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[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.

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 6 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 acute monocytic leukemia to acute lymphocytic leukemia.

Supplementary Fig. S1-S5. Fig. S1. Anti-leukemic activity of PD0332991 against Ph+ lymphoid leukemia cell lines. Fig. S2. Effect of cytokines and PD0332991 on cell growth of Ph+ lymphoid leukemia cell lines. Fig. S3. Anti-leukemic activity of PD0332991 against a Ph+ALL cell lines with a T315I mutation of BCR-ABL in vitro. Fig. S4. Anti-leukemic activity of PD0332991 against a Ph+ ALL cell line with a T315I mutation of BCR-ABL in a xenograft model using NOG mice. Fig. S5. Expression of cell cycle regulators in Ph+ lymphoid leukemia cells.

S-phase progression of the cell cycle is accelerated in tumors through various genetic abnormalities, and, thus, pharmacologic inhibition of altered cell-cycle progression would be an effective strategy to control tumors. In the current study, we analyzed the antileukemic activity of three available small molecules targeting CDK4/CDK6 against lymphoid crisis of chronic myeloid leukemia (CML-LC) and Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL), and found that all three molecules showed specific activities against leukemic cell lines derived from CML-LC and Ph+ ALL. In particular, PD0332991 exhibited extremely high antileukemic activity against CML-LC and Ph+ ALL cell lines in the nanomolar range by the induction of G0–G1 arrest and partially cell death through dephosphorylation of pRb and downregulation of the genes that are involved in S-phase transition. As an underlying mechanism for favorable sensitivity to the small molecules targeting CDK4/CDK6, cell-cycle progression of Ph+ lymphoid leukemia cells was regulated by transcriptional and posttranscriptional modulation of CDK4 as well as Cyclin D2 gene expression under the control of BCR-ABL probably through the PI3K pathway. Consistently, the gene expression level of Cyclin D2 in Ph+ lymphoid leukemia cells was significantly higher than that in Ph− lymphoid leukemia cells. Of note, three Ph+ ALL cell lines having the T315I mutation also showed sensitivity to PD0332991. In a xenograft model, PD0332991, but not imatinib, suppressed dissemination of Ph+ ALL having the T315I mutation and prolonged survival, demonstrating that this reagent would be a new therapeutic modality for relapsed CML-LC and Ph+ ALL patients after treatment with tyrosine kinase inhibitors. Mol Cancer Ther; 15(1); 94–105. ©2015 AACR.

Supplementary Table S1-S5. Table S1. Characteristics leukemia cell lines. Table S2. List of primary antibodies used for Western blot analysis. Table S3. List of Taqman probe for real-time RT-PCR analysis. Table S4. Sequences of siRNA. Table S5. Sensitivity to PD0332991 of B-precursor cell lines and their gene expression level of components for CDK4/6-cyclin D pathway and gene status of p16.

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., Tumor immune environment characterized by a T helper 1‐polarized immune profile was observed in recurrent B cell precursor acute lymphoblastic leukemia. Regulatory T cells were activated in recurrent B cell precursor acute lymphoblastic leukemia.

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., Targeting CD146 inhibits in vitro proliferation of neuroblastoma (NB) cells and in vivo growth of NB tumors. Exposure of NB cells to the anti‐CD146 antibody led to a significant reduction in survival and anchorage‐independent growth. Intraperitoneal injection of the anti‐CD146 antibody into immunodeficient mice for ~50 d led to a significant inhibition of tumor growth.

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.

Acute undifferentiated leukemia (AUL) is a rare subtype of leukemia that expresses no lineage-specific markers; no optimal treatment for AUL has been established. Here, we report a 16-year-old female with CD22-positive refractory AUL who responded well to inotuzumab ozogamicin (InO). Minimal residual disease negativity was achieved using InO, followed by HLA-mismatched unrelated bone marrow transplantation (BMT). Although grade II veno-occlusive disease/sinusoidal obstruction syndrome occurred, it improved immediately. She remained disease-free at 10 months post-BMT, without severe complications (grade III–IV). This case demonstrates the feasibility of a treatment strategy using InO against CD22-positive AUL.

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.

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.

