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Carfilzomib, lenalidomide, and dexamethasone (KRD) therapy is widely used for patients with relapse/refractory multiple myeloma (RRMM). However, the response in patients who underwent assessment for measurable residual disease (MRD) has not been elucidated in a prospective study. We aimed to clarify the response rate and outcome of KRD therapy in patients in RRMM, including those with MRD.Twenty-one consecutive RRMM patients treated with KRD at 4 Japanese Centers between September 2016 and October 2018 were enrolled and assessed for MRD in the bone marrow (cut-off: 1×10The median number of therapy lines before KRD was 3 (range=1-6), and the median number of KRD cycles was 4 (range=1-22). As the best overall response post-KRD therapy, 52% (11/21) of patients achieved a MRD negative complete response, 71% (15/21) achieved stringent complete response/complete response, and 14% (3/21) achieved a very good partial response. MRD negativity was achieved in 12 of 16 (75%) and 14 of 21 (67%) patients during and after KRD treatment, respectively. The 2-year progression-free survival and overall survival from the start of KRD therapy were 100% and 100%, respectively, in MRD-positive cases and 88% and 100%, respectively, in MRD-negative cases (median follow-up=1.8 years). Grade 3/4 toxicities were reported in 15 patients (71%), with thrombocytopenia being the most frequent toxicity (6 patients, 29%).This is the first study that prospectively assessed MRD of patients with RRMM after KRD therapy. KRD treatment achieved a high MRD negativity rate and good outcomes with manageable toxicities.

The outcome of immunosuppressive therapy (IST) and prognosis in patients with aplastic anaemia (AA) secondary to chemotherapy or radiotherapy for cancers remains unknown. A total of 43 of 2559 patients with AA referred to our hospital had previously received chemoradiotherapy for various types of solid tumours (n = 25) or haematological malignancies (n = 18). Their cancer status was complete remission (CR) in 27, non-CR in 13, and unknown in three. Small populations of glycosylphosphatidylinositol-anchored protein-deficient [GPI(-)] granulocytes were detected in 16 patients (37·2%). Of 18 patients who were treated with IST, 50% improved regardless of the presence of GPI(-) cells. The overall survival (OS) rate was significantly higher in patients with a history of solid tumours patients than in those of haematological malignancies (median OS, 87 vs. 11 months, P = 0·0003), and in patients treated with IST than in those of untreated patients (median OS, 115 vs. 20 months, P = 0·028). Cancer aggravation occurred in two of four patients who were treated with IST while in non-CR of their original cancers. Progression to myelodysplastic syndromes was observed in two patients not possessing GPI(-) cells. IST should thus be considered for patients with AA secondary to chemoradiotherapy for cancers, particularly when their original solid tumours are in CR.

Background Autologous stem cell transplantation (ASCT) is still a gold standard treatment in multiple myeloma (MM). So far, we have reported the prognostic value of minimal residual disease (MRD) detection in autografts at ASCT setting using EuroFlow next-generation flow (NGF) and next-generation sequencing (NGS) (Takamatsu et al, ASH 2018). The main problem of EuroFlow-NGF is its lower sensitivity (2×10-6) compared with that of NGS ( Methods The study enrolled 9 newly-diagnosed MM patients whose frozen autografts’ cells were preserved. The median age at ASCT was 52 (range 47-61) years and included 4 males and 5 females at ISS I (n=2), II (n=6) and III (n=1). Of there, 4 patients harbored high-risk chromosomal abnormalities including t(4;14) (n=1), t(14;16) (n=1), del17p (n=1), and t(4;14) and del 17p (n=1). All patients received bortezomib-based chemotherapy for induction together with melphalan at 200 mg/m2 for conditioning before ASCT. Two patients received consolidation therapy with carfilzomib-lenalidomide-dexamethasone (KRD) and all patients received lenalidomide maintenance until progressive disease. Frozen autografts (n=9) and primary myeloma cells (n=1) were thawed for MRD assessment by EuroFlow-NGF and NGS. The EuroFlow-NGF method was based on the previous report (Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive’s standardized NGS-MRD Assay (Seattle, WA) (Ching et al., BMC Cancer 2020). The EuroFlow-NGF method was modified to increase the sensitivity of MRD by capturing cells up to 5×107. Results Because frozen autografts were used in this study, we performed the sensitivity test using the dilution of frozen/thawed primary MM cells in an autograft by EuroFlow-NGF. The sensitivity test revealed a strong correlation between 1×10-7 and 1×10-4 of MRD level (r=0.9996, p Conclusions This modified EuroFlow-NGF method can assess MRD of frozen/thawed autografts and its sensitivity can be increased up to 4×10-7 that is comparable to NGS.

To characterize glycosylphosphatidylinositol-anchored protein-deficient (GPI[−]) and HLA-class I allele-lacking (HLA[−]) hematopoietic stem progenitor cells (HSPCs) in acquired aplastic anemia (AA), we studied the peripheral blood (PB) of 56 AA patients in remission who possessed both (n = 13, Group A) or either GPI(−) (n = 34, Group B) and HLA(−) (n = 9, Group C) cell populations. Seventy-seven percent (10/13) of Group A had HLA(−) cells in all lineages of PB cells, including platelets, while only 23% (3/13) had GPI(−) cells in all lineages, and the median percentage of HLA(−) granulocytes in the total granulocytes (21.2%) was significantly higher than that of GPI(−) granulocytes (0.28%, P

We sought to determine the efficacy of a new, inexpensive, single-tube 8-color multiparameter flow cytometry (MFC) method (SRL-Flow), which is based on the EuroFlow next-generation flow (NGF) (tube 2 only), to assess minimal residual disease (MRD)-negative status. MRD-negative status is considered a treatment milestone in multiple myeloma (MM). We used 45 bone marrow samples from patients with MM, including 11 cases treated with anti-CD38 monoclonal antibody. The SRL-Flow sample preparation protocol was identical to that of EuroFlow-NGF. The antibody panel for SRL-Flow was as follows: CD138V450/CD27V500/CD38ME (multiepitope)FITC/CD56PE/CD45PerCP-Cy5.5/CD19PE-Cy7/cytoplasmic (Cy) immunoglobulin (Ig) κAPC/CyIgλAPC-H7. To identify abnormal plasma cells (aPCs) of patients with MM who received anti-CD38 monoclonal antibody, we used a panel of anti-CD45 and anti-CD138 antibodies (Abs) rather than a panel of anti-CD45 and anti-CD38 Abs. We comparatively analyzed the total nucleated cell numbers, total PC levels, and MRD levels between the SRL-Flow and EuroFlow-NGF. High correlations (r > 0.9) in total PC and MRD levels were noted among SRL-Flow, original EuroFlow-NGF (2 tubes), and EuroFlow-NGF (tube 2 only), suggesting that SRL-Flow is an inexpensive (< $200 USD/sample as of January of 2019) alternative to EuroFlow-NGF (< $350 USD/sample) for assessing MRD in MM.

