ALBERT

Description: Background: Owing to the development of novel agents, the rate of complete response (CR) in multiple myeloma (MM) has increased. Additionally, the development of methods for measuring minimal residual disease (MRD) (e.g., multiparameter flow cytometry [MFC] and next-generation sequencing) has enabled us to stratify CR patients according to MRD levels. In this study, we hypothesized that deep response predicts better prognosis in MM. To investigate this hypothesis, we assessed the response of patients treated with carfilzomib + lenalidomide + dexamethasone (KRD) using MFC and compared survival outcomes between different groups defined by the MRD status. Methods: The response of patients with relapsed/refractory MM treated with KRD at four different centers between September 2016 and October 2018 was prospectively investigated using the EuroFlow next-generation flow (EuroFlow-NGF) method. In this method, ammonium chloride-based bulk lysis was used, followed by surface staining with antibodies against CD138-BV421, CD27-BV510, CD38 multiepitope (ME)-FITC, CD56-PE, CD45-PerCP Cy5.5, CD19-PECy7, CD117-APC, and CD81-APC C750 in tube 1 and surface/intracellular staining with antibodies against CD138-BV421, CD27-BV510, CD38 ME-FITC, CD56-PE, CD45-PerCP Cy5.5, CD19-PECy7, CD117-APC, CD81-APC C750, cytoplasmic (cy) Igκ-APC, and cyIgλ-APC C750 after permeabilization in tube 2. MRD levels were assessed using bone marrow (BM) cells after several KRD cycles, with the lower limit of detection set at 1 × 10−5. Presence of high-risk cytogenetics [del 17p, t(4;14) and/or t(14;16)] in BM cells was analyzed through FISH. Results: A total of 21 patients (12 males, 9 females) were treated with KRD and assessed for MRD levels. The median age of these patients was 66 years at KRD initiation (range 30-83 years), and 11 patients had ISS 1, 6 had ISS 2, and 4 had ISS 3. Four patients displayed high-risk chromosomal abnormalities, including del 17p (n = 3) and t(14;16) (n = 1). The median number of prior treatments was 3 (range 1-6); these included bortezomib (n=12), lenalidomide (n=19), and autologous stem-cell transplantation (n=12). The median number of KRD cycles was 4 (range 1-22). The proportion of patients achieving ≥CR and overall response (≥ partial response [PR]) was significantly higher after KRD treatment than the proportion that had been achieved by previous therapies (71% vs. 9.5%, p 〈 0.001; 100% vs. 71%, p = 0.008, respectively). Pre-KRD responses included 2 stringent CR (sCR), 7 very good PR (VGPR), 6 PR, 3 stable disease, and 3 progressive disease. Post-KRD responses included 13 sCR, 2 CR, 3 VGPR, and 3 PR. A total of 95% (20/21) of patients achieved sCR, and 5% (1/21) VGPR as best response. After KRD, response was upgraded in 19 (90%) patients and maintained in two PR (10%) patients. During and after KRD treatment, MRD negativity was achieved in 12 of 16 (75%) and in 15 of 21 (71%) patients, respectively. The median number of therapy lines after KRD was 1 (range 0-5). All 4 high-risk cytogenetic cases achieved MRD negativity. Among MRD-positive cases, both 2-year progression-free survival (PFS) and 2-year overall survival (OS) from KRD initiation were 100%. Among MRD-negative cases, 2-year PFS and OS from KRD initiation were 92% and 100%, respectively. The median follow-up was 1.8 years (range 0.5-2.5 years). One MRD-negative case showed extramedullary relapse 1.4 years after the last KRD cycle. This patient did not have high-risk cytogenetics and achieved "flow MRD negativity" after two KRD cycles, and the treatment was stopped after 7 KRD cycles due to peripheral neuropathy. Paiva et. al. also reported that only 6 of 225 (3%) MRD-negative patients relapsed. Strikingly, all 6 relapsing cases in the report had extramedullary plasmacytomas at diagnosis; all relapsed with extramedullary plasmacytomas and only 2 developed concomitant serological relapse (ASH 2017, abstract #905). Conclusions: KRD induced deep responses in relapsed/refractory MM patients who eventually displayed excellent PFS. All patients with high-risk cytogenetics achieved EuroFlow-NGF negativity. Post-remission imaging studies such as MRI/PET-CT may be necessary for patients who presented with extramedullary plasmacytomas even when they achieved flow MRD negativity. Figure Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Takamatsu:Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ono pharmaceutical: Honoraria, Research Funding; CSL Behring: Research Funding; SRL: Consultancy, Research Funding; Janssen Pharmaceutical: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Fujimoto Pharmaceutical: Honoraria; Becton, Dickinson and Company: Honoraria; Abbvie: Consultancy; Daiichi-Sankyo Company: Honoraria. Yamashita:Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Daiichi-Sankyo Company: Honoraria; TEIJIN PHARMA LIMITED: Honoraria. Murata:Celgene: Honoraria; Ono pharmaceutical: Honoraria. Yoshihara:Kyowa Kirin: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Eisai Co., Ltd.: Honoraria. Yoshihara:Chugai Pharmaceutical Co.,Ltd: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis Pharma K.K.: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Kyowa Kirin: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Celgene: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria. Nakao:Bristol-Myers Squibb: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Novartis Pharma K.K: Honoraria; Kyowa Kirin: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Ono Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; SynBio Pharmaceuticals: Consultancy. Matsue:Takeda Pharmaceutical Company Limited: Honoraria; Novartis Pharma K.K: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria.