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., キメラ抗原受容体T細胞療法におけるリンパ球採取効率化の取り組み --最適な治療戦略策定への貢献に期待--. 京都大学プレスリリース. 2022-06-20.

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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.

Introduction Due to the considerable success of cancer immunotherapy for leukemia, the tumor immune environment (TIE) has become a focus of intense research; however, there are few reports on the dynamics of the TIE in leukemia, especially in bone marrow (BM), the primary site of leukemia. Mass cytometry, which allows high-dimensional analysis with single-cell resolution, is a powerful tool for the characterization of the TIE, and enables us to examine pediatric BM samples containing only a few numbers of immune cells. Methods Primary and recurrent BM samples were collected serially from pediatric patients with BCP-ALL at Kyoto University Hospital from 2006 to 2013. Mononuclear cells were isolated by density centrifugation and viably preserved until they were used. We examined the TIE of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) by analyzing serial BM samples from patients in primary and recurrent disease phases by mass cytometry, using 39 immunophenotype markers, and transcriptome analysis. Results and Discussion The proportion of BCP-ALL cells in the samples was 87.3-97.8% (mean: 94.0%) at onset, and 81.3-98.8% (mean: 92.2%) at relapse. High-dimensional single-cell analysis by mass cytometry elucidated a dynamic shift of T cells from naïve to effector subsets, and clarified that the TIE during relapse comprised a T helper 1 (Th1)-polarized immune profile, together with the increase of effector regulatory T cells (Tregs). Th1 cells are typically known as supporters of cytotoxic T lymphocytes which would eliminate leukemia cells and work against relapse, but recently, it is reported that Th1 cells directly support ALL proliferation in vitro (Traxel et al. Oncogene. 2019; 38:2420-2431), and that the concentrations of pro-inflammatory cytokines and Th1 cytokines (IFN-γ and IL-12) were elevated in patients with ALL, which could create favorable conditions for ALL (Perez-Figueroa et al. Oncol Rep. 2016; 35: 2699-2706, Vilchis-Ordonez et al. Biomed Res Int. 2015; 2015: 386165). Therefore, there is a possibility that the Th1-polarized immune profile would work in favor of leukemia survival for relapse. Furthermore, Gene set enrichment analysis based on RNA expression identified the enrichment of six immune-related pathways were enriched at the time of relapse; chemokine activity, chemokine production, complement activation, positive regulation of cytokine production involved in immune response, positive regulation of lymphocyte chemotaxis, and positive regulation of lymphocyte migration. These expression signatures suggest that BCP-ALL cells attract lymphocytes, and upregulate immune activities at relapse. Conclusion In summary, a TIE characterized by a Th1-polarized immune profile, with the increase of effector Tregs, may be involved in the pathophysiology of recurrent ALL, and BCP-ALL cells at relapse were enriched with gene expressions related to lymphocyte attraction and activities. This information could contribute to the development of effective immunotherapeutic approaches against BCP-ALL relapse. Disclosures Ogawa: Eisai Co., Ltd.: Research Funding; Kan Research Laboratory, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding.

Intracranial germ cell tumor is sometimes associated with Down syndrome; however, no optimal treatment has been developed due to the high risk of recurrence and treatment-related mortality. Here, we report on a patient with an intracranial germinoma in the bilateral basal ganglia. The patient received 3 courses of ifosfamide-cisplatin-etoposide in combination with whole-brain irradiation (24 Gy), with no serious complications. The patient is alive and disease free 16 months after the initial diagnosis. This regimen is a feasible treatment for intracranial germ cell tumor associated with Down syndrome, although careful attention must be paid to the increased risk for severe infection.

