Chronic active Epstein-Barr virus (EBV) infection (CAEBV) is an orphan disease characterized by the proliferation and infiltration of EBV-infected T/natural killer (NK) cells into multiple organs. Although CAEBV is a heterogeneous disease with diverse clinical courses, its pathogenesis remains poorly understood. In this study, we explored the molecular mechanisms underlying CAEBV by performing a comprehensive multi-omics analysis, including genome, transcriptome, epigenome, and single-cell transcriptome and surface proteome analyses, of 65 CAEBV patients. Methylation analysis identified two distinct subtypes of NK cell-type CAEBV based on the CpG island methylator phenotype (CIMP). In CIMP-positive CAEBV, regions associated with enhancer of zeste homolog 2 binding sites and histone H3 lysine 27 trimethylation exhibited increased DNA hypermethylation, resulting in downregulation of tumor suppressor and anti-herpes virus genes. CIMP-positive CAEBV had a particularly poor prognosis and displayed a "neoplastic" phenotype with a DNA methylation pattern similar to that of extranodal NK/T-cell lymphoma, a higher tumor mutation burden, and frequent copy number alterations. In addition, both in vitro and in vivo functional assays demonstrated that 5-Azacytidine, a hypomethylating agent, was a potentially effective agent for high-risk CIMP-positive CAEBV. Finally, we established a method to effectively detect EBV-infected cells in single-cell analysis, suggesting that EBV-infected NK cells have tissue-resident properties and that innate and adaptive immunity to EBV is compromised in patients with CAEBV. The present findings provide insight into the complex molecular features of CAEBV and suggest potential molecular therapies.

Not available.

Emberger syndrome (ES), an autosomal dominant disorder characterized by congenital deafness, primary lymphedema, and predisposition to myeloid malignancies, is caused by mutations in the GATA2 gene. Although primary lymphedema is an important hallmark of ES, the pathophysiology remains unclear due to the lack of a suitable experimental model. In this study, we isolated induced pluripotent stem cells (iPSCs) from two patients with ES (i.e., ES-iPSCs) and analyzed their in vitro lymphatic differentiation potential via the mesodermal progenitor stage. KDR+ CD34+ early mesodermal progenitors generated from either ES-iPSCs or wild-type iPSCs during a 6-days serum- and feeder-free culture supplemented with bone morphogenetic protein 4 and vascular endothelial growth factor (VEGF) had almost equivalent developmental potential. However, upon co-culture with OP9 stromal cells, KDR+ CD34+ cells derived from ES-iPSCs developed into CD31+ lymphatic vessel endothelial hyaluronan receptor-1+ VEGF receptor 3+ lymphatic endothelial cells less efficiently than KDR+ CD34+ cells derived from wild-type iPSCs. Thus, patient-derived iPSCs recapitulate impairments at an early stage of lymphangiogenesis, making them a useful experimental tool for dissecting the pathophysiology of primary lymphedema in ES and developing potential therapeutic approaches.

BACKGROUND: Chimeric antigen receptor (CAR) T cells are a major new treatment option for children, adolescents, and young adults (CAYA) patients with relapsed and refractory (R/R) B cell acute lymphoblastic leukemia (B-ALL). Therefore, accumulating evidence from real-world experiences of CAR-T outcomes in various regions world-wide is important, particularly when comparing outcomes of patients with differing medical and ethnic backgrounds. OBJECTIVE: More than 5 years have passed since tisagenlecleucel was approved in Japan. Here, we report a retrospective, multi-institutional investigation examining the association between baseline parameters and clinical outcomes. The aim was to investigate real-world experience, and to better comprehend the efficacy of commercial tisagenlecleucel. STUDY DESIGN: A nationwide consortium called the Japan CAR-T Consortium conducted a retrospective, multicenter study of CAYA patients who received CAR-T cell treatment with commercial tisagenlecleucel. Forty-two patients with R/R B-ALL whose leukapheresis samples were shipped to Novartis for commercial tisagenlecleucel manufacture were included in the analysis. All infused patients were included in the response, toxicity, and survival analyses. RESULTS: The best overall response rate was 93%. The 1-year overall survival (OS) and event-free survival (EFS) rates after infusion were 82% and 56%, respectively. Twenty-seven (64%) had low disease burden (LB, defined as <5% bone marrow [BM] lymphoblasts) prior to tisagenlecleucel infusion. LB was associated with superior outcomes, with a 1-year EFS rate of 80% compared with 24% in high disease burden (≧5% BM lymphoblasts). Multivariate analysis identified an association between prior hematopoietic stem cell transplantation (HSCT) (n=23, 55%) and superior outcomes, with a 1-year EFS rate of 75% compared with 24% for patients without prior HSCT. CONCLUSION: This first analysis of CAYA patients with R/R B-ALL undergoing treatment with commercial tisagenlecleucel in Japan reports an efficacy similar to that in clinical trials and other real-world studies and confirms that LB and prior HSCT are associated with superior EFS.

Not available.

Abstract The prognosis of patients with osteosarcoma who experience recurrence or progression (R/P) is extremely poor, and more effective and less toxic therapies are needed. In the current study, the clinical data of osteosarcoma patients who experienced R/P were retrospectively analyzed to verify the reliability of O‐6‐methylguanine‐DNA methyltransferase (MGMT) protein expression or MGMT promoter methylation for predicting the response to off‐label temozolomide (TMZ)‐containing chemotherapy. Of the 30 evaluable patients, 9 (30%) showed no/low MGMT protein expression, whereas all 16 evaluable patients had unmethylated MGMT promoter irrespective of MGMT protein expression levels. Twenty‐three patients received TMZ‐containing chemotherapy for measurable lesions (n = 14) or as adjuvant therapy following resection of recurrent lesions (n = 9). Among 14 patients with radiologically measurable lesions, the objective response rate was higher in the MGMT no/low‐expression group (50.0%) than in the MGMT intermediate/high‐expression group with borderline significance (0%, p = 0.066). The 6‐month progression‐free survival (PFS) rate in patients with radiologically measurable lesions was significantly higher in the MGMT no/low‐expression group (50.0%) than in the MGMT intermediate/high‐expression group (0%, p = 0.036). In the multivariate analysis of the 23 patients receiving TMZ‐containing chemotherapy, MGMT expression and disease status before TMZ‐containing chemotherapy were significantly associated with PFS. No severe adverse effects were observed during TMZ‐containing chemotherapy. MGMT protein expression, but not MGMT promoter methylation, could predict a favorable outcome in patients receiving TMZ‐containing chemotherapy.

Abstract Early diagnosis and prompt initiation of appropriate treatment are critical for improving the prognosis of acute leukemia. Acute leukemia is diagnosed by microscopic morphological examination of bone marrow smears and flow cytometric immunophenotyping of bone marrow cells stained with fluorophore‐conjugated antibodies. However, these diagnostic processes require trained professionals and are time and resource‐intensive. Here, we present a novel diagnostic approach using ghost cytometry, a recently developed high‐content flow cytometric approach, which enables machine vision‐based, stain‐free, high‐speed analysis of cells, leveraging their detailed morphological information. We demonstrate that ghost cytometry can detect leukemic cells from the bone marrow cells of patients diagnosed with acute lymphoblastic leukemia and acute myeloid leukemia without relying on biological staining. The approach presented here holds promise as a precise, simple, swift, and cost‐effective diagnostic method for acute leukemia in clinical practice.