The loss of killer cell Ig-like receptor ligands (KIR-Ls) due to the copy number–neutral loss of heterozygosity of chromosome 6p (6pLOH) in leukocytes of patients with acquired aplastic anemia (AA) may alter the susceptibility of the affected leukocytes to NK cell killing in vivo. We studied 408 AA patients, including 261 who were heterozygous for KIR-Ls, namely C1/C2 or Bw6/Bw4, for the presence of KIR-L–missing [KIR-L(−)] leukocytes. KIR-L(−) leukocytes were found in 14 (5.4%, C1 [n = 4], C2 [n = 3], and Bw4 [n = 7]) of the 261 patients, in whom corresponding KIR(+) licensed NK cells were detected. The incidence of 6pLOH in the 261 patients (18.0%) was comparable to that in 147 patients (13.6%) who were homozygous for KIR-L genes. The percentages of HLA-lacking granulocytes (0.8–50.3%, median 15.2%) in the total granulocytes of the patients with KIR-L(−) cells were significantly lower than those (1.2–99.4%, median 55.4%) in patients without KIR-L(−) cells. KIR2DS1 and KIR3DS1 were only possessed by three of the 14 patients, two of whom had C2/C2 leukocytes after losing C1 alleles. The expression of the KIR3DS1 ligand HLA-F was selectively lost on KIR-L(−) primitive hematopoietic stem cells derived from 6pLOH(+) induced pluripotent stem cells in one of the KIR3DS1(+) patients. These findings suggest that human NK cells are able to suppress the expansion of KIR-L(−) leukocytes but are unable to eliminate them partly due to the lack of activating KIRs on NK cells and the low HLA-F expression level on hematopoietic stem cells in AA patients.

Background: Autologous stem cell transplantation (ASCT) remains the gold-standard treatment for multiple myeloma (MM). To date, we have reported the prognostic value of minimal residual disease (MRD) detection in autografts in an ASCT setting using EuroFlow next-generation flow (NGF) and next-generation sequencing (NGS) (Takamatsu et al., ASH 2018, 2020). The main problem with NGF is its lower sensitivity (2 × 10 -6) compared with that of NGS ( Methods: We reanalyzed 11 autografts in which the MRD were negative on NGF but positive (n = 7) or negative (n = 4) on NGS (Takamatsu et al., ASH 2018, abstract #258) using 5-20 mL of autografts with NGF to increase the sensitivity of MRD detection. Additionally, we enrolled 9 patients with newly diagnosed MM, from whom 5-20 mL of apheresed autografts were cryopreserved. We included 20 patients with newly diagnosed MM. The median age at ASCT was 60 (range, 45-67) years, and the patients included 12 men and 8 women at International Staging System I (n = 3), II (n = 13), and III (n = 4), 6 of whom harbored high-risk chromosomal abnormalities, including t(4;14) (n = 3), t(14;16) (n = 1), del17p (n = 1), and t(4;14) and del 17p (n = 1). All patients received bortezomib-based chemotherapy for induction followed by melphalan at a dose of 200 mg/m 2 for conditioning before ASCT. Three patients received consolidation therapy with carfilzomib-lenalidomide-dexamethasone (n = 2) or bortezomib-lenalidomide-dexamethasone (n = 1), and 18 patients received lenalidomide (n = 16), thalidomide (n = 1), or thalidomide and lenalidomide (n = 1) maintenance. Frozen autografts (n = 20) were thawed for MRD assessment using NGF and NGS. The NGF method was based on a previous report (Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD assay (Seattle, WA) (Ching et al., BMC Cancer 2020). The NGF method was modified to increase the sensitivity of MRD detection by capturing up to 6 × 10 7 cells. Results: Frozen autografts were used in this study; therefore, we performed a sensitivity test using a dilution of frozen/thawed primary MM cells in an autograft with NGF. The sensitivity test revealed a strong correlation between 5 × 10 -7 and 1 × 10 -4 MRD levels (Figure 1A; r = 0.999, P Conclusion: The modified EuroFlow-NGF method may be used to assess MRD in frozen/thawed autografts, and its sensitivity may increase up to 5 × 10 -7, which is comparable to that of NGS. Figure 1 Figure 1. Disclosures Yamashita: Janssen: Honoraria; Bristol-Myers Squibb: Honoraria; celgene: Honoraria; Takeda: Honoraria. Nakao: Novartis Pharma: Honoraria; Symbio: Consultancy; Kyowa Kirin: Honoraria; Alexion Pharma: Research Funding. Takamatsu: Adaptive Biotechnologies, Eisai: Honoraria; SRL: Consultancy; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

Background: Novel agents capable of inducing deeper responses dramatically improve the prognosis of patients with multiple myeloma (MM). Innovative technologies such as multiparameter flow cytometry (MFC) and next-generation sequencing (NGS) are utilized to assess minimal residual disease (MRD) for further stratification of patients who achieve a complete response (CR). EuroFlow-next-generation flow (EuroFlow-NGF) is one of the gold standard MFC methods. Recently, both NGF and NGS have been used in many clinical trials to assess MRD levels associated with progression-free survival (PFS) and overall survival (OS). The present study prospectively assessed MRD levels by both NGF and NGS to elucidate the prognostic impact of both methods and clarify their characteristics in MM patients in an autologous stem cell transplantation (ASCT) setting. Methods: We prospectively assessed the response in Japanese patients with newly diagnosed MM who underwent ASCT and lenalidomide-based maintenance therapy at multiple Japanese medical centers between September 2016 and July 2021. The diagnosis of MM and patients' responses to therapy were assessed using the IMWG criteria. Only patients with CR or stringent CR on days 100-365 post-ASCT were included, and bone marrow (BM) samples were obtained to assess MRD. Four milliliters of BM was divided equally. Cells derived from 2 mL BM were analyzed by the NGF method (Flores-Montero et al., Leukemia 2017) at Kanazawa University, and DNA extracted from the remaining 2 mL BM cells was processed by Adaptive Biotechnologies' standardized NGS-MRD assay (Seattle, WA) (Ching et al., BMC Cancer 2020) to assess MRD levels. MRD levels in BM were also monitored at 1-year (± 20 days) and 2-year (± 20 days) post-ASCT. The prognostic value of MRD levels in BM was assessed, and their correlation between NGF and NGS was compared at a cut-off value of 1×10 -5. Sustained MRD negativity was defined as the maintenance of MRD negativity in the BM for more than 6 months. BM cells were analyzed for high-risk cytogenetics (del(17p), t(4;14), and t(14;16)) by FISH. Results: A total of 60 patients (male = 29, female = 31) underwent bortezomib-based induction therapy, ASCT conditioned with high-dose melphalan, and lenalidomide-based maintenance. The median age was 62 years at the ASCT (range 36-71; ISS 1 [n = 13], 2 [n = 24], and 3 [n = 23]). Thirty-three percent of patients showed high-risk chromosomal abnormalities (del17p (n=11), t(4;14) (n=10), t(14;16) (n=2)), 3 patients had double hit diseases, and five patients had extramedullary diseases. With a median follow-up of 3 years, the 3-year progression-free survival (PFS) and 3-year overall survival (OS) rates were 69.2% and 94.2%, respectively. In total, 148 samples were analyzed using NGF and 138 were analyzed using NGS. The rates of MRD negativity at least once using NGF and NGS were 80% and 61%, respectively. The patients who achieved at least one MRD negativity exhibited significantly better 3-year PFS (82.9% by NGF; 84.8% by NGS) than those who did not (P < 0.0001, 0% by NGF; P = 0.005, 49.1% by NGS). Patients who sustained MRD negativity for more than 6 months also showed significantly better 3-year PFS (96.7% by NGF; 92.3% by NGS) compared with those without sustained MRD negativity (Figure; P < 0.0001, 37.1% by NGF; P < 0.01, 50.9% by NGS). The MRD levels between the NGF and NGS methods were significantly correlated with each other (r = 0.9295, P < 0.0001). Among the 17 patients who developed PD after ASCT, seven cases showed discrepancies in the MRD results and two cases in which one case was MRD-positive and the other was MRD-negative by both methods progressed with extramedullary diseases. Five of the seven cases were MRD-positive by NGS and MRD-negative by NGF. Conclusions: In this prospective comparison study of MRD assessment in BM cells using EuroFlow-NGF and NGS approaches, MRD levels highly correlated with each other, and MRD negativity and sustained MRD negativity were significantly associated with prolonged PFS. Multiple MRD assessments by NGF or NGS are essential for predicting durable remission and prolonged clinical outcomes. Figure 1 Figure 1. Disclosures Takamatsu: Bristol-Myers Squibb: Honoraria, Research Funding; Adaptive Biotechnologies, Eisai: Honoraria; SRL: Consultancy; Janssen: Consultancy, Honoraria, Research Funding. Yoshihara: Bristol-Myers Squibb: Honoraria; Janssen: Honoraria; Novartis: Honoraria. Matsumoto: Sanofi: Honoraria; Janssen: Honoraria; Ono: Honoraria; Bristol-Myers Squibb: Honoraria. Yamashita: Janssen: Honoraria; Bristol-Myers Squibb: Honoraria; celgene: Honoraria; Takeda: Honoraria. Fuchida: Takeda Pharmaceutical Co., Ltd.: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Sanofi: Honoraria; Bristol-Myers Squibb Co., Ltd.: Honoraria; Celgene Co., Ltd.: Honoraria. Hiragori: BML: Current Employment. Suzuki: Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; ONO: Honoraria; Novartis: Honoraria; Sanofi: Honoraria; Abie: Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Nakao: Symbio: Consultancy; Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Alexion Pharma: Research Funding. Durie: Amgen: Other: fees from non-CME/CE services ; Amgen, Celgene/Bristol-Myers Squibb, Janssen, and Takeda: Consultancy.

