ALBERT

Description: Cases of human herpesvirus-6 (HHV-6) associated central nervous system (CNS) dysfunction such as encephalitis after hamatopoietic cell transplantation (HCT) have been increasingly reported. However, clinical features, appropriate diagnostic methods, treatments and outcomes of HHV-6 encephalitis are not fully understood. A questionnaire was sent to transplant centers in Japan, and adult allogeneic HCT recipients with CNS dysfunction who had HHV-6 DNA detected by polymerase chain reaction (PCR) in cerebrospinal fluid (CSF) were retrospectively analyzed. We describe 23 cases with HHV-6 associated encephalitis. Median age was 38 (range; 18–63) years. Underlying diagnosis included acute leukemia (AML, N=8; ALL, N=6), lymphoma (N=5), and others (N=4); 12 of whom had refractory or relapsed disease before HCT. Patients received bone marrow or peripheral blood stem cell from HLA-matched (N=2) or mismatched (N=9) related donors, unrelated donors (N=7), or umbilical cord blood (N=5). Conditioning included high-dose TBI-containing myeloablative regimens (N=16) and reduced-intensity regimens (N=7). In majority of cases, GVHD prophylaxis contained tacrolimus and methotrexate. Median onset of CNS dysfunction was day 22 (range; days 12–614) after HCT; 17 of whom had CNS symptoms within 7 days of neutrophil engraftment. Symptoms included coma/impaired consciousness (N=21; 91%), amnesia/loss of short-term memory (N=17; 73%), seizure (N=16; 70%), and fever (N=14; 61%). All patients had HHV-6 DNA in the CSF identified by qualitative (N=9) or quantifiable (N=14) PCR. Median HHV-6 DNA levels in the CSF was 3.3 (range; 0.8–500) x 103 copies/ml. HHV-6 typing study revealed subtype B virus in all 8 cases examined. No other causative agents were detected. Analysis of the CSF samples revealed increased WBCs in 10 of 22 patients (45%). Brain MRI scans showed abnormal findings within the temporal lobe in 17 patients (73%). Treatment for HHV-6 encephalitis included ganciclovir (N=17), foscarnet (N=11), intraveneous immunoglobulin (N=13), acyclovir (N=4), vidarabine (N=1), and donor lymphocyte infusion (N=1), and 5 were switched from ganciclovir to foscarnet due to ineffectiveness or toxicities. Although the clinical status appeared to improve after treatment in 21 of 23 patients (91%), 10 had a sequela such as memory problem. These data suggest that HHV-6 may cause severe CNS disease after HCT, and detection of viral DNA in the CSF appeared to be useful for the rapid diagnosis and early anti-viral treatment. The presence of amnesia and abnormal findings within the temporal lobe may be a useful diagnostic indicator of HHV-6 associated encephalitis after HCT. In this analysis, majority of HHV-6 encephalitis cases were those who received cord blood transplant or grafts from unrelated or HLA-mismatched donors. Prospective studies are warranted to determine accurate diagnostic methods and appropriate treatment for HHV-6 encephalitis in high-risk patients.