Inversion of chromosome 16 (inv(16)) generates a fusion gene CBFB-MYH11, which is a driver mutation for acute myeloid leukemia (AML). Gene expression profiling suggests that Gata2, a hematopoietic transcription factor, is a top upregulated genes in preleukemic Cbfb-MYH11 knockin mice and is expressed in human inv(16) AML. On the other hand, we have also identified recurrent monoallelic deletions of GATA2 in relapsed human CBF-AML patients. To clarify the role of Gata2 in leukemogenesis by Cbfb-MYH11, we generated conditional Cbfb-MYH11 knockin mice with Gata2 heterozygous knockout. Gata2 heterozygous knockout reduced abnormal myeloid progenitors, which are capable of inducing leukemia in the Cbfb-MYH11 mice. Consequently, Cbfb-MYH11 mice with Gata2 heterozygous knockout developed leukemia with longer latencies than those with intact Gata2. Interestingly, leukemic cells with Gata2 heterozygous knockout gained higher number of mutations and showed more aggressive phenotype in both primary and transplanted mice. Moreover, leukemic cells with Gata2 heterozygous knockout showed higher repopulating capacity in competitive transplantation experiments. In summary, reduction of Gata2 activity affects mutational dynamics of leukemia with delayed leukemia onset in Cbfb-MYH11 knockin mice, but paradoxically results in a more aggressive leukemia phenotype, which may be correlated with leukemia relapse or poor prognosis in human patients.

Permanent alopecia is a grave late complication of multi-drug chemotherapy (CTx) plus cranial irradiation, reducing both patient self-esteem and quality of life in pediatric patients. We started to use craniospinal irradiation (CSI) using the volumetric-modulated arc whole-brain radiotherapy (VMAT-WBRT) in order to prevent permanent alopecia. We treated 5 pediatric patients with CSI using VMAT-WBRT, and report the initial clinical outcome. Five consecutive patients (4–11 years old) who received CSI using VMAT-WBRT from June 2015 to November 2018 were included into this study. One patient with embryonic carcinoma received radiotherapy (RT) with concurrent CTx; four patients with medulloblastoma (two patients with standard risk, and two patients with high risk) received RT followed by CTx. The prescribed doses of CSI were 23.4–35.2 Gy in 13–22 fractions, respectively. Optimization for VMAT-WBRT was performed to reduce doses to the hair follicles with keeping the dose coverage to the planning target volume. Although all patients experienced temporary alopecia, their hair fully recovered over the whole scalp within 8 months after finishing RT. One patient had disease progression after 6 months after completing CTx; this patient who was diagnosed as Group 3 subtype had diffuse meningeal dissemination confirmed with contrast enhanced spinal MRI before RT. The other four patients had no evidence of recurrence. Although CSI with VMAT-WBRT might be one of considerable options, more cases are needed to verify the efficacy to prevent permanent alopecia for pediatric patients who receive multi-drug CTx and cranial irradiation.

Malignant rhabdoid tumor (MRT) is a rare, highly aggressive pediatric malignancy that primarily develops during infancy and early childhood. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is dismal; therefore, a greater understanding of the biology of this disease is required to establish novel therapies. In this study, we identified a highly tumorigenic sub-population in MRT, based on the expression of CD146 (also known as melanoma cell adhesion molecule), a cell adhesion molecule expressed by neural crest cells and various derivatives. CD146+ cells isolated from four MRT cell lines by cell sorting exhibited enhanced self-renewal and invasive potential in vitro. In a xenograft model using immunodeficient NOD/Shi-scid IL-2Rγ-null mice, purified CD146+ cells obtained from MRT cell lines or a primary tumor exhibited the exclusive ability to form tumors in vivo. Blocking of CD146-related mechanisms, either by short hairpin RNA knockdown or treatment with a polyclonal antibody against CD146, effectively suppressed tumor growth of MRT cells both in vitro and in vivo via induction of apoptosis by inactivating Akt. Furthermore, CD146 positivity in immunohistological analysis of 11 MRT patient samples was associated with poor patient outcomes. These results suggest that CD146 defines a distinct sub-population in MRT with high tumorigenic capacity and that this marker represents a promising therapeutic target.