Background: Early diagnosis and prompt initiation of appropriate treatment are critical for improving the prognosis of acute leukemia. Acute leukemia is typically diagnosed through microscopic morphological examination of bone marrow smears and flow cytometric immunophenotyping of bone marrow cells stained with fluorophore-conjugated antibodies. However, these diagnostic processes require trained professionals and are time and resource-intensive. Here, we present a novel diagnostic approach using ghost cytometry (GC), a recently developed high-content flow cytometric approach that leverages machine vision-based, stain-free, high-speed morphological characterization and analysis of cells. Methods: Label-free morphological information of cells was acquired as compressive imaging temporal waveforms. These waveforms were directly analyzed using the support vector machine (SVM) algorithm without computational image production, enabling high-speed and accurate information processing. Conventional flow cytometric data modalities, such as forward-scattering (FSC), backscattering (BSC), and fluorescence signals from biological labeling, were used as ground truth labels to identify and annotate blast and normal cells in bone marrow (BM) based on flow cytometric gating schemes. Subsequently, we trained a leukemia cell classifier using the annotated GC (compressive imaging) waveforms and evaluated its classification ability by comparing the predicted results with the ground truth labels. Finally, we applied the trained classifier to unidentified GC waveforms (a test dataset not used for classifier development) to demonstrate the detection of blast cells in BM based on their morphology, without relying on biological labels. Following this protocol, we first trained and tested a machine classifier using a human leukemia NB-4 cell line spiked into BM from a healthy donor. Next, we trained and tested two classifiers using BM aspirates from patients diagnosed with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), where CD45 was used as the ground truth marker of leukemic cells. Results: The leukemia cell classifier, developed using a human NB-4 cell line spiked into BM cells from a healthy donor, demonstrated excellent classification accuracy with an ROC-AUC score of 0.9998. The high classification accuracy was reproducible across different spike-in concentrations ranging from 1% to 95%. The classifiers trained using BM cells from patients diagnosed with ALL and AML showed high ROC-AUC scores of 0.986 and 0.941, respectively. When we used the classifiers to evaluate blast cell proportions in test datasets that were not used to develop the classifiers, they were also highly accurate in identifying the percentage of blast cells: 56.8% for ALL with a decision boundary of SVM scores at 0, and 30.4% for AML with a decision boundary of SVM scores at 0.37, when compared to May-Grunwald-Giemsa staining of the same cases by medical technologists at diagnosis (60.0% for ALL and 31.2% for AML). Conclusions: Here, we present a novel diagnostic approach that leverages machine vision-based, stain-free, high-speed morphological characterization and analysis of cells. We demonstrate that the approach can detect leukemic cells from the bone marrow cells of patients diagnosed with ALL and AML, without requiring biological staining. The method presented here holds promise as a rapid, precise, simple, and cost-effective method for the diagnosis of acute leukemia in clinical settings.

Purpose: Although fertility preservation for pediatric cancer patients is becoming more widespread in Japan, some facilities do not provide sufficient information regarding fertility. This study aimed to elucidate the problems pertaining to the lack of information about fertility among patients. Methods: Based on a 2020 survey, seminars addressing fertility preservation were held from the Designated Pediatric Cancer Care Hospitals in each of the seven blocks in Japan to their partner hospital (pediatric cancer hospitals). The seminar consisted of lectures and group discussions, and a questionnaire was also administered after each seminar. Results: In the group discussions, a lack of explanations to patients and explanatory materials for children were cited as issues by many facilities. The survey results revealed a lack of material explaining fertility preservation and a lack of knowledge among health care providers. There were also many requests to use the patient explanation videos presented at the seminar. Conclusion: The results indicate that further education for health care providers by seminar and other sources and enhancement of explanatory materials are important for fertility preservation in pediatric cancer hospitals in Japan.

Treatment outcomes for children with relapsed and refractory acute lymphoblastic leukemia (R/R-ALL) remain poor, and the optimal induction therapy has not been determined. Bortezomib is a proteasome inhibitor that acts synergistically and additively with standard chemotherapy for ALL. We evaluated the efficacy and safety of combination chemotherapy with bortezomib in children with R/R-ALL. This single-arm, multicenter, phase 2 study was conducted in Japan between 2016 and 2020. Eligible patients were divided into two cohorts: a high-risk first-relapse cohort of untreated patients with high-risk first-relapsed ALL and an expansion cohort of patients with refractory ALL, including multiple relapses, relapse after allogeneic hematopoietic cell transplantation, and induction failure. All patients received a single course of chemotherapy as induction therapy. Sixteen patients (10 in the high-risk first-relapse cohort, six in the expansion cohort) were evaluable. The overall remission rate after induction therapy was 60% in the high-risk first-relapse cohort and 16.7% in the expansion cohort. All patients had minimal residual disease. Adverse events were acceptable except for interstitial lung disease and hypoxia in a patient in the expansion cohort, but addition of bortezomib to conventional chemotherapy did not produce obvious improvement in children with R/R-ALL.

There is no clear consensus on the most effective treatment for relapsed/refractory high-risk neuroblastoma (NB). We retrospectively assessed seven NB patients with relapsed/refractory disease who received high-dose carboplatin-irinotecan-temozolomide (HD-CIT). Five of seven patients showed favorable therapeutic response (complete remission or partial remission). Regarding toxicity, the cytopenia period tended to prolong when more than three cycles were repeated, but nonhematological toxicities were controllable with general supportive care. Due to its antitumor efficacy and well-tolerated nonhematologic toxicity, HD-CIT is a promising salvage chemotherapy for relapsed/refractory NB. However, it is important to pay attention to the exacerbation of hematological toxicity when repeating the regimen.

OBJECTIVE: The preset study evaluated the outcome of living-donor segmental lung transplantation for pediatric patients. METHODS: Between August 2009 and May 2021, we performed living-donor segmental lung transplantation in 6 critically ill pediatric patients, including 1 patient on a ventilator alone and another patient on a ventilator and extracorporeal membrane oxygenation (ECMO). There were 4 male and 2 female patients, with a median age of 7 years (range, 4-15 years) and a median height of 112.7 cm (range, 95-125.2 cm). The diagnoses included complications of allogeneic hematopoietic stem cell transplantation (n = 4) and pulmonary fibrosis (n = 2). All patients received bilateral lung transplantation under cardiopulmonary bypass. A basal segment and a lower lobe were implanted in 3 patients, and a basal segment and an S6 segment were implanted in the other 3 patients. In 2 patients, the right S6 segmental graft was horizontally rotated 180° and implanted as the left lung. RESULTS: Among the 9 segmental grafts implanted, 7 functioned well after reperfusion. Two rotated S6 segmental grafts became congestive, with 1 requiring graft extraction and the other venous repair, which was successful. There was 1 hospital death (14 days) due to sepsis and 1 late death (9 years) due to leukoencephalopathy. The remaining 4 patients are currently alive at 9 months, 10 months, 1.3 years, and 1.9 years. CONCLUSIONS: Living-donor segmental lung transplantation was a technically difficult but feasible procedure with acceptable outcomes for small pediatric patients with chest cavities that were too small for adult lower lobe implantation.