In immune-mediated acquired aplastic anemia (AA), the presence of an HLA allele, which is highly overrepresented or lost due to somatic mutations, may represent a specific immune pathophysiology and a clinical manifestation. HLA-B*14:02 is one of the most overrepresented class I alleles in AA and is also frequently affected by a somatic loss of expression; the inherited B*14:02 genotype was correlated with high-risk clonal evolution in two independent cohorts in the U.S. (Babushok DV et al. Blood Adv 2017; Zaimoku Y et al. manuscript in preparation). In contrast, HLA-B*14:02 is virtually absent in Japanese, in whom somatic mutations of AA have frequently been detected in HLA-B*40:02, B*54:01, and A*02:06, and occasionally in A*02:01, A*02:07, A*31:01, B*13:01, B*40:01, B*40:03, B*44:03, B*55:02, and B*56:01 (Mizumaki H et al. Haematologica 2021). A class II allele HLA-DRB1*15 is highly overrepresented in AA across various ethnic groups, including those in the U.S. and Japanese. This retrospective study in the Japanese population aimed to explore the clinical significance of disease-associated non-B*14:02 HLA class I and II alleles in AA. A total of 423 enrolled patients with AA (very severe [n = 81], severe [n = 266], transfusion dependent non-severe [n = 76]; median age 60 [range, 1-86] years) had undergone genotyping for HLA-A, HLA-B, HLA-C, and HLA-DRB1 at 2-field resolution. The HLA allele frequencies in these patients were compared to those in a Japanese HLA haplotype dataset (n = 19183; Ikeda N et al. Tissue Antigens 2016). The most overrepresented allele in AA was HLA-DRB1*15:02, followed by DRB1*15:01, B*40:02, and A*02:06 (Table); DRB1*13:02 and B*44:03, which are in linkage disequilibrium, were markedly underrepresented, consistent with a well-known protective role of DRB1*13 against autoimmune diseases. HLA-DRB1*15:02 was also significantly correlated with age and its frequency among patients aged The overall response rate to anti-thymocyte globulin-based immunosuppressive therapy at 6 months was 63% (139 of 220 treated and evaluable patients). A trend for a higher response was observed in patients harboring mutation-related HLA-B alleles (except for minor alleles B*13:01, B*40:03, and B*55:02) and the highly overrepresented or protective HLA-DRB1 alleles, but not in the HLA-A alleles (Figure D). A multivariate logistic regression revealed that the combination of the presence of any favorable alleles in HLA-B (odds ratio 3.6, P < 0.0001) or in HLA-DRB1 (odds ratio 2.3, P = 0.00085) was significantly and independently associated with a hematologic response; the tendencies for a lower or higher response in very severe disease and the presence of paroxysmal nocturnal hemoglobinuria clone did not reach statistical significance. Further, there was likely an additive effect when two favorable alleles coexisted in HLA-B or HLA-DRB1 (Figure E); the copy number of the favorable HLA-B and HLA-DRB1 alleles stratified the response rate to four groups: three or four copies, 95% (19 of 20); two copies, 72% (61 of 85); one copy, 59% (50 of 85); and zero copy, 30% (9 of 30). Only eight patients displayed clonal evolution to monosomy 7, myelodysplastic syndrome, or acute myeloid leukemia after immunosuppression without significant overrepresentation or underrepresentation of the pathogenic HLA alleles. Using a large dataset of homogeneous Japanese population with high-resolution HLA typing, we revealed, for the first time, a strong relationship between disease-associated (overrepresented, inactivated, or protecting) HLA alleles and the responsiveness to immunosuppressive therapy. Figure 1 Figure 1. Disclosures Takamatsu: Bristol-Myers Squibb: Honoraria, Research Funding; SRL: Consultancy; Adaptive Biotechnologies, Eisai: Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Yamazaki: Novartis Pharma: Honoraria; Kyowa Kirin: Honoraria; Kyowa Kirin: Research Funding. Nakao: Symbio: Consultancy; Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Alexion Pharma: Research Funding.