BACKGROUND AND AIM Acute gastrointestinal graft-versus-host disease (GI-GVHD) is a major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). There are very few studies on specific endoscopic findings in pediatric acute GI-GVHD. The aim of this retrospective case-control study was to elucidate the characteristic endoscopic findings in pediatric acute GI-GVHD that improve the diagnostic accuracy of endoscopy. METHODS All consecutive patients under 18 years of age who underwent allogeneic HSCT in Kyoto University Hospital from May 2003 to October 2014 were identified retrospectively. Patients who underwent GI endoscopy as a result of sustained GI symptoms were identified. Intestinal villous patterns were evaluated by magnification endoscopy with the water-immersion technique. The patients were diagnosed with acute GI-GVHD and non-GVHD on the basis of biopsy histology. Endoscopic findings of the two groups were compared. RESULTS Of the 171 patients who underwent HSCT, 30 underwent GI endoscopy. Of these, 17 and nine were diagnosed with acute GI-GVHD and non-GVHD, respectively. Compared with non-GVHD, acute GI-GVHD was associated significantly more often with short blunt villi in the duodenum (P = 0.013), variable defect villi and short blunt villi in the ileum (P = 0.009 and 0.035, respectively), and edema, erosion, and tortoiseshell-like mucosae in the colon (P = 0.017, 0.023, and 0.017, respectively). CONCLUSION Pediatric acute GI-GVHD was associated with several characteristic features on magnifying endoscopy with the water-immersion technique. These features will be useful for endoscopic diagnosis of pediatric acute GI-GVHD.

Patients with desmoplastic small round cell tumors (DSRCTs) have an extremely poor outcome despite the use of aggressive therapy. The current study presents the case of 16-year-old male with metastatic DSRCT, in which multimodal therapy, including intensive chemotherapies using frequent autologous stem cell support, gross resection of primary and metastatic lesions, and whole abdominopelvic intensity-modulated radiation therapy, was administered. Subsequent to these treatments, there was no evidence of active disease. However, cerebellar and pineal body lesions, and bone metastasis to the left humerus were detected 1 year and 2 months after the initial diagnosis. Combination chemotherapy with irinotecan and temozolomide was initially effective against the central nervous system (CNS) metastatic lesions; however, the patient succumbed due to progressive CNS disease after seven courses of combination chemotherapy. Additional studies are required to accumulate information regarding CNS recurrence of DSRCT.

We report on three cases of pediatric acute lymphoblastic leukemia presenting with bone pain and arthralgia as initial symptoms. At the first visit, their primary signs were recurrent bone pain and arthralgia, without significant peripheral blood abnormalities. It took 2-4 months to confirm the diagnosis from the onset of arthralgia due to this atypical presentation of the disease. Definitive diagnosis was obtained by bone marrow examination, and in all cases, complete remission was achieved by chemotherapy. As a feature of imaging, MRI exhibited diffuse bone marrow signal changes in T1-weighted images, and FDG-PET showed extensive abnormal bone marrow uptakes. In cases 2 and 3, it was difficult to diagnose by bone marrow aspiration from the iliac bone, but definitive diagnosis was obtained by bone marrow aspiration from the tibia, in which FDG-PET showed increased uptake. FDG-PET was therefore considered useful for the selection of bone marrow aspiration sites. In cases presenting with recurrent migratory bone pain and arthralgia, we need to consider performing bone marrow aspiration and imaging, such as MRI and FDG-PET, for early diagnosis and treatment of leukemia.

A 16-year-old boy was incidentally found to have hyperleukocytosis during a school physical examination. He was diagnosed with atypical chronic myeloid leukemia in chronic phase. Although treatment with hydoxyurea was started, his white blood cell count increased and he eventually developed lethal intracranial hemorrhage. Although very rare, intracranial hemorrhage should be considered as a possible complication in patients with atypical chronic myeloid leukemia, even in chronic phase, if they have hyperleukocytosis and thrombocytopenia.

LT has contributed to an elevation in cure rates for patients with unresectable HB; however, patients with recurrent HB after LT have poor prognosis. To analyze the prognostic and therapeutic factors that influence the clinical outcome of patients with HB receiving LT, we retrospectively analyzed 24 patients with HB who underwent LT between 1997 and 2015. The 5-year OS rate of all patients was 69.6±9.7%. The 5-year OS rate of 11 patients receiving salvage LT for recurrent tumor after a primary resection was comparable to that of 13 patients receiving primary LT. Among 12 evaluable patients receiving primary LT, six of 10 patients with a decline of serum AFP >95% at LT are currently alive and in remission, whereas two patients with a decline of AFP ≤95% experienced post-LT relapse. Among 9 evaluable patients receiving salvage LT, all three patients with any decline of AFP at LT are currently alive in remission, and three of six patients with no response to pre-LT salvage chemotherapy are also alive and in remission. Response to chemotherapy may be a reliable marker for prediction of post-LT relapse, even for patients receiving salvage LT.