INTRODUCTION: Relapsed and refractory B-cell acute lymphoblastic leukaemia (R/R-B-ALL) is linked to a significant relapse rate after allogeneic haematopoietic cell transplantation (allo-HCT) in children, adolescents and young adults (CAYA). No standard treatment has been established to prevent relapse after allo-HCT for R/R-B-ALL, which is an unmet medical need. The administration of blinatumomab after allo-HCT is expected to enhance the antileukaemic effect on residual CD19-positive blasts by donor-derived CD3-positive T-cells. METHODS AND ANALYSIS: The goal of this multicentre, open-label, uncontrolled, phase I-II clinical trial is to assess the safety and effectiveness of post-transplant maintenance therapy with blinatumomab for CAYA patients (25 years old or younger) with CD19-positive R/R-B-ALL who have received allo-HCT beyond first complete remission (CR) and have CR with haematological recovery between 30 and 100 days after allo-HCT. Eighty-five paediatric institutions in Japan are participating in this study. Forty-one patients will enrol within 2.25-year enrolment period and follow-up period is 1 year. The primary endpoints are the treatment completion rate for phase I study and the 1-year graft-versus-host disease-free/relapse-free survival rate for phase II study, respectively. ETHICS AND DISSEMINATION: This research was approved by the Central Review Board at National Hospital Organization Nagoya Medical Center (Nagoya, Japan) on 21 January 2022 and was registered at the Japan Registry of Clinical Trials (jRCT) on 3 March 2022. Written informed consent is obtained from all patients and/or their guardians. The results of this study will be disseminated through peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: jRCTs041210154.

Pediatric colorectal cancer (CRC) is extremely rare, with little information about genetic profiles compared with adult CRC. Here, a 13-year-old male with advanced CRC underwent cancer gene panel testing, which detected 4 genetic abnormalities ( MET amplification in addition to TP53 , SMAD4 , and CTNNA1 mutations) that might be associated with a poor prognosis. Based on high-level MET amplification, he received a multikinase inhibitor, cabozantinib, after failure of first-line and second-line chemotherapy, resulting in transient disease stabilization. Tailored targeted therapy based on molecular profiling can be an effective treatment strategy for rare cancers such as pediatric CRC.

BACKGROUND: Patients with osteosarcoma who experience relapse or progression [R/P] have a poor prognosis. METHODS: Data from 30 patients who experienced R/P among 59 with a diagnosis of high-grade osteosarcoma, who were younger than 40 years old between 2000 and 2019, were retrospectively analyzed to identify prognostic and therapeutic factors influencing their outcomes. RESULTS: The 5-year overall survival [OS] rates after the last R/P of patients experiencing first [n=30], second [n=14], and third [n=9] R/P were 50.3%, 51.3%, and 46.7%, respectively. Multivariate analysis did not identify any independent risk factors affecting OS. The 5-year PFS rate of the 30 patients after first R/P was 22.4%, and multivariate analysis identified histologic subtype and curative local surgery as independent risk factors influencing PFS. Long [>6 mo] partial response was observed in three patients treated using temozolomide+etoposide, irinotecan+carboplatin, or regorafenib. CONCLUSIONS: OS rate in the patients with osteosarcoma experiencing R/P included in this study was markedly higher than that reported previously, mainly due to the surgical total removal of tumors, even after subsequent R/P. The recent establishment of salvage chemotherapy or molecular targeted therapy may also increase survival rates in a subgroup of patients.

Detailed case reports of autologous recovery of hematopoiesis after hematopoietic stem cell transplantation with myeloablative conditioning are scarce. We present a rare case of a 3-year-old male with relapsed KMT2A -rearranged acute lymphoblastic leukemia who experienced autologous recovery following secondary engraftment failure after cord blood transplantation with myeloablative conditioning. Similar to prior reports, we detected unusual chromosomal abnormalities, which differed at each bone marrow examination. He remains alive without relapse of acute lymphoblastic leukemia 8 months after cord blood transplantation. As the rate of recurrence or late occurrence of secondary malignant neoplasm remains unclear, careful follow-up is required, especially in pediatric patients.

Malignant rhabdoid tumor (MRT) is a highly aggressive pediatric malignancy with no effective therapy. Therefore, it is necessary to identify a target for the development of novel molecule-targeting therapeutic agents. In this study, we report the importance of the runt-related transcription factor 1 (RUNX1) and RUNX1-Baculoviral IAP (inhibitor of apoptosis) Repeat-Containing 5 (BIRC5/survivin) axis in the proliferation of MRT cells, as it can be used as an ideal target for anti-tumor strategies. The mechanism of this reaction can be explained by the interaction of RUNX1 with the RUNX1-binding DNA sequence located in the survivin promoter and its positive regulation. Specific knockdown of RUNX1 led to decreased expression of survivin, which subsequently suppressed the proliferation of MRT cells in vitro and in vivo. We also found that our novel RUNX inhibitor, Chb-M, which switches off RUNX1 using alkylating agent-conjugated pyrrole-imidazole polyamides designed to specifically bind to consensus RUNX-binding sequences (5'-TGTGGT-3'), inhibited survivin expression in vivo. Taken together, we identified a novel interaction between RUNX1 and survivin in MRT. Therefore the negative regulation of RUNX1 activity may be a novel strategy for MRT treatment.

BACKGROUND: Living-donor lobar lung transplantation (LDLLT) remains a life-saving option for pediatric patients with respiratory failure. However, the long-term survival and post-transplant quality of adult lobar grafts transplanted into children are unknown. Therefore, this study aimed to evaluate the outcomes of pediatric LDLLT and post-transplant graft growth. METHODS: We retrospectively reviewed the prospectively collected clinical data of 25 living-donor lung transplantations performed in 24 pediatric recipients aged ≤17 years. The annual pulmonary function test data and computed tomography scans of 12 recipients, followed up for >5 years without significant complications, were used to evaluate growth in height, graft function, and radiological changes. The Kaplan-Meier method and simple linear regression were performed for analysis. RESULTS: Bilateral lower lobe transplantation was performed in 12 patients, unilateral lower lobe transplantation in 12, and bilateral middle lobe transplantation in 1. The median volumetric size matching at transplantation was 142% (range, 54%-457%). The 5- and 10-year overall survival rates were 87.7% and 75.1༅, respectively. Chronic lung allograft dysfunction occurred in 2 patients. During a median follow-up of 6 years, the median increases in height and vital capacity were 14.4% (range, 0.80%-43.5%) and 58.5% (range, 6.7%-322%), respectively. Graft weight was positively correlated with graft volume (r2=0.622, p<0.001) after the graft volume exceeded the original lobar volume in the donor. CONCLUSIONS: This study shows that pediatric LDLLT offers satisfactory long-term survival, with the growth of mature adult lobes transplanted into growing children.

Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy has facilitated progress in treatment of refractory/relapsed diffuse large B-cell lymphoma (DLBCL). A well-known adverse event after CAR-T therapy is cytokine release syndrome(CRS). However, the etiology and pathophysiology of CRS-related coagulopathy remain unknown. Therefore, we conducted a prospective cohort study to comprehensively analyze coagulation/ fibrinolysis parameters present in peripheral blood of adult DLBCL patients treated with tisagenlecleucel in a single institution. Samples were collected from 25 patients at 3 time points: before lymphocyte-depletion chemotherapy and on days 3 and 13 after CAR-T infusion. After infusion, all patients except 1 experienced CRS, and 13 required the administration of tocilizumab. A significant elevation in the plasma level of total plasminogen activator inhibitor 1 (PAI-1), which promotes the initial step of coagulopathy (mean, 22.5 ng/mL before lymphocyte-depletion and 41.0 on day 3, P = .02), was observed at the onset of CRS. Moreover, this suppressed fibrinolysis-induced relatively hypercoagulable state was gradually resolved after CRS remission with normalization of total PAI-1 to preinfusion levels without any organ damage (mean values of soluble fibrin: 3.16 µg/mL at baseline, 8.04 on day 3, and 9.16 on day 13, P < .01; and mean PAI-1: 25.1 ng/mL on day 13). In conclusion, a hypofibrinolytic and relatively hypercoagulable state concomitant with significant total PAI-1 elevation was observed at the onset of CRS even in DLBCL patients with mild CRS. Our results will facilitate understanding of CRS-related coagulopathy, and they emphasize the importance of monitoring sequential coagulation/fibrinolysis parameters during CAR-T therapy.

The purpose of this study is to establish a treatment with appropriate intensity for children (<16 years old at diagnosis) with de novo acute myeloid leukemia (excluding acute promyelocytic leukemia and myeloid leukemia associated with Down syndrome) according to a risk stratification based on recurrent leukemic cytogenetic abnormalities and flow-cytometric minimal residual disease at end of initial induction chemotherapy and to validate the safety and efficacy of gemtuzumab ozogamicin (GO)-combined post-induction chemotherapy for the non-low-risk (non-LR) patients. The primary endpoint of this phase III study is three-year disease-free survival rate, which will be compared between the GO and non-GO arms of the non-LR (intermediate-risk and high-risk [HR]) patients. All HR patients will be allocated to allogeneic hematopoietic stem cell transplantation in first remission. This trial has been registered at the Japan Registry of Clinical Trials (jRCTs041210015).

As chimeric antigen receptor (CAR) T cell therapy targeting CD19 has shown favorable outcomes in patients with relapsed or refractory (r/r) mature B cell lymphomas and B cell acute lymphoblastic leukemia (B-ALL), an increasing number of patients are waiting to receive these treatments. Optimized protocols for T cell collection by lymphapheresis for chimeric antigen receptor (CAR) T cell therapy are urgently needed to provide CAR T cell therapy for patients with refractory and progressive disease and/or a low number of lymphocytes owing to prior chemotherapy. The predicted efficiency of CD3+ cell collection in apheresis can guide protocols for apheresis, but a clinically applicable model to produce reliable estimates has not yet been established. In this study, we prospectively analyzed 108 lymphapheresis procedures for tisagenlecleucel therapy at 2 centers. The apheresis procedures included 20 procedures in patients with B cell acute lymphoblastic leukemia and 88 procedures in patients with diffuse large B cell lymphoma, with a median age at apheresis of 58 years (range, 1 to 71 years). After lymphapheresis with a median processing blood volume of 10 L (range, 3 to 16 L), a median of 3.2 × 109 CD3+ cells (range, .1 to 15.0 × 109 cells) were harvested. Collection efficiency 2 (CE2) for CD3+ cells was highly variable (median, 59.3%; range, 11.0% to 199.8%). Multivariate analyses revealed that lower hemoglobin levels, higher circulating CD3+ cell counts, and higher platelet counts before apheresis significantly decreased apheresis CE2. Based on multivariate analyses, we developed a novel formula that estimates CE2 from precollection parameters with high accuracy (r = .56; P < .01), which also suggests the necessary processing blood volume. Our strategy for lymphapheresis should help reduce collection failure, as well as achieve efficient utilization of medical resources in clinical practice, thereby allowing delivery of CAR T cell therapy to more patients in a timely manner.

Ponatinib is effective in adults with Philadelphia chromosome-positive (Ph+) leukemia, resistant or intolerant to second-generation tyrosine kinase inhibitors, but there are limited data on its use in children. The clinical courses of nine pediatric patients with Ph+ acute lymphoblastic leukemia (Ph+ ALL) and four with chronic myeloid leukemia (CML) who received ponatinib therapy were retrospectively reviewed. The median age at the start of ponatinib therapy was 12 years (range 8-16 years). Nine patients were male and four were female. Six patients received ponatinib alone, three received ponatinib with prednisolone, one received ponatinib with rituximab intrathecal therapy, and one received ponatinib with conventional chemotherapy. Two patients received ponatinib both alone and in combination with chemotherapy. The median dose and duration of ponatinib were 16.9 mg/m2 (7-34.3) and 1.1 months (0.2-22.7), respectively. Six patients with Ph+ ALL and two with CML responded to ponatinib. One of the eight patients who received ponatinib alone had grade 4 increased lipase levels. Grade 3 non-hematologic toxicities included elevated alanine aminotransferase levels (25%), elevated aspartate aminotransferase levels (25%), elevated gamma-glutamyl transferase levels (12.5%), hypertension (12.5%), and polymorphic erythema (12.5%). Ponatinib may be safe and effective in pediatric patients with Ph+ leukemia.

The prognosis of patients with osteosarcoma recurring at extrapulmonary/extraosseous sites, especially those with unresectable tumors, is generally dismal due to high resistance to chemotherapy. The present study describes a pediatric patient with osteosarcoma recurring to the liver and stomach. Complete remission was achieved by long-term systemic chemotherapy with temozolomide+etoposide, local irradiation of the stomach, and radical surgical removal of multiple liver metastases following percutaneous transhepatic portal embolization. Second-line multimodal therapy, consisting of salvage chemotherapy and curative local treatment of metastases, may enhance disease-free survival of patients with osteosarcoma experiencing relapse to uncommon sites.

Lineage switch is a rare event at leukemic relapse. While mostly known to occur in KMT2A-rearranged infant leukemia, the underlying mechanism is yet to be depicted. This case report describes a female infant who achieved remission of KMT2A-MLLT3-rearranged acute monocytic leukemia, but six months thereafter, relapsed as KMT2A-MLLT3-rearranged acute lymphocytic leukemia. Whole exome sequencing of the bone marrow obtained pre-post lineage switch revealed two somatic mutations of PAX5 in the relapse sample. These two PAX5 alterations were suggested to be loss of function, thus to have played the driver role in the lineage switch from AML to ALL.

With the advancement of immunosuppressive strategies, the outcome of liver transplantation during childhood has dramatically improved. On the other hand, Epstein–Barr virus (EBV) infection and associated post-transplant lymphoproliferative diseases (PTLD), such as malignant lymphoma, are serious complications that contribute to morbidity and mortality, and are still an important issue today. Recently, an early diagnosis by quantitative PCR and PET-CT testing, and treatment with rituximab (an anti-CD20 antibody) has been established, and long-term remission has been achieved in many cases. However, the optimal immunosuppression protocol after remission of PTLD needs to be determined, and it is hoped that a treatment for refractory PTLD (e.g., PTL-NOS) will be proposed.