Description: The existence of T cells capable of inhibiting in vitro hematopoiesis has been shown in aplastic anemia (AA), although whether such inhibition is mediated by a specific immune reaction involving an HLA allele remained unknown. We isolated a CD4+ Vβ21+ T-cell clone that was most dominant among Vβ21+ T cells in the bone marrow (BM) of an AA patient whose HLA-DRB1 alleles included 1501 and 0405. The T-cell clone named NT4.2 lysed an autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) and phytohemagglutinin-stimulated lymphocytes (PHA-blasts) as well as allogeneic LCLs sharing HLA-DRB1*0405. Cytotoxicity against LCL cells and PHA-blasts by NT4.2 was blocked by anti–HLA-DR monoclonal antibody (MoAb) or anti-CD3 MoAb. NT4.2 also lysed autologous BM mononuclear cells enriched with CD34+ cells that had been cultured for one week in the presence of colony-stimulating factors as well as allogeneic CD34+ cells of a normal individual carrying HLA-DRB1*0405, cultured in the same way. Moreover, NT4.2 strongly inhibited colony formation by hematopoietic progenitor cells derived from cultured CD34+ cells sharing HLA-DRB1*0405. These results indicate that the AA patient has T cells capable of killing hematopoietic cells in an HLA-DRB1*0405-restricted manner and that such cytotoxic T cells may contribute to the pathogenesis of AA.

Description: Background: The rate of complete response (CR) in multiple myeloma (MM) has dramatically increased because of the development of novel agents. In addition, the development of methods for measuring minimal residual disease (MRD), such as multiparameter flow cytometry and next-generation sequencing, has made it possible to stratify CR patients according to the MRD extent. EuroFlow next-generation flow (EuroFlow-NGF) is considered one of the gold standard methods for evaluating the negative status of MRD in MM. The automated gating strategy of EuroFlow-NGF has been shown to detect MRD as accurately as the manual gating strategy by experts. Oberle et al. (Haematologica, 2017) have found that daratumumab persisted on the surface of myeloma cells treated with it and that the anti-CD38 multi-epitope antibody used in EuroFlow-NGF has partial cross-reactivity with daratumumab, leading to generally lower mean fluorescence intensities of CD38. Therefore, MRD levels may have been underestimated in patients who were treated with anti-CD38 monoclonal antibodies (mAbs) using the automated gating strategy, leading to inappropriate management of the patients. Because no studies have examined the correlation of MRD extent between the manual and automated gating strategies in patients with MM who have received anti-CD38 mAbs, we compared MRD detection between the two gating strategies of EuroFlow-NGF in patients with MM. Methods: The study included bone marrow samples from 51 patients with MM (27 male and 24 female patients), including 13 patients treated with anti-CD38 mAb (12 treated with daratumumab and 1 treated with isatuximab). The median patient age was 70 years (range, 32-92 years) at MRD assessment. The disease statuses at MRD assessment were stringent CR in 26 patients (51%), CR in 7 (14%), very good partial response in 13 (26%), partial response in 1 (2%), and progressive disease in 4 (8%). The sample preparation protocol, Ab panel, and automated gating strategy of EuroFlow-NGF have been reported previously (Flores-Montero et al. Leukemia 2017). Briefly, we performed the EuroFlow-NGF method, which involved ammonium chloride-based bulk lysis, followed by surface staining using antibodies against CD138-BV421, CD27-BV510, CD38 multiepitope (ME)-FITC, CD56-PE, CD45-PerCP Cy5.5, CD19-PECy7, CD117-APC, and CD81-APC C750 in tube 1 and surface/intracellular staining using antibodies against CD138-BV421, CD27-BV510, CD38 ME-FITC, CD56-PE, CD45-PerCP Cy5.5, CD19-PECy7, CD117-APC, CD81-APC C750, cytoplasmic (cy) Igκ-APC, and cyIgλ-APC C750 after permeabilization in tube 2. For data analysis, events from both eight-color tubes (tubes 1 and 2) were merged, and the values of all parameters per tube were mathematically calculated using the merge and calculation functions of Infinicyt software (Cytognos SL, Salamanca, Spain). Automatic identification and enumeration of total plasma cells (tPCs) and abnormal plasma cells (MRD) were performed using the automatic gating function of Infinicyt software as described previously (Flores-Montero et al. Leukemia 2017). We compared the total nucleated cell number, tPC ratio, and MRD ratio between the manual (by experts) and automated gating strategies of EuroFlow-NGF. Results: In patients with MM who did not receive any anti-CD38 mAb therapy, we observed high correlations for both the tPC (r = 0.959, P 〈 0.0001) (Figure A) and MRD (r = 0.974, P 〈 0.0001) (Figure B) ratios between the manual and automated gating strategies of EuroFlow-NGF. On the other hand, in patients with MM who received anti-CD38 mAb therapy, we did not observe good correlations for both the tPC (r = 0.349, P = 0.2) (Figure A) and MRD (r = 0.292, P = 0.3) (Figure B) ratios between the two strategies owing to a lower fluorescence intensity of CD38 on PCs. In addition, when the MRD threshold was set to 10-5, the discordance of MRD positivity/negativity between the two strategies was significantly higher in patients who received anti-CD38 mAb therapy than in those who did not receive anti-CD38 mAb therapy [4/13 (31%) vs. 1/38 (3%), P = 0.012]. Conclusion: Although the automated gating strategy of EuroFlow-NGF could be a viable alternative to the manual strategy for the assessment of MRD in MM, we may have to utilize the manual strategy to obtain precise MRD results for patients with MM who received anti-CD38 mAbs. Figure Disclosures Takamatsu: Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ono pharmaceutical: Honoraria, Research Funding; CSL Behring: Research Funding; SRL: Consultancy, Research Funding; Janssen Pharmaceutical: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Fujimoto Pharmaceutical: Honoraria; Becton, Dickinson and Company: Honoraria; Abbvie: Consultancy; Daiichi-Sankyo Company: Honoraria. Yoroidaka:Ono Pharmaceutical: Honoraria. Yamashita:Janssen Pharmaceutical K.K.: Honoraria; Daiichi-Sankyo Company: Honoraria; Kyowa Kirin: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; TEIJIN PHARMA LIMITED: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria. Murata:Celgene: Honoraria; Ono pharmaceutical: Honoraria. Nakao:Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Ohtsuka Pharmaceutical: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria. Matsue:Novartis Pharma K.K: Honoraria; Ono Pharmaceutical: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Janssen Pharmaceutical K.K.: Honoraria.