Background: Autologous stem cell transplantation (ASCT) in conjunction with novel therapeutic drugs can dramatically improve response rates and the prognoses of patients with multiple myeloma (MM). However, most patients with MM ultimately relapse due to minimal residual disease (MRD). Next-generation multiparameter flow cytometry (MFC) (EuroFlow-NGF) and next-generation sequencing (NGS) are currently the standard methods to assess MRD. Aims: To compare the prognostic value of MRD detection in autografts and bone marrow (BM) cells using 8-color MFC (EuroFlow-NGF) and NGS (Adaptive Biotechnologies), and also MRD levels between fresh and cryopreserved autografts using NGF. Methods: The study enrolled 52 newly-diagnosed MM patients who underwent ASCT. The median age ASCT was 61 (range 41-69) years and included 29 males and 23 females at ISS I (n = 17), II (n = 23), and III (n = 12). Of these, 18 patients harbored high-risk chromosomal abnormalities including t(4;14) (n = 15), del17p and t(4;14) (n = 2), and complex (n = 1). Bortezomib-based chemotherapy was used for induction together with melphalan at 140 mg/m2 (n = 1) and 200 mg/m2 (n = 51) for conditioning before ASCT. 39 of 52 (75%) patients received maintenance therapy until progressive disease. The best responses achieved post-ASCT included 30 sCR, 4 CR, 15 VGPR, and 3 PR. Forty autografts, one from each MM patient, were analyzed using NGF and NGS protocols, and BM cells at pre/post-ASCT and autografts derived from 16 patients were analyzed using NGS. The EuroFlow-NGF method uses standard sample preparation; large numbers of cells are evaluated using an optimized 8-color antibody panel that facilitates accurate identification of discrimination between phenotypically aberrant plasma cells (aPCs) and their normal counterparts (Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD Assay (Seattle, WA) (Martinez-Lopez et al., Blood 2014). Eight additional autografts were used to assess MRD in both fresh and cryopreserved samples by NGF. Results: MRD was evaluated in 48 of 52 autografts (92%) using NGF and in 44 of 52 autografts (85%) using NGS. We identified aPCs in autografts based on multivariate analysis of individual cell populations (e.g., CD56+, CD19−, CyIgκ+, and CD117+). As the results of NGF revealed a strong correlation with respect to MRD in fresh vs. thawed autografts (r = 0.999, P < 0.0001), MRD was subsequently evaluated in thawed autografts. The sensitivity of NGF was 1 × 10−5-2 × 10−6; the sensitivity of NGS was 1 × 10−6. 28 of 48 (58%) of the autografts were MRD-positive by NGF; 30 of 44 (68%) of the autografts were MRD-positive by NGS. MRD levels in autografts using NGF and NGS correlated with one another (r = 0.69, P < 0.0001; Fig. 1A). MRD negative in autografts by NGF cases (MRDNGF (-)) and MRDNGS (-) tended to show better progression-free survival (PFS) than MRDNGF (+) (P = 0.195) and MRDNGS (+) (P = 0.156), respectively. Furthermore, MRDNGS (-) showed significantly better overall survival (OS) than MRDNGS (+) (P = 0.03) (Fig. 1C) while MRDNGF (-) showed better OS than MRDNGF (+) (P = 0.09) (Fig. 1B). Our data revealed only a minimal correlation between MRD in the autografts (median 1.1 × 10−5,range 0-7.29 × 10−4) and in the BM cells at pre-ASCT (median 5.05 × 10−3,range 6 × 10−6-2.64 × 10−1; r = 0.09, P = 0.7) or at post-ASCT (median 2.11 × 10−4,range 0-9.09 × 10−3; r = 0.14, P = 0.6); MRD detected in the autografts was > 27 times lower than that detected in pre-ASCT BM cells, and MRD detected in the post-ASCT BM cells was > 3 times lower than that detected in pre-ASCT BM cells except for one case in which the ratio was increased by two times. Interestingly, while MRD was detected in all BM cells at pre-ASCT (n = 16), 4 of 16 (25%) of these autografts were MRDNGS-negative. The median of MRD levels of the 4 cases in pre-ASCT and post-ASCT BM cells were 4.14 × 10−4 (range 6-583 × 10−6)and 1.8 × 10−5 (range 0-27 × 10−6), respectively. Conclusion: Although EuroFlow-NGF is a rapid and accurate method for detecting MRD, NGS was more sensitive and provided greater prognostic value than EuroFlow-NGF. Disclosures Takamatsu: Adaptive Biotechnologies: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical: Consultancy, Honoraria, Research Funding; Ono pharmaceutical: Honoraria, Research Funding; SRL: Consultancy, Research Funding. Takezako:Bristol-Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Janssen: Research Funding; Abbvie: Research Funding. Nakao:Symbio: Consultancy; Kyowa Kirin: Honoraria; Alexion: Research Funding; Novartis: Honoraria.

[Background] HLA-class I allele-lacking (HLA[-]) leukocytes are detected in approximately 30% of patients with acquired aplastic anemia (AA), and are thought to represent the involvement of cytotoxic T lymphocyte attack against hematopoietic stem cells (HSCs) in the development of AA, based on the high response rate to immunosuppressive therapy (IST) in patients with such aberrant leukocytes. Similar to glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient (GPI[-]) leukocytes in patients with paroxysmal nocturnal hemoglobinuria (PNH), HLA(-) leukocytes in AA patients are often clonal or oligoclonal and expand to account for more than 50% of the total leukocytes. Despite such overwhelming proliferation, somatic mutations in driver genes as well as telomere shortening that portend clonal evolution are rarely detected in HLA(-) granulocytes, suggesting the genetic stability of HLA(-) HSCs and the persistence of the immune pressure on HSCs that favors expansion of HLA(-) HSCs (Imi, et al. Blood Adv). However, recent studies from the United States have shown a higher incidence of clonal evolution to secondary myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) in AA patients with HLA(-) leukocytes than in those without such leukocytes, a finding inconsistent with the results of our previous study. Given the high prevalence of HLA(-) leukocytes in AA patients, it is critical to determine whether or not the presence of the aberrant leukocytes is associated with clonal evolution. We therefore addressed this issue by studying the prognosis of a large number of AA patients with or without HLA(-) leukocytes who had been followed for a long term period. We also studied the clonal composition of granulocytes in AA patients with HLA(-) cells, wherein aberrant clones other than HLA(-) cells might be responsible for clonal evolution to MDS/AML. [Methods] We retrospectively analyzed the clinical characteristics of 633 AA patients and peripheral blood samples were examined for the presence of HLA(-) leukocytes using a high-sensitivity flow cytometry (FCM) assay, droplet digital PCR, single-nucleotide polymorphism arrays, or next generation sequencing (NGS) between 2010 and 2020. GPI(-) cells were detected using a high-sensitivity FCM assay as previously described. [Results] HLA(-) granulocytes were detected in 127 (20.1%) of the 633 patients with a median clone size of 16.9% (range, 0.04%-100%); the aberrant granulocytes accounted for greater than 50% of the total granulocytes in 29 (22.8%) of 127 patients. Eighty-nine (70.0%) of the 127 patients possessed aberrant clones other than HLA(-) clones, which included 0.005% to 91.6% GPI(-) cells (n=86), del(13q) cells (n=3), t(1;10) cells (n=1), t(9;13) cells (n=1), inv12 cells (n=1), and trisomy 8 cells (n=1). The prevalence of GPI(-) cells was not significantly different between patients with and without HLA(-) cells (67.7% vs 65.4%). Eighty-five of 102 (83.3%) patients with HLA(-) cells responded to IST, whereas 231 of 318 (72.6%) without HLA(-) cells responded (p90% of granulocytes, suggesting that these few escape clones were enough to sustain the hematopoietic function of the patients. The prognosis survey revealed no clonal evolution to MDS/AML in any of the 127 AA patients with HLA(-) leukocytes after a follow-up period of the median 5 years. In contrast, 15 of 234 (6.4%) patients without HLA(-) cells who were trackable evolved to MDS/AML during a median 5 year follow-up. [ Conclusions] The presence of HLA(-) leukocytes and concomitant aberrant clones was not associated with clonal evolution to MDS/AML in Japanese AA patients, even in those possessing a large (>50% of the total granulocyte) HLA(-) cell population. The discrepancy between our results and the data from the United States may be due to the difference in the race and mechanism underlying HLA loss. These data suggest that HSC clones that escape immune attack, such as HLA(-) and GPI(-) clones, are healthy enough to support hematopoiesis for a long term in AA patients. Disclosures Ishiyama: Novartis: Honoraria; Alexion: Research Funding. Yamazaki:Novartis: Honoraria; Kyowa Kirin: Honoraria, Research Funding. Ogawa:Eisai Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Nakao:Alexion: Research Funding; Kyowa Kirin: Honoraria; Novartis: Honoraria; Symbio: Consultancy.

