identifying and distinguishing PD-L1+ DLBCL.

Description: Background & Objective The therapy of multiple myeloma (MM) has improved greatly over the last decade. However, the rise in health care costs has become a big problem. Therefore, it is necessary to consider the cost-effectiveness including Quality of Life (QoL). In this study, we assessed the real-world cost-effectiveness of MM treatment. Methods & Patients A total of 209 MM patients who were newly diagnosed and treated only at Japanese Red Cross Medical Center (JRC-MC) from January 2006 to December 2015 were registered for this retrospective research. Those patients who were treated at other institute than JRC-MC were excluded. All of the costs for MM treatment in hematology department were analyzed. The costs were calculated based on the health care costs spent in hematology department. Overall survival was evaluated by the Kaplan-Meier method. All of the costs were calculated in US dollars based on average value from 2006 to 2015 (1USD = 100.294 Japanese yen). Result First, the cost paid for treatment of 84 patients who died from symptom onset to death was about 0.14 million USD. We also calculated the total annual health care cost for their patients from 2006 to 2015. Annual cost of all MM patients at 2006 was 0.454 million USD, and the cost at 2015 was 7.505 million USD. The cost/patient-year of 2006, when novel agents were not obtainable was 21,656 USD, and that cost of 2015, when three new agents were available was 55,608 USD. These results showed that the costs of MM treatment became 2.56 times higher in the past 10 years. Individual costs for bortezomib, thalidomide, and lenalidomide is shown in Fig.1. Since 2011 to 2014, the ratio of cost of novel agents exceeded 50%. Secondly, overall survival was evaluated by statistical analysis. The median survival from diagnosis was 56 (range, 0-117) months 95% CI (45.12-66.88). We divided patients into 5 subgroups, according to the time of diagnosis. (1)2006: 21 patients, (2)2007-2008: 26 patients, (3)2009-2010: 38 patients, (4)2011-2012: 42 patients, (5)2013-2015: 82 patients. In Japan, the novel agents such as bortezomib, thalidomide, lenalidomide were approved the year 2006, 2008, 2010 respectively. Median survival of subgroup (1), (2), and (3) were 56 months 95% CI (42.66-69.33), 55 months 95% CI (19.12-90.87) and 54 months 95% CI (36.42-71.57) respectively. Median survival of subgroup (4) and (5) have not been reached. Then we investigated the treatment costs in these groups. In each subgroup the average annual cost was 332, 518, 507, 588 and 631 USD respectively. Compared to subgroup (1), the cost of other groups were significantly high. These results showed that the impact of three novel agents on medical cost were significant. We also investigated the relationship between health care costs and the length of hospital stay or autologous stem cell transplantation. There were no statistically significant differences among them, however, there was tendency that the length of hospital stay shortened by time; Average length of total hospital stay the subgroup (1), (2), and (3) were 205, 125 and 56 days respectively. Subgroup (4) and (5) showed also shortened length of hospital stays, although observation years subgroup (4) and (5) were short (date was not shown). Among 5 subgroups the cases of received autologous stem cell transplantation were not stastically differences (total 56 patients). Conclusion We revealed the real-world costs of MM treatment in a single-institute, by following up individual patients sequentially. Health care costs for treatment of MM increased drastically, by introduction of three novel agents. We also showed that the introduction of three novel agents shorten hospital stay, which leads to improve QoL. Therefore, bortezomib, thalidomide and lenalidomide had the significant impact of QoL. After the further approval of novel agents in Japan, health care costs will be even higher but on the other hand, they might bring positive impacts on OS and QoL. Our study clearly indicated the importance of considering the balance of cost and effectiveness for the MM treatment is important. Character count: 3477(excluding spaces) Disclosures No relevant conflicts of interest to declare.

Description: Introduction Pediatric acute myeloid leukemia (AML) comprises less than 20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology. Approximately 40% of patients still have a relapse after first-line therapies, and the expected long-term survival rate decreases following relapse. Stem cell transplantation (SCT) was a conclusive strategy for de novo AML patients with a high risk and relapsed or refractory patients with standard and intermediate risk in a recent clinical trial. AML is a molecularly and clinically heterogeneous disease caused by various genetic alterations. Thus, it is difficult to accurately evaluate risk stratification even if known representative molecular markers, including KIT, FLT3-ITD, t(8;21)/RUNX1-RUNX1T1, and KMT2A (also known as MLL)-rearrangements, are used. Methods We investigated differences in the genetic background between SCT and non-SCT groups in participants of the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial. Among 369 patients with de novo AML, 175 patients received SCT. A standardized form was used to record clinical variables, including patient demographic information. The clinical data of patients in each risk group were followed for 3 years after the date of final registration. JPLSG performed a central review of morphologic classification and karyotyping based on the World Health Organization Classification, French-American-British classification, and cytogenetic analysis using conventional G-banding. Molecular characterization included mutational analyses of KIT (exons 8 and 17), N- and K-RAS (exons 1 and 2), NPM1 (exon 12), CEBPA (exon 12), FLT3-ITD, NUP98-NSD1,and CBFA2T3-GLIS2 gene rearrangement, as well as KMT2A- partial tandem duplication (MLPA methods). We also evaluated the gene expression of MECOM (also known as EVI1) and PRDM16 (also known as MEL1) because their high expressions are known poor prognostic markers. Overall survival (OS) was defined as the time from AML diagnosis to death or censorship at the last follow-up. Event-free survival (EFS) was defined as the time from AML diagnosis to treatment failure, relapse, death, or last follow-up. Results The 3-year OS among SCT patients (n = 175) was approximately 50%. It was significantly worse than that of non-SCT patients (n = 194, 90%; P 〈 0.001). Among 137 CBF-AML patients, 44 patients (32%) received SCT and their 3-year OS was 80%. On the other hand, among 232 non-CBF-AML patients, 131 patients (57%) received SCT, and their 3-year OS was only 35%. This result indicated that SCT is beneficial for relapsed CBF-AML, whereas most non-CBF patients who received SCT did not obtain the clinical benefit. In terms of the molecular characteristics, among 58 patients with high EVI1 expression, 39 patients (67%) received SCT, and their 3-year OS was approximately 35%. EVI1 expression was especially useful when using with MLL rearrangement because prognosis of patients with both MLL rearrangement andhigh EVI1 expression were extremely poor. On the other hand, among 84 patients with high PRDM16 expression, 63 patients (75%) received SCT, and their 3-year OS was approximately 20%. Although all AML patients with FLT3-ITD were assigned to receive SCT in the AML-05 trial, FLT3-ITD(+) patients withlow PRDM16 expression had a better outcome than FLT3-ITD(+) patients with high PRDM16 expression (3-year OS: 70% vs. 21%; P 〈 0.001). This result indicated that FLT3-ITD itself might not necessarily be associated with poor prognosis. PRDM16 gene expression is a useful marker to select patients needing SCT, particularly for patients with FLT3-ITD. Conclusion SCT was beneficial for patients with CBF-AML, whereas SCT was insufficient to rescue patients with non-CBF AML who relapsed, particularly patients with both FLT3-ITD(+) and high PRDM16 expression. New strategies, such as gemtuzumab ozogamicin or haploidentical SCT, are urged to rescue high risk/refractory patients. Disclosures No relevant conflicts of interest to declare.