Description: Abstract 4707 Reduced-intensity stem cell transplantation (RIST) has come to be generally accepted as a method of allogeneic stem cell transplantation (SCT) for patients considered ineligible for myeloablative preparative regimens because of advanced age or comorbidities. We have recently reported unmanipulated nonmyeloablative HLA-haploidentical SCT using a conditioning treatment consisting of fludarabine, busulfan and anti-T-lymphocyte globulin and graft-versus-host disease (GVHD) prophylaxis consisting of tacrolimus and methylprednisolone (1 mg/kg) (Biol Blood Marrow Transplant 2006; 12:1073). In that study, the incidence of severe GVHD was only 10%. One of the mechanisms for such a low incidence of GVHD may have been caused by a reduced intensity of conditioning. Less intensive regimens should be associated with lower toxicity, a lower release of inflammatory cytokines, and potentially less GVHD; however, the mechanisms remain to be determined. Thus, using a murine MHC-haploidentical BMT model, BDF1(H-2b/d)®B6C3F1 (H-2b/k), that we established, we examined the influence of an intensity of conditioning treatment on GVHD. Recipient mice received T-cell-depleted bone marrow (5×106) and spleen cells (2×107) after total body irradiation (TBI) 13 Gy (myeloablative group) or 5 Gy (RIST group). Both groups of mice rapidly achieved donor engraftment. Recipients in the RIST group showed significantly fewer GVHD signs than those in the myeloablatve group. Histopathological examination of the myeloablative group on day 14 revealed various pathological changes in intestine (in particular large intestine) compatible to GVHD. In contrast, intestine samples from the RIST group showed few pathological changes with much less infiltration of donor T cells. Consequently, all recipients in the myeloablative group had died of GVHD by day 60, while all recipients survived for more than 3 months. These results clearly showed that the intensity of conditioning treatment influenced on the severity of GVHD and survival of recipients. Next, we investigated the mechanisms by which reduced intensity of conditioning ameliorated GVHD. Transplantation was performed using spleen cells that were labeled with the fluorescent cytoplasmic dye, carboxyfluorescein diacetate succinimidyl ester (CFSE), and cells in secondary lymphoid organs were analyzed by flow cytometry. The number of donor T cells in mesenteric lymph nodes on day 7 of the RIST group was significantly lower than that of the myeloablative group. In addition, a significantly increased number of host CD4+ T cells were recruited to secondary lymphoid organs on day 4 in the RIST group compared with the myeloablative group. An increased number of donor or host regulatory (Foxp3+CD4+) T cells were also observed in the RIST group. The levels of IFN gamma or IL-4 in lymphoid organs of the RIST group were higher than those of the myeloablative group. These results strongly suggest that host immune cells that survived conditioning treatment or cytokine milieu in secondary lymphoid organs contributed to the suppression of donor T cells during the initiation of GVHD. In addition, the expression of Th1 chemokine receptor, CXCR3, on donor T cells in secondary lymphoid organs and the expression levels of CXCL9, CXCL10, and CXCL11, ligands for CXCR3, in the large intestines were relatively lower in the RIST group, suggesting that the migration ability of donor T cells into GVHD target organs was negatively influenced by the intensity of conditioning. In conclusion, we showed that reduced intensity of conditioning improved the severity of GVHD, and that recipient immune cells, including regulatory T cells, together with reduced expression of inflammatory cytokines or chemokines, contributed to the improvement of GVHD in RIST. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4117 Related haploidentical donors, as cord blood, can be alternative donor sources in stem cell transplantation (SCT). Severe GVHD, however, has interfered the progress of haploidentical SCT (haploSCT). To deal with this strong GVHD, T cell depletion has usually been used in US and European countries. In order to pursue the controllable GVL effect by T cells, we have performed unmanipulated haploSCT using myeloablative or reduced intensity preconditioning regimen accompanied with intensified GVHD prophylaxis, including steroids. In this meeting, we will summarize our experience of haploSCT for more than ten years. From August 1998 to September 2010, we have performed 351 cases of haploSCT (all cases were HLA 2–3 antigen mismatched in GVH direction). Patients' characteristics are sex: male 186, female 168, age: 16–65 years old (median 39), disease: AML/MDS 149, ALL 81, ML 67, others 54. Eighty-three percent of cases underwent SCT in non-complete remission (non-CR) status. Patients under 45 years old underwent myeloablative preconditioning regimen consisting of FLU/CA/CY/TBI 8Gy (haplo-full, n=100), and patients over 45 years old or with comorbidities or repetitive SCT (including second to fifth SCT) underwent reduced intensity preconditioning regimen consisting of FLU/(CA)/BU/ATG or FLU/(CA)/MEL/ATG (haplo-mini, n=251). High dose Ara-C (CA) was optional to reduce tumor burden. As ATG, ATG (Fresenius) 8mg/kg, or thymoglubulin (genzyme) 2–4mg/kg were integrated into conditioning treatments mainly for reduced-intensity transplantation. GVHD prophylaxis consisted of taclolimus (TAC), methylprednisolone (mPSL) 2mg/kg/day, short term MTX, and mycophenolate mofetil (MMF) 15mg/kg/day in haplo-full, and TAC, and mPSL 1mg/kg/day in haplo-mini, respectively. For elderly patients over 50 years old in haplo-mini, MMF was added. Hematopoietic engraftment in haploSCT was as rapid as that in HLA-identical SCT, except 10 cases of graft rejection. The median time to reach a neutrophil account of 〉0.5 × 109/l was 10 days for haplo-mini and 13 days for haplo-full. Platelet recovery was achieved in 66 % and 60% of patients undergoing haplo-mini and haplo-full, respectively. The median time to reach a nontransfused platelet count of 3 20 × 109/l was 22 days for haplo-mini and 33 days for haplo-full. Sixty percent of haplo-mini patients and 54 % of haplo-full patients did not develop acute GVHD. Acute GVHD (grade II-IV) was observed in 20% for haplo-mini and 36 % for haplo-full. Overall survival at five years was 30% for haplo-full and 40% for haplo-mini, respectively. If limited to CR cases, overall survival reached over 60% in haplo-mini. There is no difference in survival rate among patients' diseases. In multivariate analysis on survival using variables, including disease status before transplantation, haplo-full vs haplo-mini, mismatches in GVH direction, mismatches in HVG direction, patients' age, and the number of transplantation times, the disease status (CR) was found to be only a significantly favorable factor (P= 0.0026). Unmanipulated haploSCT is feasible and effective for refractory diseases. ATG dose used in haplo-mini is critical, and rather low compared with that of European cases reported so far. Although it should be too early to refer long term outcome, unmanipulated haploSCT could be considered as an option to control refractory diseases. Disclosures: No relevant conflicts of interest to declare.

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: Abstract 4203 The major drawback of HLA-haploidentical stem cell transplantation is graft-versus-host disease (GVHD): however, the GVHD can be overcome using substantial T cell depletion method (Perugia), the use of a high dose ATG (Beijing), or the posttransplantation cyclophophamide method (Johns Hopkins). These transplant procedures are found to be effective for the achievement of donor engraftment and immunological tolerance: however, graft-versus-leukemia (GVL) effects are not enough strong to prevent disease relapse. To harness GVL effect of HLA-haploidentical grafts, our regimen for unmanipulated haploidentical myeloablative transplantation is designed for GVH reaction to remain to some extent in the short time after transplantation using a small dose of ATG 2 mg/kg, coupled with GVHD prophylaxis containing a low dose of steroid (methylprednisolone 1 mg/kg). We hypothesize that allogeneic immunological response by donor T cells under low cytokine milieu exerts potent GVL effect without GVHD. This regimen was applied to patients with hematologic disease in an extremely high risk. From August 2008 to June 2012, 23 patients with high-risk or refractory hematologic diseases underwent unmanipulated peripheral blood stem cells using a graft from HLA-haploidentical related donor (11 cases were HLA 2 antigen-mismatched and 12 HLA 3 antigen-mismatched in the GVH direction). Patients' characteristics are sex: male 11, female 12, age: 17–46 years old (median 34), disease: AML/MDS 7, ALL 10, ML 5, others 1. All patients except for 5 underwent transplantation in non-complete remission (non-CR) status, including 5 patients in induction failure, and 10 in resistant relapse. Eleven patients with leukemia who underwent in non-CR state had a median of 66% blasts in the bone marrow (range, 10.6 – 97.0 %). Patients received a conditioning treatment consisting of fuludarabine, cytarabine, cyclophsphamide, TBI 8Gy and ATG (thymoglobuline) 2 mg/kg. GVHD prophylaxis consisted of tacrolimus and methylprednisolone (1 mg/kg). One patient dies of pneumonia on day 3. Among 22 patients that could be evaluated, the engraftment rate was 95.5% (one patient had graft rejection because of donor-specific HLA antibody). Neutrophil (〉0.5×109/l) and platelet (〉20×109/l) engraftment was achieved on day 11 and on day 32, respectively. All patients who underwent transplantation in non-CR achieved CR after transplantation. Eleven patients (52.4%) had no GVHD. Nine patients (42.9%) developed grade II-III acute GVHD. One patient died of cardiac failure on day 46, 1 VOD on day 74, and 1 GVHD on day 235. Two patients died of relapse on days 70 and 548. Four of the 8 patients with leukemia who had more than 50% blasts in the bone marrow at the time of transplantation survive in CR. The overall survival is 60% at 3 years (Figure 1). Our regimen for HLA-haploidentical transplantation using a small dose of ATG coupled with low dose of steroid exerts a strong GVL effect even for patients who had chemorefractory disease, and transplantation-related toxicities, including GVHD, are acceptable. As a result, patients, of whom the majority had a high tumor burden, achieved a high rate of long-term survival. Thus, our strategy for haploidentical stem cell transplantation is promising, although our results will have to be confirmed in a large-scale study. Disclosures: No relevant conflicts of interest to declare.