BK virus-associated hemorrhagic cystitis (BKV-HC) is a common and major cause of morbidity in recipients of allogeneic hematopoietic stem cell transplantation. A 32-year-old woman developed severe BKV-HC on day 24 after cord blood transplantation (CBT). Despite supportive therapies - such as hyperhydration, forced diuresis, and urinary catheterization - macroscopic hematuria and bladder irritation persisted for over a month. Hyperbaric oxygen (HBO) therapy at 2.1 atmospheres for 90 min per day was started on day 64 after CBT. Macroscopic hematuria resolved within a week, and microscopic hematuria was no longer detectable within 2 weeks. Hematuria did not recur after 11 sessions of HBO therapy, and no significant side effects were observed during or after treatment. HBO therapy could thus be useful in controlling refractory BKV-HC after CBT.

[Background] Leukocytes that lack HLA class I alleles derived from hematopoietic stem progenitor cells (HSPCs) that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations are often detected in acquired aplastic anemia (AA) patients. The presence of HLA class I allele-lacking leukocytes provides compelling evidence that cytotoxic T lymphocytes (CTLs) are involved in the development of AA. Our recent study showed that, among several HLA-class I alleles that are likely to be lost as a result of 6pLOH, HLA-B*40:02 is the most frequently lost allele in AA. Therefore, HLA-B*4002 is thought to play a critical role in the autoantigen presentation by HSPCs to CTLs. We previously identified the T-cell receptor (TCR) sequences from bone marrow (BM) CD8+ T cells in two CsA-dependent AA patients possessing B4002-lacking leukocytes (Case 1, Espinoza et al, Blood Adv, 2018) and B5401-lacking leukocytes (Case 2, Elbadry et al, Haematologica, 2019) by single-cell T-cell receptor (TCR) sequencing. Identifying the TCRs specific to antigens presented by these HLA class I alleles should allow us to screen autoantigens in AA. [Method] We established B4002+ or B5401+ K562 cell lines expressing CD80 and CD137L for the screening of antigen-specific T cell responses. To identify ligands of the TCR, we transfected peripheral blood (PB) T cells with a retrovirus vector containing different TCR cDNA derived from BM T cells and examined their responses to B4002+CD80+CD137L+ or B5401+CD80+CD137L+ K562 cells. Specific responses of each TCR transfectant to K562 cells or iPSC-derived CD34+ cells were determined using an enzyme-linked immunosorbent assay for detecting IFN-γ. Deep TCR sequencing of a current PB sample taken from the same patients was performed to determine whether or not T cells with specific TCRs persisted after successful immunosuppressive therapy (IST). [Results] In Case 1, two TCR transfectants (TCR-K1 and TCR-K2 which were the third- and second-most frequent TCRs in the BM T cells, respectively) secreted greater IFN-γ levels (1730 pg/mL and 2157 pg/mL, respectively) in response to B4002+CD80+CD137L+ K562 cells than those secreted by the other six transfectants (710 to 1184 pg/mL, respectively). TCR-K1 and TCR-K2 did not respond to an A2402+ counterpart (Figure). Notably, deep TCR sequencing of a current PB sample taken from Case 1 nine years after BM sampling revealed the persistence of the TCR-K1 sequence, suggesting that TCR-K1 may be responsible for CsA dependency of this patient. Deep TCR sequencing of other three AA patients with B4002-lacking leukocytes revealed decreased diversity of the T cell repertoire in CD8+ T cells but failed to reveal the same TCR motifs as Case 1. In Case 2, two TCR transfectants (TCR-K3 and TCR-K4) showed a specific response to B5401+CD80+CD137L+ K562 cells. Furthermore, these 2 TCR transfectants secreted higher amounts of IFN-γ (1.7 and 2.0 folds for TCR-K3 and TCR-K4, respectively) in response to wild-type iPSC-derived CD34+ cells than to B5401(-) CD34+ cells. [Conclusions] Our results suggest that these TCR transfectants recognized some intrinsic antigens derived from K562 cells in a B4002 or B5401-restricted manner. These TCR transfectants are the ideal tools for screening libraries of cDNA expressed by B4002+ COS/293T cells to identify autoantigens in AA. Figure Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Chugai Pharmaceutical Co.,Ltd: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria; Kyowa Kirin: Honoraria; Novartis Pharma K.K: Honoraria.

[Background] Olfactomedin 4 (OLFM4), a member of the olfactomedin-related protein family, is constitutively expressed in bone marrow (BM) cells and many gastrointestinal organs and is involved in a variety of cellular functions, including proliferation, differentiation, apoptosis, and cell adhesion in tumor cells. Previous studies have suggested that OLFM4 supports the survival of leukemic stem cells derived from iPS cells of patients with chronic myeloid leukemia. Our recent study revealed a somatic mutation of OLFM4 in HLA allele-lacking granulocytes of a patients with acquired aplastic anemia (AA) in long-term remission (Blood advances, 2(9):1000-1012, 2018). The OLFM4 gene is located at chromosome 13q14.3, which is a commonly deleted region in AA patients with del(13q) who show a good response to immunosuppressive therapy and a high prevalence of increased glycosylphosphatidylinositol-anchored protein(GPI-AP)-deficient cells (Haematologica, 97(12):1845-9, 2012). There may be common mechanisms underlying the preferential commitment between GPI-AP- and del(13q) hematopoietic stem and progenitor cells (HSPCs), like insensitivity to inhibitory cytokines such as TGF-β, in immune-mediated BM failure. To confirm this hypothesis, we studied the effect of OLFM4 knockout on the erythroid differentiation of erythroid leukemia cell lines induced by TGF-β. [Methods] We established OLFM4 knockout (KO) K562 and TF-1 cells using a CRISPR-Cas9 system. OLFM4-knockdown (KD) cells were also prepared using siRNA to validate the results of OLFM4-KO cells. The OLFM4 mRNA and protein levels were determined using quantitative polymerase chain reaction, flow cytometry (FCM), Western blotting, immunocytochemistry, and immunofluorescence methods. The erythroid differentiation was assessed by measuring the expression of glycophorin A (GPA) with FCM, GATA-1 protein expression using Western blotting, and iron staining of the cells. [Results] The OLFM4 KO cells showed slower proliferation than wild-type (WT) cells. Both OLFM4-KO cells and OLFM4-KD cells showed a higher GPA expression than WT cells (median fluorescence intensity [MFI] of K562: 2924 and 2143 vs. 1469 and TF-1: 950 and 870 vs. 694, respectively). OLFM4-KO cells showed erythroid morphology, an elevated expression of GATA-1, and positivity for iron granules, suggesting that OLFM4 KO promoted the erythroid differentiation of K562 and TF-1 cells in RPMI1640 containing 10% fetal bovine serum (Figure 1). When WT cells were cultured in a serum-free culture medium (Steampro34) with or without TGF-β (6 ng/ml) for 8 days, the GPA expression was induced in both TF-1 (MFI: 4358 vs. 883), and K562 cell lines (33440 vs. 25655). The OLFM4 protein levels in these cell lines were significantly decreased by the TGF-β treatment in a dose-dependent manner, suggesting that TGF-β directly downregulated the OLFM4 expression in WT cells; the relative expression of OLFM4 was 1, 0.64, and 0.12 while that of TF-1 was 1, 0.12, and 0.05 at 0, 2, and 6 ng/ml of TGF-β, respectively (Figure 2). [Conclusion] OLFM4 prevents K562 and TF-1 cells from differentiating into erythroid cells in response to TGF-β. The erythroid differentiation of these leukemic cells may be mediated by the downregulation of OLFM4 induced by TGF-β. Haploinsufficiency of OLFM4 due to either a loss of function mutation or del(13q) may be related to the mechanisms underlying the preferential commitment of the mutant HSPCs to erythroid cells in patients with immune-mediated BM failure where TGF-β is abundantly present. Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Takeda Pharmaceutical Company Limited: Honoraria; Novartis Pharma K.K: Honoraria; Kyowa Kirin: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; SynBio Pharmaceuticals: Consultancy.

