ALBERT

Description: We retrospectively analyzed results for 154 patients with acquired severe aplastic anemia who received bone marrow transplants between 1993 and 2000 from unrelated donors identified through the Japan Marrow Donor Program. Patients were aged between 1 and 46 years (median, 17 years). Seventy-nine donor-patient pairs matched at HLA-A, -B, and -DRB1 loci, as shown by DNA typing. Among the 75 mismatched pairs, DNA typing of 63 pairs showed that 51 were mismatched at 1 HLA locus (18 HLA-A, 11 HLA-B, 22 HLA-DRB1) and 12 were mismatched at 2 or more loci. Seventeen patients (11%) experienced either early or late graft rejection. The incidence of grade III/IV acute graft versus host disease and chronic graft versus host disease was 20% (range, 7%-33%) and 30% (range, 12%-48%), respectively. Currently, 99 patients are alive, having survived for 3 to 82 months (median, 29 months) after their transplantations. The probability of overall survival at 5 years was 56% (95% confidence interval, 34%-78%). Multivariate analysis revealed the following unfavorable factors: transplantation more than 3 years after diagnosis (relative risk [RR], 1.86; P = .02), patients older than 20 years (RR, 2.27; P = .03), preconditioning regimen without antithymocyte globulin (RR 2.28; P = .04), and HLA-A or -B locus mismatching as determined by DNA typing. Matching of HLA class I alleles and improvement of preparative regimens should result in improved outcomes in patients with severe aplastic anemia who receive transplants from unrelated donors.

Description: Many studies have shown the presence of minimal residual disease (MRD) following therapy for childhood acute lymphoblastic leukemia (ALL) to be an important prognostic marker. We have also shown a significant relationship between survival outcomes in patients enrolled in the previous ALL 911 study and molecular MRD levels 5 weeks (time point 1, TP1) and 12 weeks (TP2) following the initiation of chemotherapy (Leukaemia and Lymphoma2002; 43: 1001). The aim of this study was to evaluate if polymerase chain reaction (PCR)-based MRD assay is sufficiently dependable for tailoring therapy, and if augmented therapy can reduce MRD levels to those associated with a favourable outcome. The subjects were under 18 years of age, and had newly diagnosed precursor B or T-cell ALL. Patients below one year old and those with t(9;22) were excluded. Written informed consent was obtained from patients or their legal guardians. The ALL 941-based protocol (45thASH, San Diego, 2003) utilized PCR-based MRD assay using immunoglobulin & T-cell receptor gene rearrangements. MRD was detected by nested PCR, with screening of rearrangements using multiplex PCR primers as described previously (Leukaemia and Lymphoma2002; 43: 1001). Patients were initially stratified into 3 risk groups (in ascending order: SR, HR, and HHR) according to leukocyte count and age at time of diagnosis. The MRD+/+ patients with levels ≥ 10−3 at both TP1 and TP2 received augmented therapy 14 weeks after initiation, and the remainder continued to receive the initial risk-adapted protocols. A total of 311 patients with a median age of 5.3 years (range 1.0–16.8) were eligible for this study. There were 4 (1.3%) non-responders and no deaths in induction. Of the 307 patients stratified, 169 (55%) were SR, 107 (35%) were HR, and 31 (10%) were HHR. The 2nd stratification by MRD level at TP2 was possible for 72.3% (222/307; insufficient DNA=28; missing time-points=25; no marker=32). Out of the 222 patients stratified, 125 (56.3%) were MRD−/−, 58 (26.1%) were MRD+/−, and 38 (17.4%) were MRD+/+. At the point of analysis, the median follow-up time was 63 months (range 33–89). The overall 5-year event–free survival (EFS) rate of the 307 patients was 80.1% (SE 2.5), higher than the EFS of the ALL941 study, which was 76.2% (SE 2.1) (p=0.167). The 5-year EFS rates according to the 1st stratification were 85.5% (SE 4) for SR, 76.1% (SE 4.5) for HR, and 64.6% (SE 9.2) for HHR, while the equivalent rates for the 2nd stratification were 87.0% (SE 3.1) for MRD−/−, 75.5% (SE 7.7) for MRD+/−, and 75.3% (SE 6.4) for MRD+/+. From the 95 patients whose MRD levels were measured at 5 consecutive points from TP1 to TP5 (5, 12, 18, 24, and 30 weeks after the start of therapy), 21 subjects with MRD+/+ received an augmented chemotherapy, and MRD levels became undetectable in 9 patients at TP3, 5 patients at TP4, and 4 patients at TP5. The corresponding cumulative 5-year relapse rates of those patients were 11%, 50%, and 50%, respectively. Thus, negative MRD status at TP3, but not at TP4 or TP5, seems to be associated with a favourable outcome. Our results confirm the strong performance of MRD-based treatment interaction in a multi-institutional study without adversely affecting the outcome in childhood ALL. Moreover, present findings suggest that an augmented therapy could reduce MRD to levels associated with a favourable outcome. To improve the applicability and accuracy of MRD assay, new MRD-PCR targets and RQ-PCR-based MRD detection are needed in subsequent studies. [Acknowledgment: This study was partly supported by grants from the Children’s Cancer Association of Japan (CCAJ)].

Description: BACKGROUND: Minimal residual disease (MRD) level after induction (Time Point1:TP1) and before consolidation therapy (Time Point2:TP2) has a strong impact in prediction of outcome for childhood acute lymphoblastic leukemia and it has clinical utility of a prognostic factor to stratify the risk groups in many current studies. We measured MRD levels on various time points to evaluate their prognostic significance in MRD-based augmented therapy. PATIENTS & METHODS: From June 2004 to September 2009, we prospectively assigned 333 consecutive children with ALL, 1〜19 years of age, to receive one of three treatment protocols on the stratification based on National Cancer Institute (NCI) risk criteria. NCI standard risk:SR, NCI high risk:HR and those with a white blood cell count≧1000x109 per L was defined as high-high risk:HHR. Patients were stratified again at TP2, patients with MRD level over 10-3 were assigned to salvage arm, treated in augmented therapy. Among 333 patients, 326 were eligible and 245 were MRD quantifiable. Then, we re-evaluated those samples by RQ-PCR according to the guideline of Euro MRD. Finally 167 cases were analyzed at 7 time points of MRD quantification in the CCLSG ALL2004 study. RESULTS: The overall 5-year event free survival (5-EFS) rate for ALL2004 was 82.5±2.1% (n=326), and 5-EFS of SR group (n=267), HR group (n=86) and HHR group (n=33) were 84.8±2.5%, 80.1±4.3% and 74.7±7.8%, respectively. In the SR group, 5-EFS of standard therapy group (n=124) with TP2 MRD

Description: Purpose It is difficult to decide whether children with leukemia who could not achieve complete remission (CR) after relapse or primary induction failure should undergo transplantation. Nonetheless, allogeneic hematopoietic stem cell transplantation (HSCT) is a possible approach for refractory acute leukemia including acute lymphoblastic leukemia (ALL). Even after refractory to conventional chemotherapy, a graft versus leukemia (GVL) effect could be expected to some extent. This approach is considered to be experimental because the mortality rate of HSCT is extremely high. A previously conducted large-scale study showed that age younger than 10 years was a factor of good prognosis; however, the details in children are unclear. The purpose of this retrospective analysis was to describe the outcomes and risk factors of HSCT for children with refractory ALL. Patients and Methods The data was collected through the Transplant Registry Unified Management Program (TRUMP) system, the registry of The Japan Society for Hematopoietic Cell Transplantation. In total, 325 patients with ALL younger than 21 years old when HSCT was performed between January 2001 and December 2015 and who harbored blasts in peripheral blood and/or bone marrow were analyzed. Both myeloablative regimens and reduced-intensity conditioning regimens were analyzed. Patients were classified as having poor-risk cytogenetics with either t(4;11), t(9;22), t(8;14), hypodiploidy or near triploidy, or more than 5 cytogenetic abnormalities. Other ALL cytogenetic findings were classified as other abnormalities or normal. Myeloablative conditioning was defined as total body irradiation (TBI) of 〉8 Gy and the administration of 8 mg/kg of busulfan (BU), 〉140 mg/m2 of melphalan, or 〉10 mg/kg of thiotepa. All other regimens were analyzed as reduced-intensity conditioning HSCT, including low-dose TBI (≤8 Gy) and low-dose BU (≤8 mg/kg). The graft included bone marrow, peripheral blood stem cells, or cord blood. Overall survival (OS) was used as a primary outcome because for HSCT during relapse, post-HSCT CR was not always achieved or reliably documented. Results The median follow-up time of survivors was 1145 days (range 110-3710). The median age was 11 years. Thirty-five percent of patients had a pre-HSCT performance status (PS) of 0, which corresponds to a Karnofsky PS of ≥90. The rate of pre-HSCT fungal infection was 14%. Fifty-nine patients had more than 25% marrow blasts when HSCT was performed. When HSCT was performed, 10%, 60%, and 30% of patients exhibited primary induction failure, first relapse, and second or later relapse, respectively. Ninety-one percent of patients had neutrophils and 67% exhibited platelet recovery by day 100. The cumulative incidence of grade II-IV acute graft-versus-host disease (aGVHD) on day 100 was 43%. The cumulative incidence of chronic GVHD (cGVHD) at 3 years after HSCT was 19%. Two hundred and forty-seven patients died. The causes of death were leukemia progression (57%), followed by graft failure (11%), GVHD (10%), hemorrhage (6%), and infection (5%). The 3-year OS rate in all the patients was 22% (95% confidence interval (CI), 18-27). Age, white blood cell count at diagnosis, prior history of central nervous disease, disease status, conditioning regimens, donor-recipient human leukocyte antigen match, graft type, or year in which HSCT was performed did not affect the OS. Grade ≥2 aGVHD did not affect OS. Whereas Patients with chronic GVHD had better 3-year OS (49%, 95% CI 35-61%) compared to that in patients without chronic GVHD (22%, 95% CI 16-29%) (p = 0.001) (Figure 1). Multivariate analysis showed