Background Hepatoblastoma is a rare childhood malignant tumor that originates from immature hepatic cells. Aminopeptidase-N(CD13), an ectopeptidase that promotes tumor invasion and metastasis, is expressed in fetal stage hepatic progenitor cells, although its role in hepatoblastoma remains unclear. Methods The expression pattern of CD13 was investigated on immunohistochemistry in 30 tissue samples from 27 hepatoblastoma patients (16 with predominantly embryonal [pE] histology and 14 with predominantly fetal [pF] histology). Immunoreactive score (IRS) was used to quantify staining data, and the relationship between CD13 expression, clinicopathological factors, and clinical outcome was investigated. The biological function of CD13 was also examined in the hepatoblastoma cell lines Huh6 and HepG2. Results All specimens stained positive for CD13, with higher CD13 expression in pE than in pF hepatoblastoma samples (median IRS, 4; range, 2–9 vs 2; range, 1–4). Strong CD13 expression was correlated with vascular invasion. Five year event-free survival and overall survival were better in patients with CD13low than in those with CD13high tumors (100% vs 51.0%, P = 0.026; and 100% vs 74.0%, P = 0.114, respectively). A CD13-neutralizing antibody and the potent CD13 inhibitor, Ubenimex, suppressed invasive activity in HepG2 cells in vitro. Conclusions CD13 expression is associated with hepatoblastoma invasiveness and could be a novel prognostic marker for hepatoblastoma.

A 14-year-old girl with multiple intra-abdominal tumors was diagnosed with stage III Burkitt's lymphoma. She achieved complete remission after multi-drug chemotherapy, but she relapsed after six courses. Autologous peripheral blood stem cells (PBSC) or allogeneic PBSC harvested from an HLA-identical sibling were insufficient, and her family did not agree to bone marrow collection from the sibling. Although the patient relapsed nine times (the relapses involved intra-abdominal organs or bone) during the following 4 years 7 months, treatment with rituximab monotherapy or in combination with ifosphamide, carboplastin, and etoposide, or local irradiation (33.8–40.0 Gy) to treat the bone metastases, proved effective, resulting in complete or partial remission. At the time of writing, the patient was in a 10th cycle of remission lasting 1 year 6 months and had not required transplantation. Thus, a chemotherapy regimen including rituximab might be effective for Burkitt's lymphoma in patients experiencing multiple relapse.

Parosteal osteosarcoma (POS) is conventionally a low-grade sarcoma with limited metastatic potential; however, the tumor occasionally transforms into a high-grade dedifferentiated POS, which commonly metastasizes to distant organs. The present report describes a rare pediatric case of conventional POS with no dedifferentiated component yet had multiple pulmonary metastases at initial diagnosis. Following limb-sparing surgery and osteosarcoma-oriented neoadjuvant chemotherapy, the patient received total resection of pulmonary metastases. Despite no treatment for pulmonary recurrence 1 year after adjuvant chemotherapy, the patient is alive with stable disease 4 years and 6 months after the initial diagnosis.

Mucosal-associated invariant T (MAIT) and invariant natural killer T (iNKT) cells are T cell subpopulations that possess innate-like properties. We examined the impact of post-hematopoietic stem cell transplantation (HSCT) MAIT and iNKT cell recovery on the clinical outcomes of 69 patients who underwent allogeneic HSCT at Kyoto University Hospital. Multivariate analyses identified the absolute number of MAIT cells (

Leukemia is the most common pediatric cancer and accounts for approximately one third of childhood malignancies. There are germline genetic alterations that significantly increase the risk of developing hematopoietic malignancies in childhood. In this review, we describe a number of these hereditary disorders and their clinical features. These predispositions to cancer syndromes can be attributed to DNA repair/genetic instability, RAS pathway dysfunction, bone marrow failure, telomeropathies, immunodeficiencies, transcription factor abnormalities, pure familial leukemia, and aneuploidy. We focus especially on acute myeloid leukemia associated with Down syndrome, but also include other hereditary syndromes in this review. Recent advances in high-throughput genotyping technology have identified new genetic variations prone to human leukemia. Understanding of the mechanism of leukemia development in these hereditary syndromes allows us to gain insight into leukemogenesis in general and suggests therapeutic strategies based on these findings.

Introduction. Core binding factor acute myeloid leukemia (CBF-AML) is caused by the dysfunction of a heterodimeric protein complex composed of the transcription factor RUNX1 and its partner CBFb. An inversion of chromosome 16 generates a fusion between CBFB and MYH11. The encoded fusion protein, CBFb-smooth muscle myosin heavy chain (SMMHC), contributes to the pathogenesis of CBF-AML. Our previous reports suggest that CBFB-MYH11 contributes to leukemogenesis by up-regulation of genes such as Gata2, which is an essential hematopoietic transcription factor. On the other hand, we recently identified recurrent monoallelic deletions of GATA2 on chromosome 3 in relapsed CBF-AML patients (Sood et al., Leukemia 30:501-504, 2016). From these findings we propose two hypotheses; 1) up-regulation of GATA2 contributes to leukemogenesis by CBFB-MYH11 in the initiation phase; 2) GATA2 deficiency contributes to the relapse of CBF-AML. Methods. Two datasets (GSE19194 and GSE102388) from microarray and RNA-Seq were used to determine Gata2 expression level in Cbfb-MYH11 preleukemic murine hematopoietic cells. Cbfb-MYH11 conditional knock-in (Cbfb+/56M), Gata2 conditional knockout (Gata2+/f), and Mx1-Cre transgenic mice were crossed to generate Gata2+/fCbfb+/56MMx1-Cre mice. Mice were injected with pIpC to induce the expression of Cbfb-MYH11 and/or knockout of Gata2 through Cre-recombinase activation. For transplantation assays, spleen cells obtained from leukemic mice were injected into irradiated recipient mice through tail vein. For in vitro colony forming assays, colonies were counted after 10 days in culture. Cell apoptosis was determined by Annexin V and 7AAD staining. Results. To test the first hypothesis, we determined the expression level of Gata2 in preleukemic cells in the Cbfb-MYH11 expressing mice. Data from both microarray and RNA-seq experiments revealed that Gata2 was highly expressed in the preleukemic hematopoietic cells of the Cbfb-MYH11 mice, as compared to those of the WT mice, and this finding was confirmed by qRT-PCR. Based on published ChIP-seq data, Gata2 is likely a direct transcriptional target of CBFb-SMMHC. Next, we determined the impact of Gata2 deficiency on leukemogenesis by Cbfb-MYH11. qRT-PCR showed reduced Gata2 expression in bone marrow cells from Gata2+/fCbfb+/56MMx1-Cre mice 12 days after pIpC injection (0.029±0.0092 vs 0.076±0.014; p=0.0089). Colony forming ability was decreased for the pre-leukemic bone marrow cells in Gata2+/fCbfb+/56MMx1-Cre mice when compared to Cbfb+/56MMx1-Cre mice (mean 37.2±6.35 vs. 74.23±8.335; p=0.0002). In addition, the Gata2+/fCbfb+/56MMx1-Cre mice had a smaller abnormal myeloid population in the bone marrow, which is capable of inducing leukemia, when compared with Cbfb+/56MMx1-Cre mice (mean 0.43±0.14% vs. 1.42±0.34%; p=0.0092). Most significantly, Gata2+/fCbfb+/56MMx1-Cre mice developed leukemia with a much longer latency than Cbfb+/56MMx1-Cre mice (median survival 215 days vs 125 days; p=0.0007). To test hypothesis 2, we compared the phenotype of the end stage mice for each genotype. Gata2+/fCbfb+/56MMx1-Cre mice had higher WBC count in peripheral blood than Cbfb+/56MMx1-Cre mice (mean 92,000±20,429 cells/ul vs. 35,644±12,001 cells/ul; p=0.0243), which is a poor prognostic marker in human leukemia. Leukemic cells from Gata2+/fCbfb+/56MMx1-Cre mice also had lower percentage of Annexin V positive cells than Cbfb+/56MMx1-Cre mice in short term culture (31.0±7.1 vs. 68.9±6.5%; p=0.0117). More importantly, upon transplantation, the recipient mice transplanted with Gata2+/fCbfb+/56MMx1-Cre leukemia cells developed leukemia much faster than recipient mice transplanted with equal numbers of Cbfb+/56MMx1-Cre leukemia cells (median survival 35.5 vs. 91.0 days; p Conclusions. Our findings suggest that Gata2 plays important but distinct roles in two different stages of Cbfb-MYH11 leukemia. Reduction of Gata2 activity delays leukemia development in primary Cbfb-MYH11 knockin mice, while contributing to a more aggressive phenotype in leukemic phase as shown in primary leukemic mice and transplanted recipients, which may be correlated with leukemia relapse in human patients. We are analyzing data from whole exome sequencing and RNA-seq to understand the mechanism underlying the observed phenotypes, and the findings will be presented at the annual meeting. Disclosures No relevant conflicts of interest to declare.

A 5-year-old boy with glioblastoma relapsed soon after postoperative irradiation in combination with temozolomide. Second-line chemotherapy was also ineffective; therefore, the bevacizumab and irinotecan were given after a third gross-total resection of the tumor. Treatment was interrupted for 1 month due to development of posterior reversible encephalopathy syndrome, but was re-initiated at a lower dose of bevacizumab with prolonged intervals between treatments. The patient was alive and disease free 2 years after initial diagnosis. Bevacizumab and irinotecan are a promising regimen for pediatric cases of recurrent glioblastoma after gross-total resection, although the optimal treatment schedule must be determined on a patient-by-patient basis.

HAX1 was identified as the gene responsible for the autosomal recessive type of severe congenital neutropenia. However, the connection between mutations in the HAX1 gene and defective granulopoiesis in this disease has remained unclear, mainly due to the lack of a useful experimental model for this disease. In this study, we generated induced pluripotent stem cell lines from a patient presenting for severe congenital neutropenia with HAX1 gene deficiency, and analyzed their in vitro neutrophil differentiation potential by using a novel serum- and feeder-free directed differentiation culture system. Cytostaining and flow cytometric analyses of myeloid cells differentiated from patient-derived induced pluripotent stem cells showed arrest at the myeloid progenitor stage and apoptotic predisposition, both of which replicated abnormal granulopoiesis. Moreover, lentiviral transduction of the HAX1 cDNA into patient-derived induced pluripotent stem cells reversed disease-related abnormal granulopoiesis. This in vitro neutrophil differentiation system, which uses patient-derived induced pluripotent stem cells for disease investigation, may serve as a novel experimental model and a platform for high-throughput screening of drugs for various congenital neutrophil disorders in the future.

Dasatinib, a second-generation tyrosine kinase inhibitor, is a highly effective treatment for Bcr-Abl-positive leukemia. However, the mechanism by which dasatinib induces cell death is unclear, particularly in vivo. Autophagy is a lysosomal degradation mechanism essential for cell survival and differentiation. Autophagy also protects cells from the effects of drugs, including those used to treat leukemia. Here, we report that dasatinib induces autophagy in Bcr-Abl-positive leukemia cell lines and further show the induction of autophagy in an immunodeficient mouse model of human Bcr-Abl-positive leukemia with central nervous system (CNS) infiltration. Autophagy was induced in bone marrow (BM) as well as cerebrospinal fluid (CSF). This study is the first to show that autophagy induction is one of the mechanisms underlying cell death in leukemic cells that infiltrate the CNS. Thus, autophagy may represent a novel therapeutic target for the treatment of Bcr-Abl leukemia with CNS infiltration.

There are few reports on the clinical significance of MRD before HSCT in pediatric ALL. We retrospectively analyzed the clinical significance of FCM-based detection of MRD (FCM-MRD) before allogeneic HSCT in pediatric ALL. Of 38 pediatric patients who underwent allogeneic HSCT for the first time between 1998 and 2014, 33 patients were in CR and five patients were in non-CR. The CR group was further divided into two groups based on the pretransplant FCM-MRD level: the MRD(neg) (