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.

[Background] In cases of immune-mediated bone marrow (BM) failure, such as acquired aplastic anemia (AA) and AA/PNH, aberrant hematopoietic stem progenitor cells (HSPCs) that acquire resistance to immune attack are thought to survive and support hematopoiesis in convalescent patients. Two representative progenies of such "escape" HSPC clones are HLA class I allele-lacking (HLA[-]) leukocytes and glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells. The mechanism underlying the immune selection of HLA(-) HSPCs is the failure of cytotoxic T lymphocyte (CTL) to recognize target antigens that are presented by particular HLA class I alleles of HSPCs. However, the mechanisms underlying the immune selection of GPI(-) HSPCs remain unclear. In addition, whether or not immune pressure that persists after immunosuppressive therapy (IST) contributes to the development and maintenance of clonal hematopoiesis by HLA(-) or GPI(-) HSPCs that are often seen in patients in long-term remission is also unknown. Phenotypical analyses of HSPCs that can be obtained from peripheral blood (PB) of AA patients who possess HLA(-) or GPI(-) leukocytes may provide a hint to elucidate these unsolved issues. [Objectives/Methods] We analyzed PB lineage-CD45dimCD34+CD38+ HSPCs of 15 AA patients who had 1%-99% HLA-A2(-) or HLA-A24(-) granulocytes (Gs) using flow cytometry (FCM). PB samples from 1 patient with severe AA were obtained before IST while the other 14 patients were in remission at the time of sampling; 10 were on cyclosporin (CsA) and eltrombopag (EPAG) (n=1), CsA and anabolic steroids (n=3), CyA (n=4), anabolic steroids (n=1) and EPAG alone (n=1); and 4 were free of therapy. We also determined the percentages of HLA(-) cells in different CD34+ subsets of BM, including HSCs (CD38-CD90+CD45RA-), MPPs, CMPs, GMPs, MEPs and CLPs for patients whose BM cells were available. Six AA/PNH patients whose GPI(-) Gs were 4-99% of the total Gs were subjected to the same PB HSPC analysis. For a separate group of seven AA patients who responded to CsA and had both HLA(-) and GPI(-) G populations, the percentages of each population were serially determined over one to 12 years. [Results] FCM identified 0.01%-0.4% (median 0.01%) CD45dimCD34+CD38+ HSPCs in the mononuclear cell population of the 15 AA patients, values that were significantly lower than those of seven healthy volunteers (0.19-0.78%, median 0.58%, P [Conclusions] Immune selection that favors the survival of HLA(-) HSPCs or HPCs takes place even in AA patients who have been in remission for many years after successful IST and may contribute to clonal hematopoiesis by HLA(-) HSPCs. Given no signs of selection at the HSPC stage and the reciprocal increases in the percentage of GPI(-) Gs in AA/PNH patients responding to CsA therapy, the proliferation of GPI(-) HSPCs may not be affected by immune pressure in convalescent patients. Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Novartis Pharma K.K: Honoraria; SynBio Pharmaceuticals: Consultancy; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Bristol-Myers Squibb: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Alaxion Pharmaceuticals: Honoraria; Kyowa Kirin: Honoraria.

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.

The authors would like to correct the error in the publication of the original article. The corrected detail is given below for your reading.

[Background] Hematopoietic stem progenitor cells (HSPCs) with PIGA mutations are thought to acquire a survival advantage over normal HSPCs under immune attack against HSPCs and produce glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells in patients with acquired aplastic anemia (AA). Various underlying mechanisms of the survival advantage of PIGA-mutated HSPCs have been proposed; however, it remains still unclear how PIGA-mutated HSPCs are immunologically selected in AA. Approximately 15% of AA patients with increased GPI(-) cells possess another aberrant leukocyte subset that lacks the expression of the HLA-class I allele due to a copy number-neutral loss of heterozygosity of the HLA haplotype, which occurs in the short arm of chromosome 6 (6pLOH) as a result of uniparental disomy, or HLA allelic mutations. The presence of HLA-class I allele-lacking leukocytes (HLA-LLs) is considered to be the most compelling evidence to support the involvement of cytotoxic T lymphocytes (CTLs) in the development of bone marrow failure. Charactering GPI(-) leukocytes and platelets in AA patients with HLA-LLs may provide an insight into the mechanism underlying the immune selection of PIGA-mutated HSPCs. [Patients and Methods] We investigated the presence of GPI(-) leukocytes, erythrocytes, and platelets in 63 patients with AA using high-sensitivity flow cytometry (FCM). For the platelet analysis, platelet rich plasma (PRP) was obtained by centrifuging anticoagulated blood at 1000 rpm for 7 minutes with the brake turned off. Thirty microliters of PRP was incubated with monoclonal antibodies specific to CD55-PE, CD59-PE, CD41a-APC and HLA-A2 or A24-FITC for 20 minutes at room temperature in the dark. To prevent doublets, samples were diluted 1 to 100 in PBS and filtered with mesh immediately before the FCM analysis. Thirty of the 63 patients were heterozygous for the HLA-A allele with A24 and A2, and thus the presence of both HLA-LLs and HLA-A allele-lacking platelets could be evaluated by FCM. The lack of the HLA-A allele due to 6pLOH or allelic mutations in all HLA-LL(+) patients was confirmed by a droplet digital PCR or deep sequencing. [Results] Increased GPI(-) granulocytes, which accounted for 0.01-99.8% of the total granulocytes, were detected in 37 (58.7%) patients while HLA-A24 or A2-lacking granulocytes accounted for 0.39-98.3% of the total granulocytes in 20 (66.7%) of the 30 patients. Eight patients possessed both GPI(-) cells and HLA-LLs. In all 8 of these patients, the two aberrant cell populations were mutually exclusive. The analyses of different cell lineages revealed HLA-A allele-lacking cells in all lineages of cells, including granulocytes (Gs), monocytes (Ms), T cells (Ts), B cells (Bs), NK cells (NKs), and platelets (Ps) in 7 of the 8 patients; the remaining one patient had the GMTP pattern. In contrast, the lineage diversity of GPI(-) in the 8 patients was more restricted; GMTBNKP was only detected in 2 patients; the combinations in the other 6 patients were GT (n=1), GMBNKP (n=2), GMTNKP (n= 1) and GMTBP (n= 2). In Case 1, GPI(-) cells were not detected in T cells while HLA-A24(-) cells were detected in all lineages of cells including T cells (Figure 1). The limited lineage diversity of GPI(-) cells was also evident in 6 patients who did not possess HLA-LLs (GMP, GMBP, GMBNKP, GMTNKP, GMTBP) with GPI(-) granulocytes>10% while the GMTBNKP pattern was common in 10 HLA-LL(+) patients who did not possess GPI(-) cells, regardless of their percentage of HLA-A allele-lacking granulocytes. Longitudinal follow-up of 5 patients over a period of 8-27 years showed a decline in the percentage of GPI(-) granulocytes (39.2 to 0.00%, 11.4 to 0.04%, 3.50 to 0.30%, 1.77 to 0.00% and 0.79 to 0.11%) and a reciprocal increase in the percentage of HLA-A allele-lacking granulocytes (80.0 to 95.2%, 92.0 to 99.1%, 24.0 to 24.4%) in 3 patients who had been placed under observation; in two patients (Cases 2 and 3) whose GPI(-) granulocyte percentages had been >10%, the PNH clones were completely replaced by HLA-LL clones during 6 and 8 years, respectively (Figure 2). [Conclusions] The limited diversity of the blood cell lineage and spontaneous decline of GPI(-) cells that coexisted with HLA-LLs suggest that GPI(-) cells are derived from the PIGA-mutated hematopoietic progenitor cells that were allowed to proliferate as a bystander in the environment where the CTL attack against HSPCs is taking place. Disclosures Nakao: Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria.