that other than cGVHD, low PS (relative risk (RR): 2.53), blasts in bone marrow greater than 25% (RR: 1.43), T cell phenotype (RR: 1.86), high-risk or normal cytogenetics (RR: 1.42), and a history of HSCT (RR: 1.90) were significant adverse pre-HSCT variables (Table 1). Patients who had 0 or 1 (n = 113), 2 (n = 113), and 3-5 pre-HSCT variables (n = 99) had 42% (95% CI, 32-51), 17% (95% CI, 10-25), and 6% (95% CI, 2-13) 3-year OS, respectively (Figure 2). Conclusion Some patients with refractory pediatric ALL achieved relatively long survival following HSCT in the relapsed period, especially when a GVL effect was obtained. A scoring system using pre-HSCT variables should help decide whether HSCT should be performed or not. HSCT is worth considering for children who have undergone ≤2 pre-HSCTs. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Epstein-Barr virus (EBV) is a double-stranded DNA virus that infects 〉95% of the human population and is associated with a substantial risk of cancer development. Most infections in children and adolescents are asymptomatic or result in infectious mononucleosis; however, in some patients, EBV is associated with various hematological malignancies including Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), and extranodal NK/T-cell lymphoma. EBV infection is also present in a portion of epithelial cell neoplasms such as gastric cancer and nasopharyngeal carcinoma. Despite the large population risk of cancer associated with EBV, it is poorly understood why only a small subset of EBV-infected individuals develop neoplasms, while others do not. Patients and Methods We designed a target enrichment system to capture several EBV strains including the Akata strain, which is responsible for the majority of EBV infections in Japan. We analyzed the genomes of EBV strains in 139 patients with various EBV-associated diseases and 17 EBV-positive cell lines. Next-generation sequencing reads were aligned to the Akata reference genome to analyze nucleotide variations, copy number alterations, and structural variations including sequence insertions in the human genome. The institutional review board of Nagoya University Graduate School of Medicine approved this study. Results We identified a median of 645 single nucleotide variants (SNVs) in the EBV genomes, 78% of which affected coding sequences. SNVs in coding sequences were significantly biased toward synonymous variants, suggesting negative selection pressure. The SNVs detected in noncoding sequences were enriched in two evolutionarily conserved viral noncoding RNAs (EBER1 and EBER2), particularly in the PAX5-binding domain of EBER2. However, most SNVs identified in the EBV genome do not seem to affect the development of neoplasms, as hierarchical clustering of EBV genomes from neoplastic and non-neoplastic diseases based on SNVs revealed no significant association between the EBV strain and disease type. In addition to SNVs, we identified frequent intragenic deletions in the EBV genomes of patients with EBV-positive DLBCL (10/14, 71%), extranodal NK/T-cell lymphoma (10/23, 43%), chronic active EBV infection (27/77, 35%), and other EBV-associated neoplasms (2/7). Such deletions were also identified in several EBV-associated cell lines (6/17), but not in non-neoplastic diseases such as infectious mononucleosis (0/4) and post-transplant lymphoproliferative disorders (0/14), suggesting a unique role of these mutations in the neoplastic proliferation of EBV-infected cells. Frequent deletions were detected in BamHI A rightward transcripts microRNA clusters (31/156), which suppress viral transcription factors (BZLF1 and BRLF1) required for the lytic reactivation of EBV. Deletions also were associated with several genes essential for virus production (20/156). These observed deletions are thought to upregulate lytic cycle-associated genes, some of which benefit neoplasms by inducing genomic instability and immune escape and mitigate cell damage caused by the production of viral particles. In fact, deletion of one essential gene, BALF5, resulted in upregulation of the lytic cycle and promotion of lymphomagenesis in a xenograft model. Discussion Although the essential roles of several latency-associated genes, such as LMP-1 and EBNA-2, in EBV-mediated immortalization and transformation of human lymphocytes have long been discussed, our finding raises the possibility that lytic cycle-associated genes also contribute to lymphomagenesis. This agrees with reports that lytic cycle-associated genes are expressed in Burkitt lymphoma, DLBCL, and chronic active EBV infection, and that BZLF1-deficient lymphoblastoid cells exhibit significantly impaired tumorigenicity in mice. In addition, essential gene deletions lead to the protection of EBV-infected cells from lysis. Further studies are warranted to exploit these findings for the design of novel therapeutics for EBV-associated neoplasms. Disclosures Kiyoi: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Phizer Japan Inc.: Research Funding; Sanofi K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria. Nakamura:Roche/Chugai,: Research Funding; Kyowa-Kirin: Research Funding.