Description: Idarubicin (IDA) is one of the key drugs for treating hematological malignancies. Severe myelosuppression is one of the major adverse events of IDA. Interestingly, when IDA is administered in 2 consecutive courses of therapy, IDA tends to exert a stronger bone marrow suppressive effect in the second course compared with the first course while other anthracyclines such as daunorubicin tend to milder suppressive effect in the second course compared with the first course (Wiernik PH et al, Blood, 1992). In order to clarify this unique characteristics, in vivo and in vitro studies regarding the pharmacokinetic behavior were performed. When RLN-B2 (a rat liver cell line) was precultured in the presence of IDA, higher carbonyl reducing enzymes (CREs) activity was observed compared with non-precultured cells, suggesting the anthracycline-reducing enzymes was induced in the cells incubated in the presence of IDA. We examined the in vivo effects of IDA administration on the induction of CREs and subsequent enhanced formation of the 13-OH metabolite, idarubicinol (IDAol) which is more active compared with IDA and has a remarkably long half-life in the blood. The rats (F344) preadministered IDA showed higher enzymatic activity than that from non-preadministered rats (p 〈 0.05). At 4 hours after IDA administration, the production of IDAol was facilitated in the preadministered group compared with the non preadministered group (p 〈 0.05). Clinically, the duration of leucopenia was compared between IDA and mitoxantrone, an CREs independent anthraquinone, in combination with enocytabine, 6-mercaptopurine and etoposide. In 2 consecutive therapy, namely remission-induction and first consolidation therapy, in 30 cases of acute myelogenous leukemias, the duration of leucopenia was substantially equal in IDA group while it was substantially shorter in consolidation therapy compared with induction therapy in mitoxantrone group. These results suggest that CREs was induced by IDA pretreatment in vitro and in vivo, resulting in increased IDAol, which could potentiate the myelosuppressive and probably antitumor effects of IDA in contrast to other anthracyclines. This unique PK/PD characteristics of pharmacokinetic self-potentiation could be important in the safe and effective use of IDA in the therapy for hematological malignancies.

Description: The existence of T cells capable of inhibiting in vitro hematopoiesis has been shown in aplastic anemia (AA), although whether such inhibition is mediated by a specific immune reaction involving an HLA allele remained unknown. We isolated a CD4+ Vβ21+ T-cell clone that was most dominant among Vβ21+ T cells in the bone marrow (BM) of an AA patient whose HLA-DRB1 alleles included 1501 and 0405. The T-cell clone named NT4.2 lysed an autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) and phytohemagglutinin-stimulated lymphocytes (PHA-blasts) as well as allogeneic LCLs sharing HLA-DRB1*0405. Cytotoxicity against LCL cells and PHA-blasts by NT4.2 was blocked by anti–HLA-DR monoclonal antibody (MoAb) or anti-CD3 MoAb. NT4.2 also lysed autologous BM mononuclear cells enriched with CD34+ cells that had been cultured for one week in the presence of colony-stimulating factors as well as allogeneic CD34+ cells of a normal individual carrying HLA-DRB1*0405, cultured in the same way. Moreover, NT4.2 strongly inhibited colony formation by hematopoietic progenitor cells derived from cultured CD34+ cells sharing HLA-DRB1*0405. These results indicate that the AA patient has T cells capable of killing hematopoietic cells in an HLA-DRB1*0405-restricted manner and that such cytotoxic T cells may contribute to the pathogenesis of AA.

Description: Administration of cyclosporine A (CsA) after autologous stem cell transplantation elicits an autoimmune syndrome with pathology similar to graft-versus-host disease (GVHD). This syndrome, termed autologous GVHD, is associated with the appearance of autoreactive T cells directed at major histocompatibility class (MHC) class II antigens. In the rat model of autologous GVHD, clonal analysis reveals that the effector T cells are highly conserved and recognize a peptide from the invariant chain peptide presented by MHC class II. Although human autologous GVHD effector T cells share a similar phenotypic specificity, clonality of the response in humans has not been determined. To examine the human effector T-cell response, the T-cell repertoire of peripheral blood lymphocytes was assessed by complementarity-determining region 3 (CDR3) size distribution analysis and T-cell clonotype analysis in 26 patients treated with CsA after transplantation. Autologous GVHD developed in 3 of 4 patients with human leukocyte antigen (HLA)-DRB1*0701, and clonal expansions of β-chain variable region (BV)16+ T cells were shared. Clonal expansions within BV15+ and BV22+ T cells were also detected in 4 of 6 patients with HLA-DRB1*1501 and in 3 of 4 patients with HLA-DRB1*0401, respectively. Sequencing of BV16 cDNA for which the CDR3 size pattern exhibited apparent clone predominance revealed an identical CDR3 peptide sequence in 2 different patients, one with HLA-DRB1*0701 and the other with HLA-DRB1*1502. These findings indicate that the discrete antigen-driven expansion of T cells is involved in autologous GVHD.