【Background】The use of novel agents with different mechanisms of action has resulted in high complete response (CR) rates and improved the survival of patients with multiple myeloma (MM). Obtaining minimal residual disease (MRD) negativity following treatment is an important determinant for longer survival in patients with MM. The EuroFlow MM-MRD panel (EuroFlow panel) using the Infinicyt software (Cytognos, Salamanca, Spain) is a two-tube six-color antibody panel. By the addition of two drop-in antibodies (CD138, CD27; vendor of user's choice) into both tubes, an eight-color configuration, which has been validated by the EuroFlow consortium is obtained. The DuraClone RE PC kit (DuraClone panel) is a recently developed one-tube eight-color dry antibody panel using the Kaluza software (Beckman Coulter, Miami, USA) for manual identification of abnormal plasma cells (PCs) to detect MRD in MM. Although both methods are available, a comparison between them has yet to be performed. This study aimed to compare the EuroFlow panel and the DuraClone panel analysis of residual abnormal plasma cells by eight-color flow cytometry. 【Methods】Patients with International Myeloma Working Group defined symptomatic MM treated at Kanazawa University Hospital and Kameda Medical Center in Japan from January 2017 to July 2018 were included. Bone marrow samples were obtained as part of routine clinical practice for evaluating treatment response when appropriate. Bone marrow aspirates (4 mL; first pull of bone marrow aspiration) anti-coagulated with ethylenediamine tetraacetic acid were evenly split (2 mL each) for the EuroFlow panel and the DuraClone panel. PC quantification by the EuroFlow panel was performed at Kanazawa University Hospital and the DuraClone panel was performed at Kameda Medical Center. The monoclonal antibodies used in the EuroFlow and the DuraClone panels are detailed in Table 1. Identification of neoplastic PCs required ≥ 20 cells. Sample processing and measurement of PCs were performed within 48 hours of collection. The correlation of total leukocyte acquisition, total plasma cells and MRD were analyzed for both methods. Qualitative comparison of MRD negativity was also performed. Spearman's correlation coefficient was used for evaluating the correlation of paired data. The Bland-Altman plot was used for detection of fixed bias and proportional bias. 【Results】A total of 79 samples were analyzed. We first assessed the number of total leukocytes using the EuroFlow panel and the DuraClone panel. The median number of total leukocytes acquired with the EuroFlow panel was significantly higher than with the DuraClone panel (median: 8,505,172 cells, interquartile range [IQR]: 6,164,094 - 9,105,097 cells and median: 2,858,026 cells, IQR: 1,466,004 - 4,284,926 cells, respectively; p < 0.01), but the percentage of total plasma cells showed good concordance between both methods (regression coefficient = 0.91, p < 0.01). The median percentage of abnormal plasma cells was 0.0035% (IQR: 0.0005 - 0.05) and 0.0046% (IQR: 0.0005 - 0.05) using the EuroFlow panel and the DuraClone panel, respectively. Figure 1A shows scatter plots comparing the percentage of abnormal cells analyzed using the EuroFlow panel and the DuraClone panel. Both methods showed a high degree of concordance (regression coefficient = 0.90, p < 0.01). Bland-Altman plots also showed good agreement between both methods. There was no statistically significant fixed bias with a mean difference of 10% (95% confidence interval [CI]: -12~14%). In addition, no significant proportional bias was detected between the two methods (regression coefficient: -0.01, p = 0.85; Figure 1B). Qualitative analysis of MRD negativity also showed substantial agreement between the two methods (kappa = 0.7); 11 of 79 (13.9 %) samples showed discrepancy for determination of MRD negativity (< 1 × 10-5). Nine of these samples were MRD negative by the EuroFlow panel, but MRD positive by the DuraClone panel, and two samples were MRD positive by the EuroFlow panel, but MRD negative by the DuraClone panel. However, all discrepancies occurred near the limit of detection of both methods (1.0 × 10-5). 【Conclusion】Our findings indicate that the DuraClone panel has good overall concordance with the EuroFlow panel for the detection of abnormal PCs in MM samples. The one-tube DuraClone panel enables to reduce processing time and test cost compared with the two-tube EuroFlow panel. Disclosures Takamatsu: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Bristol-Myers Squibb: Research Funding; Ono: Research Funding. Nakao:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria.