Description: Abstract 4470 Background: Despite intensive multi-modal therapies, prognosis of refractory or relapsed pediatric solid tumor is dismal because majority of those tumors already get acquired chemo-resistance. Therefore new break through approaches are expected in order to improve their survival. The efficacy of allogeneic hematopoietic stem celltransplantation(SCT) is primarily attributed to a T/NK- cell-mediated response to HLA disparity between donor and tumor cell. Non–T-cell depleted (non TCD) haploidentical hematopoietc stem cell transplantation as immunothrapy is attractive challenge for refractory tumor, however, there are several reports describing graft-versus-tumor (GVT) effects in patients with solid tumors. The major problems of non-TCD haplo-SCT are lethal graft-versus-host disease (GVHD), graft failure (GF) and high-risk of early death. Previously we reported the safety profile from the retrospective study assessing GVHD prophylaxis that was conducted with anti-human thymocyte immunoglobulin (ATG), tacrolimus, methotrexate and prednisolone in non-TCD haplo-SCT (Mochizuki, Kikuta, Clin Transplant,2010 DOI:10.1111/j.1399-0012.2010.01352.x). We started clinical study of non-TCD HLA haploidentical hematopoietc stem cell transplantation for refractory or relapsed pediatric solid tumor as cell-mediated immune therapy since July, 2007. Objectives/Methods: This study presents a series of transplant experiments aiming to evaluate the efficacy and feasibility of non-TCD HLA haploidentical hematopoietc stem cell transplantation for refractory or relapsed pediatric solid tumor. Seven cases (3males, 4females) with refractory or relapsed pediatric solid tumor were enrolled on this study between July 2007 to December 2010. One patient had second transplantation due to tumor progression 1year after transplantation. Among 7 patients, there are 3 cases of relapsed neuroblastoma, 1 case of relapsed Mesenchymal Chondrosarcoma, 1 case of relapsed Ewing sarcoma family tumor, 1 case of refractory alveolar soft part sarcoma with multiple lung metastasis, and 1 case of refractory primitive neuroectodermal tumor. Conditioning regimens consisted with fludarabine30mg/m2 at day-9 to -5+Melphalan70mg/m2 at day-4 to-3+rabbit ATG 1. 25mg/kg at day-2 to -1. The GVHD prophylaxis was conducted with tacrolimus (0.03mg/kg/day, start on day-1), methotrexate (10mg/m2, 7mg/m2, 7mg/m2 on day+1, +3, +6) and predonisolone (1mg/kg/day, day 0–29, taper on day30 without GVHD). HLA disparities were 3/8 in 3, 4/8 in 5. Donors included father(1), mothers(5), siblings(1), mother’s younger brother(1). Four pts received peripheral blood stem cells and 4 pts received bone marrow. Result: All of seven patients achieved primary engraftment but secondary graft rejection was observed in one patients. Median follow up period after transplantation were 14 months (range 11–40 months). Incidence of acute GvHD was 3/7 cases (grade±:1, gradeII:1, grade III:1), chronic GvHD was observed in 5(83%) of 6 evaluable patients. Three cases were underwent DLI for tumor progression. Treatment-related mortality(TRM) was not observed and reactivation of VZV, interstitial pneumonia, and HHV6 related limbic system encephalitis were recognized as major complication within 100 days after transplantation. Five cases of patients had tumor progression after transplantation, and two children were dead by tumor progression, other 3 cases had additional treatment. Two cases are alive and well, with no evidence of disease 13 and 14 months after transplantation. Graft versus Tumor effect was clearly observed in three cases. Conclusion: The survival rate of relapsed or refractory pediatric solid tumor is under 20%, however, the two-year probability of overall survival was 71.4% with no TRM in this study. These results indicated the feasibility and the possibility of efficacy of non-TCD HLA haploidentical hematopoietc stem cell transplantation for relapsed or refractory pediatric solid tumor. Disclosures: No relevant conflicts of interest to declare.