In this study, we performed a retrospective analysis of a cohort of Japanese paediatric patients with B-cell precursor (BCP)-acute lymphoblastic leukaemia (ALL) treated with a Berlin-Frankfurt-Münster (BFM)95-based protocol, to clarify the incidence, clinical characteristics, and risk factors of osteonecrosis (ON) in comparison to the ALL-02 protocol. We identified a high frequency of ON with the BFM95-based protocol compared to the ALL-02 protocol. The incidence of symptomatic ON with the BFM95-based protocol is comparable to previous studies in Western countries. We believe that the type of treatment regimen has more impact on the incidence of symptomatic ON in paediatric ALL than ethnicity.

Due to the considerable success of cancer immunotherapy for leukemia, the tumor immune environment has become a focus of intense research; however, there are few reports on the dynamics of the tumor immune environment in leukemia. Here, we analyzed the tumor immune environment in pediatric B cell precursor acute lymphoblastic leukemia by analyzing serial bone marrow samples from nine patients with primary and recurrent disease by mass cytometry using 39 immunophenotype markers, and transcriptome analysis. High-dimensional single-cell mass cytometry analysis elucidated a dynamic shift of T cells from naïve to effector subsets, and clarified that, during relapse, the tumor immune environment comprised a T helper 1-polarized immune profile, together with an increased number of effector regulatory T cells. These results were confirmed in a validation cohort using conventional flow cytometry. Furthermore, RNA transcriptome analysis identified the upregulation of immune-related pathways in B cell precursor acute lymphoblastic leukemia cells during relapse, suggesting interaction with the surrounding environment. In conclusion, a tumor immune environment characterized by a T helper 1-polarized immune profile, with an increased number of effector regulatory T cells, could contribute to the pathophysiology of recurrent B cell precursor acute lymphoblastic leukemia. This information could contribute to the development of effective immunotherapeutic approaches against B cell precursor acute lymphoblastic leukemia relapse.

Neuroblastoma, the most common extracranial solid tumor of childhood, is thought to arise from neural crest-derived immature cells. The prognosis of patients with high-risk or recurrent/refractory neuroblastoma remains quite poor despite intensive multimodality therapy; therefore, novel therapeutic interventions are required. We examined the expression of a cell adhesion molecule CD146 (melanoma cell adhesion molecule [MCAM]) by neuroblastoma cell lines and in clinical samples and investigated the anti-tumor effects of CD146-targeting treatment for neuroblastoma cells both in vitro and in vivo. CD146 is expressed by 4 cell lines and by most of primary tumors at any stage. Short hairpin RNA-mediated knockdown of CD146, or treatment with an anti-CD146 polyclonal antibody, effectively inhibited growth of neuroblastoma cells both in vitro and in vivo, principally due to increased apoptosis via the focal adhesion kinase and/or nuclear factor-kappa B signaling pathway. Furthermore, the anti-CD146 polyclonal antibody markedly inhibited tumor growth in immunodeficient mice inoculated with primary neuroblastoma cells. In conclusion, CD146 represents a promising therapeutic target for neuroblastoma.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for the hematologic manifestations of Diamond-Blackfan anemia (DBA). However, data regarding the optimal conditioning regimen for DBA patients are limited. We retrospectively compared the outcomes of DBA patients who underwent HSCT using either myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC) regimens. The patients belonged to a cohort treated at our hospitals between 2000 and 2018. HSCT was performed in 27 of 165 patients (16.4%). The median age at the time of HSCT was 3.6 years. Stem cell sources included bone marrow for 25 patients (HLA-matched sibling donors, n = 5; HLA-mismatched related donors, n = 2; HLA-matched/mismatched unrelated donors, n = 18) or cord blood for 2 patients. MAC or RIC regimens were used in 12 and 15 patients, respectively. Engraftment was successful in all 27 patients who underwent HSCT. Three patients who underwent HSCT using MAC regimens developed sinusoidal obstruction syndrome. The 3-year overall survival (OS) and failure-free survival rates (FFS) post-transplantations were 95.2% and 88.4%, respectively, with no significant differences between MAC and RIC regimens. Our data suggest that HSCTs using RIC regimens are effective and obtain engraftment with excellent OS and FFS for young DBA patients.

Bone marrow necrosis (BMN) describes necrosis of the myeloid tissues without cortical bone involvement. Imatinib, a tyrosine kinase inhibitor, can trigger BMN during the treatment of malignant disease. In such cases, it is necessary to reduce imatinib dose or discontinue its administration, which could influence treatment outcomes. Here, we report a 6-year-old boy with Philadelphia chromosome-positive acute lymphoblastic leukemia, who developed BMN in response to imatinib. We replaced imatinib with dasatinib, and necrotic lesions gradually disappeared and were never exacerbated. In Philadelphia chromosome-positive acute lymphoblastic leukemia with BMN, tyrosine kinase inhibitor replacement may allow continued chemotherapy without intensity reduction.

Post-transplant lymphoproliferative disorders (PTLDs) are life-threatening neoplasms after organ transplantation. Because of their rarity and multiple grades of malignancy, the incidence, outcomes, and clinicopathological features affecting patient survival after liver transplantation (LT) remain unclear. We reviewed 1,954 LTs in 1,849 recipients (1990-2020), including 886 pediatric (<18 years of age) and 963 adult recipients. The following clinicopathological factors were studied: age, sex, liver etiologies, malignancy grades, Epstein-Barr virus status, performance status (PS), Ann Arbor stage, international prognostic index, and histopathological diagnosis. Of 1,849 recipients, 79 PTLD lesions (4.3%) were identified in 70 patients (3.8%). After excluding 3 autopsy cases incidentally found, 67 (45 pediatric [5.1%] and 22 adult [2.3%]) patients were finally enrolled. Comorbid PTLDs significantly worsened recipient survival compared with non-complicated cases (P < 0.001). The 3-year, 5-year, and 10-year overall survival rates after PTLD diagnosis were 74%, 66%, and 58%, respectively. The incidence of PTLDs after LT (LT-PTLDs) was significantly higher (P < 0.001) with earlier onset (P = 0.002) in children, whereas patient survival was significantly worse in adults (P = 0.002). Univariate and multivariate analyses identified the following 3 prognostic factors: age at PTLD diagnosis ≥18 years (hazard ratio [HR], 11.2; 95% confidence interval [CI], 2.63-47.4; P = 0.001), PS ≥2 at diagnosis (HR, 6.77; 95% CI, 1.56-29.3; P = 0.01), and monomorphic type (HR, 6.78; 95% CI, 1.40-32.9; P = 0.02). A prognostic index, the "LT-PTLD score," that consists of these 3 factors effectively stratified patient survival and progression-free survival (P = 0.003 and <0.001, respectively). In conclusion, comorbid PTLDs significantly worsened patient survival after LT. Age ≥18 years and PS ≥2 at PTLD diagnosis, and monomorphic type are independent prognostic factors, and the LT-PTLD score that consists of these 3 factors may distinguish high-risk cases and guide adequate interventions.

Myeloid leukemia of Down syndrome (ML-DS) is associated with good response to chemotherapy, resulting in favorable outcomes. However, no universal prognostic factors have been identified to date. To clarify a subgroup with high risk of relapse, the role of minimal residual disease (MRD) was explored in the AML-D11 trial by the Japanese Pediatric Leukemia/Lymphoma Study Group. MRD was prospectively evaluated at after induction therapy and at the end of all chemotherapy, using flow cytometry (FCM-MRD) and GATA1-targeted deep sequencing (GATA1-MRD). A total of 78 patients were eligible and 76 patients were stratified to the standard risk (SR) group by morphology. In SR patients, FCM-MRD and GATA1-MRD after induction were positive in 5/65 and 7/59 patients, respectively. Three-year event-free survival (EFS) and overall survival (OS) rates were 93.3% and 95.0% in the FCM-MRD-negative population, and 60.0% and 80.0% in the positive population. Three-year EFS and OS rates were both 96.2% in the GATA1-MRD-negative population, and 57.1% and 71.4% in the positive population. Adjusted hazard ratios for associations of FCM-MRD or GATA1-MRD with EFS were 10.98 (p = 0.01) and 27.68 (p < 0.01), respectively. Detection of MRD by either FCM or GATA1 after initial induction therapy represents a significant prognostic factor for predicting ML-DS relapse.

The anti-CD19 chimeric antigen receptor (CAR) T cells showed excellent effect against acute lymphoblastic leukemia (ALL) in bone marrow (BM) in clinical trials. However, it remains to be elucidated whether the CD19 CAR T cell therapy is effective for ALL cells in central nervous system (CNS) because the patients with isolated or advanced CNS disease were excluded from clinical trials of systemic intravenous (i.v.) delivery of CAR T cells. Therefore, the preclinical evaluation for the efficacy of CAR T cell therapy against ALL cells in CNS is essential for clinical application. We evaluated the effect and adverse reaction of CD19 CAR T cells against ALL in CNS using a xenograft mouse model by i.v. or intra-cerebroventricular (i.c.v.) delivery of CAR T cells. Injection of piggyBac CD19 CAR T cells by i.v. had partial effects, whereas all CAR T i.c.v.-delivered mice had eliminated ALL in CNS. Although some CAR T i.c.v.-delivered mice showed transient changes of clinical symptoms during the first few days after treatment, none of CAR T i.c.v.-delivered mice displayed fatal adverse events. In this study, we demonstrated that direct delivery into CNS of CAR T cells is a possible therapeutic approach with the xenograft mouse model.

Shwachman-Diamond syndrome (SDS), an autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, and skeletal abnormalities, is caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene, which plays a role in ribosome biogenesis. Although the causative genes of congenital disorders frequently involve regulation of embryogenesis, the role of the SBDS gene in early hematopoiesis remains unclear, primarily due to the lack of a suitable experimental model for this syndrome. In this study, we established induced pluripotent stem cells (iPSCs) from patients with SDS (SDS-iPSCs) and analyzed their in vitro hematopoietic and endothelial differentiation potentials. SDS-iPSCs generated hematopoietic and endothelial cells less efficiently than iPSCs derived from healthy donors, principally due to the apoptotic predisposition of KDR+CD34+ common hemoangiogenic progenitors. By contrast, forced expression of SBDS gene in SDS-iPSCs or treatment with a caspase inhibitor reversed the deficiency in hematopoietic and endothelial development, and decreased apoptosis of their progenitors, mainly via p53-independent mechanisms. Patient-derived iPSCs exhibited the hematological abnormalities associated with SDS even at the earliest hematopoietic stages. These findings will enable us to dissect the pathogenesis of multiple disorders associated with ribosomal dysfunction.

Cryotherapy is a conventional method for preventing melphalan-induced oral mucositis (OM) in adult patients. We retrospectively examined the clinical benefits of cryotherapy in 41 pediatric patients undergoing autologous stem cell transplantation using a melphalan-etoposide-carboplatin regimen. Twenty-two patients received cryotherapy. The cumulative incidence of grade 3-4 OM was significantly lower in the cryotherapy group (57.1%) than in the noncryotherapy group (89.5%; P = .041). Multivariate analyses identified cryotherapy and the melphalan dose as independent factors for the lower occurrence of OM. The present study demonstrates the clinically significant efficacy of cryotherapy for preventing melphalan-induced OM in pediatric patients.

Growing teratoma syndrome is a well-recognized condition associated with both intracranial and extracranial nongerminomatous germ cell tumors (NGGCTs), which mostly manifest as rapid growth of cystic and solid components during or within several months after treatment. Here, we report a patient with NGGCT who experienced slow growth of intracranial growing teratoma syndrome with intraventricular lipid accumulation over 10 years without any clinical symptoms. Considering the clinicopathologic heterogeneity of this syndrome, long-term clinical and radiologic follow-up is required for all patients with intracranial NGGCT.

Continuous deep sedation (CDS) is used to alleviate unbearable and otherwise refractory symptoms in patients dying of cancer. No data are available concerning CDS in children from Japan to date. This study primarily aimed to describe experience in CDS in child cancer patients at Kyoto University Hospital. The secondary aims were to identify the characteristics of patients who received CDS, and to assess ability in daily living at the end of life. A retrospective chart review was performed for child cancer patients who died at the institute between 2008 and 2017. The data of 35 patients were analyzed. Nine (26%) patients had received CDS. Indications for CDS were dyspnea (56%), agitation (22%), seizures (22%), and pain (11%). Midazolam was used in all nine cases. In eight (89%) patients, opioids were also prescribed. In seven (78%) patients, CDS was performed for < 48 hours. In all nine cases, consent was obtained from the parent(s) but not from the children. CDS was more likely in patients with solid tumors (p = 0.018) and those who had received no respite sedation (p = 0.002). Patients without central nervous system symptoms tended to maintain their capacity for oral intake and verbal communication until a few days prior to death. This is the first report on CDS in child cancer patients from Japan. In the CDS literature, cross-study differences are evident for incidence, target symptoms, duration, and the decision-making process. Further international discussion is warranted concerning indications for CDS and the decision-making process.

The prognosis of patients with relapsed osteosarcoma is extremely poor and the optimal treatment remains to be identified. Here, we retrospectively analysed the clinical outcomes of nine patients with relapsed osteosarcoma treated with temozolomide/etoposide. Of the two patients who received temozolomide/etoposide as palliative therapy for unresectable tumours, one remained alive with stable disease for >4 years. The remaining seven patients received temozolomide/etoposide as adjuvant therapy following resection of relapsed metastatic disease; of these, one was free from disease for 41 months. Potentially beneficial effects were observed in two of three O6-methylguanine-DNA methyltransferase protein-negative patients, whereas all five O6-methylguanine-DNA methyltransferase-positive patients experienced subsequent relapse. None of the patients experienced severe adverse effects requiring hospitalization. Temozolomide/etoposide is a feasible candidate as salvage therapy for relapsed osteosarcoma. Further studies are needed to verify the utility of O6-methylguanine-DNA methyltransferase protein expression as a biomarker for predicting the response to this treatment.

Granulocyte transfusion (GTX) is a therapeutic option for severe bacterial or fungal infection in patients with sustained neutropenia after chemotherapy or stem cell transplantation. However, high molecular weight hydroxyethyl starch (HES), which has been used for selective sedimentation of red blood cells during apheresis, is not easily available in many countries including Japan. In this study, we evaluated the efficiency of granulocyte collection using medium molecular weight HES (130 kDa) in combination with the Spectra Optia apheresis system. Apheresis was performed for 2 consecutive days from seven donors and the mean total neutrophil yield from the first and second apheresis was 5.27 ± 3.10 × 1010 and 2.91 ± 2.92 × 1010, respectively. Infusion of concentrates from the first apheresis resulted in a significant neutrophil count increase and concentrates from the second apheresis were enough for maintenance of the neutrophil counts in all the recipients. Although the number of cases is limited, our results clearly show that sufficient number of granulocytes can be harvested by using medium molecular weight HES and this strategy is a safe and effective clinical practice in countries where high molecular weight HES is not available.

Runt-related transcription factor (RUNX) play pivotal roles in leukemogenesis and inhibition of RUNX has now been widely recognized as a novel strategy in anti-leukemic therapies. However, the role of RUNX remains elusive in acute promyelocytic leukemia (APL). Here, we demonstrate that targeting RUNX1-NFATC2 axis is effective strategy to suppress drug-resistant (DR)-APL cells. Considering the well-established roles of RUNX and NFATC2 in T cell immunity, we also apply targeting RUNX-NFATC2 strategy to suppress T cell activation and xenogeneic graft-versus-host disease (GVHD). Firstly, to investigate whether RUNX1 is essential for APL proliferation and maintenance, we examined cell proliferation of all-trans retinoic acid (ATRA) resistant APL cell line, NB4, using shRNA-mediated knockdown of RUNX1. Profound knockdown of RUNX1 in NB4 cells led to growth suppression and apoptotic cell death. We found that NFATC2 is one of the most consistently up-regulated genes in RUNX1-high expressing APL cells derived from previously reported human clinical samples (GSE2550, GSE13159, GSE61804), and also has RUNX1-binding regions in the promoter from chromatin-immunoprecipitation and sequencing (ChIP-Seq) data (GSE22178, GSE31221). ChIP qPCR assay confirmed the actual binding of RUNX1 in the NFATC2 promotor region of NB4. In addition, luciferase reporter experiments showed NFATC2 promoter significantly increased its reporter activity by RUNX1 over-expression. These results confirmed RUNX1 directly upregulated NFATC2 transcriptional activity. Consistent with these findings, silencing of RUNX1 suppressed the expression of NFATC2. Besides, silencing of NFATC2 in NB4 cells suppressed cell growth and induced apoptotic cell death. Next, the efficacy of our novel RUNX inhibitor: chlorambucil-conjugated pyrrole-imidazole polyamide (Chb-M'), which specifically binds to the consensus RUNX-binding sequence, was examined for NB4. Chb-M' was remarkably more effective against NB4 (IC50 value at nM level) than ATRA and arsenic trioxide. Consistent with association between RUNX1 and NFATC2, Chb-M' suppressed the expression of NFATC2 of NB4. Furthermore, Chb-M′ suppressed NB4 proliferation and NFATC2 expression in xenograft tumor model. Since RUNX and NFATC2 are key regulators of T cell function, we next investigate whether targeting RUNX is also effective for T cell mediated diseases including GVHD. Previous reports showed that RUNX1 directly regulate Th1 cytokine genes such as IL2 and IFNG (Ono M et. al. Nature 2007). From public ChIp-Seq data (ENCODE project. Nature 2012), RUNX1 also binds to NFATC2 promotor region in mouse and human primary CD4 T cells and T-ALL cell line, Jurkat. To study the efficacy of Chb-M' for allo-reactive T-cell activation and proliferation, we performed mixed lymphocyte reaction from different healthy donors using thymidine uptake assay. Compared to control, Chb-M' significantly reduced T cell proliferation. To examine the effect of Chb-M' for T cell cytokine expression, we stimulated peripheral blood mononuclear cells (PBMC) with PMA-ionomycin and measured cytokine expression by quantitative PCR. Results showed that Chb-M'moderately but significantly reduced IL2 , TNF , IFNG and NFATC2 mRNA expression. To investigate whether RUNX is essential for cytokine and NFATC2 expression, we silenced RUNX family by shRNA-knockdown of Jurkat E6.1 cell line. Knockdown of RUNX family reduced IL2 , TNF and NFATC2 expression. These results indicated targeting RUNX-NFATC2 axis is also effective for T cell activation and cytokine expression. Finally, we examined in vivo effect of Chb-M' for xenogeneic GVHD mouse model by transplanting human PBMC onto immunodeficient mice. Compared to control, mice injected by Chb-M' showed almost no sign of GVHD assessed by clinical score and pathological score of lung and liver. Analysing peripheral blood of GVHD mice showed that especially CD4 T cell was decreased and GVHD-associated cytokines including TNF-α and GM-CSF were reduced in Chb-M' injected mice. Taken together, we have shown that RUNX transcriptionally upregulated NFATC2, which is essential for APL proliferation and T cell activation. RUNX-NFATC2 axis can be a novel therapeutic target against DR-APL and GVHD. Disclosures No relevant conflicts of interest to declare.

小児ALLのマイクロRNA(miR)発現解析の先行研究に基づき、独自のlenti-virus vectorを用いてALL細胞株のmiR-146aknock downを行ったところ、コロニー形成能の抑制を認め、NOTCH1 pathwayの関与が示唆された。しかし、マウスへの生着性には有意な差を認めなかった。そこで、改めて、次世代シークエンサー用いて小児ALLのmRNAとmiRの網羅的発現解析を行ったところ、miR発現によってのみ規定される新たな予後不良群（miR-low cluster；MLC）を同定した。MLCパターンを示すALL細胞の分子遺伝学的基盤を解析中である。

マイクロRNA(miR)は、造血器腫瘍において治療反応性マーカーや治療標的として研究が進められているが、本研究では小児急性リンパ性白血病のmiR発現解析結果に基づき、miR-146aに着目し、細胞内でのmiR発現レベルを可視化出来るmiR sensing vector並びに、活性を抑制できるmiR knock down vectorを用いて種々の急性リンパ性白血病細胞株のmiR発現を解析した。発現はおしなべて高く、同一株内であってもその発現に多様性があることを見出した。また、miR-146aのノックダウンによってコロニー形成能を30-50％抑制しことから、その分子機構を解析している。

すべての血液細胞を生み出すことが出来るヒト造血幹細胞の性質については未だ不明な点が多く、その生物学的特徴を明らかにすることで、骨髄移植など造血幹細胞治療をより効率的に行うことが出来るようになると考えられる。造血幹細胞表面には特徴的なマーカーが発現しているが、我々は近年、造血幹細胞において重要な働きを担っているマイクロRNA-126という遺伝子を可視化できるシステムを開発し、従来、造血幹細胞が存在しないと考えられている細胞分画にマイクロRNA-126が強く発現している細胞群を見いだした。この細胞群は新たなヒト造血幹細胞ソースになる可能性を秘めており、生物学的特徴につきさらに研究を進めている。

筋ジストロフィー症に代表される筋変性疾患に対する根本的治療法は存在しない。新規治療として遺伝子治療が模索されているが、一部の筋変性疾患のみが対象となり、普遍性を持つ新しい治療展開が期待されている。近年、ヒト ES 細胞に加えて、ヒト iPS 細胞が作成され、その多能性幹細胞が有する骨格筋分化能を用いて、筋変性疾患に対する新規診療基盤の確立が模索されている。その1つは骨格筋幹細胞(satellite 細胞)を作成し、移植医療への展開を進める方向であるが全身投与という難問が存在する。一方、近年の iPS 細胞作成技術の確立により、筋ジストロフィー症患者由来ヒト疾患特異的 iPS 細胞から骨格筋幹細胞、成熟細胞を作成することが可能となった。その手法を用い、骨格筋幹細胞(satellite 細胞)細胞死、枯渇を抑制する新規の分子を見い出す、ジストロフィン遺伝子に対して効率的なエクソンスキッピングを引き起こす分子を同定する、などの新規の治療基盤開発が現実のものとなった。我々は、既に開発済みのマウス ES 細胞、マウス iPS 細胞を用いて骨格筋幹細胞(satellite 細胞)を作成する基盤技術(Mizuno Y FASEBJ 24:2245:2010, Chang H FASEBJ 23:1907:2009)をもとに、ヒト ES 細胞、ヒト iPS 細胞より、特異的に骨格筋を作成する基盤技術を開発した。(Awaya T, Heike T et al. PloS One 7:e51638 2012)。さらに、7名の筋ジストロフィー患者からの iPS 細胞作成にも着手しており(内6名に関しては iPS 細胞を作製済み)、疾患特異的 iPS 細胞を用いた研究体制が整備済みである。

筋ジストロフィー症に代表される筋変性疾患に対する根本的治療法は存在せず、その転帰は不幸な経過をたどる。新規治療の試みとして遺伝子治療が模索されているが、現在の方法では特異的な遺伝子変異を持つ一部の筋変性疾患のみが対象となり、普遍性を持つ新しい治療展開が期待されている。根本的治療法がなく不幸な転帰をたどる患者さんに明るい未来をもたらす治療法を確立するには、ES細胞より骨格筋幹細胞であるsatellite細胞形成に至る機構を明らかにした上で、大量培養にて作成するシステムの構築を行ない、新しい治療体系の確立が必須である。本研究では、ES細胞よりsatellite細胞を特異的に形成する基盤技術開発を行った。ES細胞をembryoid bodyの形で培養し、マトリジェル上で培養を継続することにより筋繊維の発現を確認した。さらに、詳細な観察にて、Pax3,Pax7を発現する骨格筋幹細胞であるsatellite細胞の発現も確認した。このsatellite細胞は、SM/C-2.6というマウスsatellite細胞を認識する抗体で分離可能であり、分離したSM/C-2.6陽性細胞は、in vitro, in vivoにおいて骨格筋組織へ分化し得ることを確認した。また、in vivoに移植したSM/C-2.6陽性細胞は長期にわたって存在すること、筋障害を加えることにより増幅すること、二次移植が可能であることにより、satellite細胞という骨格筋幹細胞であることを確認した。現在、マウスES細胞で得られた知見をもとに、霊長類ES細胞においてもその成果を検討し、筋ジストロフィー症に対する治療基盤技術の開発を目的として、研究を継続している。

中枢神経系を含む生体各組織には、組織の再生をつかさどる組織幹細胞が存在することが知られるようになった。従来組織幹細胞は、その由来組織へと分化する能力を有する細胞と理解されてきた。しかし、近年の報告では、由来組織の枠を超えて様々な組織へと分化する能力である可塑性を有することが明らかとなった。造血組織である骨髄よりは、ES細胞に匹敵する多能性を持つMAPC(multipotent adult stem cell)が同定され、基礎的、臨床的見地より注目を集めている。しかし、その分離、同定には困難を伴い、基礎的検討、臨床的応用を視野に入れた場合、効率性、再現性、安定性、容易性を担保とした樹立方法の確立が切望されている。我々は、種々組織幹細胞同定に共通して用いられるsphere培養法に、従来の造血組織由来幹細胞樹立方法を組み合わせることにより、簡便に骨髄由来多能性幹細胞の樹立に成功した。このsphere培養の確立には数週間の時間を要するが、再現性をもってsphereの確立を確認した。このsphere細胞を分化させると、従来の間葉系幹細胞に相当した分化能力(骨細胞、脂肪細胞)を有する。興味あることに、この幹細胞は、従来の骨髄由来組織幹細胞では困難とされてきた心筋細胞や神経細胞への分化を含む、多くの組織細胞へと効率よく分化し得た。心筋障害を惹起したマウスへの同細胞の移植においても、心機能の改善を誘導した。

より鋭敏なヒト造血幹細胞移植モデルマウスを作製する目的で開発した NOD/SCID/g_c^(NOG)マウスは、従来のNOD/SCIDマウスに比べて遥かに効率よくヒト造血細胞が生着する。臍帯血由来CD34陽性細胞10万個を移植すると3ヶ月後には骨髄、脾臓においてヒト血球は70%にも達した。また、このマウスに生着した細胞を詳細に検討してみると、骨髄球系、赤芽球系、巨核球系、リンパ球系のすべての血球が分化、増殖していることがわかった。特筆すべきこととしてこのマウスモデルではヒトT細胞、NK細胞が分化、増殖し、これらのリンパ球はサイトカイン産生能や細胞障害能を有するなど従来のマウスモデルでは全く見られなかった特徴を有していることを確認した。このマウスを用いることでヒト造血幹細胞移植に酷似した動物モデルを作製することが可能になった。臍帯血由来のヒト造血幹細胞分画としてCD34陽性細胞を分離し、Stem cell factor, IL-6, soluble IL-6 receptor, Flt-3 ligand, Thrombopoietinの存在下に1週間培養した。細胞数は60〜80倍に増え、コロニー形成数も10倍以上に達した。増幅した細胞をNOGマウスに移植し、4週後ないし8週後において解析すると顆粒球系のみならず、B細胞、T細胞など多系列に分化する細胞の生着を認めた。ヒト細胞の割合は現在までのところ有意な差を認めていないものの、我々の培養系が多分化能を保持したまま増殖を可能にしていることが示された。

骨格筋再生における造血幹細胞の関与について検討した。放射線照射したマウスに、GFPトランスジェニックマウス由来のc-kit陽性、Sca-1陽性、Lin陰性の造血幹細胞分画を移植し、放射線障害により惹起される骨格筋再生過程における関与について検討した。その結果、移植1週間において、GFP陽性の骨格筋を認めるようになり、c-kit陽性、Sca-1陽性、Lin陰性の細胞分画に骨格筋への分可能を有する細胞群が存在することが明らかとなった。しかし、このGFP陽性骨格筋は、次第にGFP陰性の骨格筋へと置換され、短期における骨格筋再生現象しか観察されない。その後、移植後6カ月を経過した移植マウスの骨格筋においても、GFP陽性骨格筋は認められない。しかし、このマウスにおいて、骨格筋よりsingle fiberを単離し、single fiberに付着するように存在する骨格筋幹細胞satellite細胞を観察するに、GFP陽性satellite細胞の存在、およびin vitroの分化検討におけるGFP陽性骨格筋の形成を確認した。この結果、c-kit陽性、Sca-1陽性、Lin陰性の細胞分画には、短期の骨格筋再生に寄与する細胞群とともに、長期にわたって骨格筋再生に寄与するsatellite細胞へと分化し得る細胞群が存在することが明らかとなった。より精密な細胞分画の同定、効率よくsatellite細胞へと分化する機構の開発に向けて、検討を進めている。