Background: Autologous stem cell transplantation (ASCT) in conjunction with novel therapeutic drugs can dramatically improve response rates and the prognosis of patients with multiple myeloma (MM). However, most patients with MM are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper responses are required. Next-generation sequencing (NGS) and multiparameter flow cytometry (MFC) methods have been used to assess MRD. However, the lack of standardization of conventional MFC approaches has had a negative impact on its reproducibility. Recently, a next-generation MFC method (EuroFlow, NGF) has been developed by the EuroFlow Consortium and the International Myeloma Foundation (IMF) for a highly sensitive and standardized detection of MRD in MM. Aims: To compare the prognostic value of MRD detection in autografts in MM between NGS (Adaptive) and 8-color MFC method (EuroFlow, NGF), and also MRD levels between fresh and cryopreserved autografts. Methods: A total of 39 newly-diagnosed MM patients who underwent ASCT were enrolled in this study. Median age 60 at ASCT (range 41-69); males 22, females 17; ISS 1 (n=10), 2 (n=19), 3 (n=10). 10 patients showed high-risk chromosomal abnormalities (t(4;14) (n=9), del17p & t(4;14) (n=1)). The induction regimen was bortezomib-based chemotherapy. All patients received melphalan 200 mg/sqm as conditioning regimen before ASCT. 34 of 39 (87%) patients received maintenance therapy until progressive disease. The best response post-ASCT was as follows: 23sCR, 2CR, 12VGPR, 2PR. 39 autografts, one from each MM patient, were analyzed using NGF and NGS methods. The NGF method was based on a standardized lyse-wash-and-stain sample preparation protocol, the measurement of high numbers of cells and an optimized 8-color, 2-tubes, antibody panel, for accurate identification of plasma cells (PCs) and discrimination between phenotypically aberrant (aPC) and normal PC (nPC) (J Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD Assay (Seattle, WA) (Martinez-Lopez et al., Blood 2014). To assess the correlation of MRD levels between fresh and cryopreserved autografts using NGF, 6 additional MM patients' autografts were used. Results: MRD levels in all 39 autografts were assessed using EuroFlow, while those in 32 of 39 (82%) were assessed with NGS due to limited availability of material for calibration. We identified abnormal plasma cells (aPC) in autografts based on multivariate analysis of individual cells from each patient (e.g. CD56+, CD19-, CyIgκ+, CD117+). Since there was a good correlation in MRD levels between fresh and thawed frozen autografts detected by EuroFlow (R=0.943, P=0.02), we assessed the MRD levels in thawed frozen autografts. For the MM MRD in autografts, the events from tube 1 and tube 2 were combined and a median of 7.3×106 (range: 2.2×106-37.6×106) events was acquired. The sensitivity of EuroFlow was 1×10-5-2×10-6 while that of NGS was 10-7 due to the high number of DNA derived from autografts (Takamatsu et al., Ann Oncol 2017). 21 of 39 (54%) cases were MRD positive by 8-color MFC while 22 of 32 (69%) cases were MRD positive by NGS. The correlation of MRD levels between 8-color MFC and NGS was relatively high (Fig. 1A). MRD negative by NGF (MRDMFC (-)) cases tended to show better PFS than MRDMFC (+) cases (P=0.145) (Fig. 1B) while MRD negative by NGS (MRDNGS (-)) cases showed significantly better PFS than MRDNGS (+) cases (P=0.03) (Fig. 1C). Furthermore, MRDMFC (-) MRDNGS (-) cases showed significantly better PFS than MRDMFC (-) MRDNGS (+) cases (P=0.01), but the PFS of MRDMFC (-) MRDNGS (+) cases was not different from that of MRDMFC (+) MRDNGS (+) cases (P=0.70). MRDMFC (-) and MRDNGS (-) cases showed better OS than MRDMFC (+) (P=0.14) and MRDNGS (+) (P=0.08) cases, respectively. Conclusions: Although EuroFlow is a fast and accurate method for detecting MRD of MM in autografts, in this study the NGS platform had a higher sensitivity and prognostic value than EuroFlow. The homogenous nature of the mobilized autograft relative to the focal nature of myeloma in bone marrow might provide a better sample to assess MRD. Figure 1. Figure 1. Disclosures Takamatsu: Celgene: Honoraria, Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria. Nakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

[Background] Acquired aplastic anemia (AA) is a rare syndrome characterized by pancytopenia and bone marrow hypoplasia. The cytotoxic T lymphocyte (CTL) attack against autologous hematopoietic stem progenitor cells (HSPCs) is thought to be responsible for bone marrow failure in the majority of AA cases; however, little is known about the target antigens of the CTLs. HLA class I-allele lacking leukocytes (HLA-LL) due to copy-number neutral loss of heterozygosity in the short arm of chromosome 6 (6pLOH) or somatic loss-of-function mutations in HLA class I genes are detected in approximately 20% of patients with newly diagnosed AA, and the presence of HLA-LL represents compelling evidence to support that CTLs specific to HSPCs are involved in the development of AA. Our recent studies using single nucleotide polymorphism array (SNP-A) genotyping and droplet digital polymerase chain reaction (ddPCR) revealed that HLA-B*40:02 is the most frequently lost among all class I alleles that are lost as a result of 6pLOH (Zaimoku, et al. Blood 2017). Various somatic loss-of-function mutations in B*40:02 revealed by deep sequencing in the study substantiated the important role of HLA-B4002 in the autoantigen presentation of AA. However, in the other 6pLOH(+) AA patients who did not possess HLA-B4002, which accounted for 20% of the total AA cases involving patients possessing HLA-LL, the allele in the missing haplotype that was responsible for the autoantigen presentation was largely unknown because the lost fragment of chromosome 6p usually contained 2 or more HLA class I alleles. [Objectives/Methods] To identify class I alleles other than HLA-B*40:02 that are critically involved in the auto-antigen presentation of AA, we screened a total of 624 patients for the presence of HLA-LL using monoclonal antibodies specific to class I HLA alleles, SNP-A, and ddPCR, and performed targeted deep sequencing of HLA class I genes by using SeqCap EZ Choice pobes (Roche) and MiSeq sequencer (Illumina). The paired fractions, including granulocytes that lacked an HLA-A allele and granulocytes that retained the HLA-A allele, as well as CD3+ T cells, were sorted using monoclonal antibodies specific to HLA-A alleles with a BD FACSAria Fusion system (BD Biosciences), and were subjected to DNA extraction. All DNA samples of granulocytes and control cells (CD3+ T cells or buccal mucosa cells) were prepared for targeted deep sequencing. [Results] One hundred and fourteen patients were found to be positive for HLA-LL and 62 (54.4%) of the 114 HLA-LL(+) patients did not carry B*40:02 (severe, n=30; non-severe, n=32; male, n=38; female, n=24; median age, 62 [range, 6-93] years). Apart from B*40:02 (45.6%), A*02:06 (24.6%) was the second-most frequent HLA class I allele in the lost haplotype. The targeted deep sequencing of 20 patients with HLA-LL revealed 6pLOH alone in 11 patients, and somatic loss-of-function mutations plus 6pLOH in 9 patients; none of the patients were positive for somatic loss-of-function mutations alone. Of note, somatic loss-of-function mutations were found in only 5 alleles (A*02:06 in four, B*40:01 in two, B*40:03, A*31:01, and B*54:01 in one each) out of 27 different alleles contained in the lost haplotype. Among the 9 patients with somatic loss-of-function mutations, the median number of mutations per patient was 1 (range, 1-2); these included a missense mutation (n=1), frameshift deletions (n=3) and nonsense mutations (n=7) (Figure). Four patients had a breakpoint of 6pLOH in between the HLA-A and C loci; their lost alleles were A*02:06 (n=2) and A*31:01 (n=2), and the occurrence of 6pLOH in the four patients was therefore attributed to the two HLA-A alleles. Sixty-six percent of the HLA-LL(+) B*40:02(-) patients had at least one of the five alleles in the lost haplotypes. The frequencies of each "high risk" allele found in patients possessing HLA-LL are summarized in Table. [Conclusions] In addition to B*40:02, five class I alleles including HLA-A*02:06, A*31:01, B*54:01, B*40:03 and B*40:01 are thought to play an essential role in the auto-antigen presentation by the HSPCs of Japanese AA patients. The frequencies of the six class I alleles in general Japanese population are much higher than those in the general Caucasian populations but similar to the frequencies in East Asian populations. The higher frequencies of the six alleles in comparison to Caucasian countries may account for the higher incidence of AA in East Asia. Disclosures Takamatsu: Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria; Celgene: Honoraria, Research Funding. Nakao:Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria.