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•We identified a novel miRNA signature in patients with BCP-ALL at high risk of relapse, which is independent of known genetic subtypes.•This miRNA signature is often newly acquired at relapse and is associated with the upregulation of MYC target genes.

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 (METamplification in addition to TP53, SMAD4, and CTNNA1mutations) that might be associated with a poor prognosis. Based on high-level METamplification, 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.

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 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.

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 gene in preleukemic Cbfb-MYH11knockin mice and is expressed in human inv(16) AML. On the other hand, we have also identified recurrent monoallelic deletions of GATA2in relapsed human CBF-AML patients. To clarify the role of Gata2in leukemogenesis by Cbfb-MYH11, we generated conditional Cbfb-MYH11knockin mice with Gata2heterozygous knockout. Gata2heterozygous knockout reduced abnormal myeloid progenitors, which are capable of inducing leukemia in the Cbfb-MYH11mice. Consequently, Cbfb-MYH11mice with Gata2heterozygous knockout developed leukemia with longer latencies than those with intact Gata2. Interestingly, leukemic cells with Gata2heterozygous knockout gained higher number of mutations and showed more aggressive phenotype in both primary and transplanted mice. Moreover, leukemic cells with Gata2heterozygous knockout showed higher repopulating capacity in competitive transplantation experiments. In summary, reduction of Gata2activity affects mutational dynamics of leukemia with delayed leukemia onset in Cbfb-MYH11knockin mice, but paradoxically results in a more aggressive leukemia phenotype, which may be correlated with leukemia relapse or poor prognosis in human 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.

•This study provides an overall picture of driver mutations in ML-DS, including novel and frequent IRX1and ZBTB7Amutations, and RUNX1-PTD.•CDKN2Aand TP53deletions and/or mutations are associated with a poor prognosis in ML-DS.

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.

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.

Transient abnormal myelopoiesis (TAM) is a clonal preleukemic disorder that progresses to myeloid leukemia of Down syndrome (ML-DS) through the accumulation of genetic alterations. To investigate the mechanism of leukemogenesis in this disorder, a xenograft model of TAM was established using NOD/Shi-scid, interleukin (IL)-2Rγnull mice. Serial engraftment after transplantation of cells from a TAM patient who developed ML-DS a year later demonstrated their self-renewal capacity. A GATA1 mutation and no copy number alterations (CNAs) were detected in the primary patient sample by conventional genomic sequencing and CNA profiling. However, in serial transplantations, engrafted TAM-derived cells showed the emergence of divergent subclones with another GATA1 mutation and various CNAs, including a 16q deletion and 1q gain, which are clinically associated with ML-DS. Detailed genomic analysis identified minor subclones with a 16q deletion or this distinct GATA1 mutation in the primary patient sample. These results suggest that genetically heterogeneous subclones with varying leukemia-initiating potential already exist in the neonatal TAM phase, and ML-DS may develop from a pool of such minor clones through clonal selection. Our xenograft model of TAM may provide unique insight into the evolutionary process of leukemia.

Transient abnormal myelopoiesis (TAM) is a clonal preleukemic disorder that progresses to myeloid leukemia of Down syndrome (ML-DS) through the accumulation of genetic alterations. To investigate the mechanism of leukemogenesis in this disorder, a xenograft model of TAM was established using NOD/Shi-scid, interleukin (IL)-2Rγnullmice. Serial engraftment after transplantation of cells from a TAM patient who developed ML-DS a year later demonstrated their self-renewal capacity. A GATA1mutation and no copy number alterations (CNAs) were detected in the primary patient sample by conventional genomic sequencing and CNA profiling. However, in serial transplantations, engrafted TAM-derived cells showed the emergence of divergent subclones with another GATA1mutation and various CNAs, including a 16q deletion and 1q gain, which are clinically associated with ML-DS. Detailed genomic analysis identified minor subclones with a 16q deletion or this distinct GATA1mutation in the primary patient sample. These results suggest that genetically heterogeneous subclones with varying leukemia-initiating potential already exist in the neonatal TAM phase, and ML-DS may develop from a pool of such minor clones through clonal selection. Our xenograft model of TAM may provide unique insight into the evolutionary process of leukemia.

Drug-induced hypersensitivity syndrome (DIHS) is a rare but severe disorder due to a systemic hypersensitivity reaction. We report on a case with DIHS-like symptoms following human herpes virus 6 (HHV-6) infection complicated with encephalopathy.

•This study analyzed lymphapheresis procedures for CAR-T cell therapy.•High CD3+cell and platelet counts and anemia reduced efficiency of lymphapheresis.•A clinically applicable model for efficient lymphapheresis is newly established in this study.

Introduction

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.

We studied the relationship between the mucociliary transport time and the ciliary beat frequency in the chicken nares following water deprivation and topical administrations of cocaine, atropine, and Newcastle disease virus (NDV). Dehydration produced progressive decelerations of mucociliary flow but not ciliary beat frequency. In vivo 5% cocaine resulted in decreased sinus mucociliary transport time, while application of 20% cocaine to in vitro tissues caused ciliary paralysis within 5 min. Topical atropine was seen to affect both mucociliary flow and the ciliary beat frequency. NDV infection induced deceleration of the turbinate clearance time after 72 h, but did not change ciliary beat frequency. By day 12, mucociliary flow remained unchanged although ciliary beat frequency decreased significantly. Our results support the general concept that mucus is more important than are the cilia in normal nasal mucociliary function.

The influence of lipopolysaccharide (LPS) of Haemophilus influenzaeon the ciliary activity of human nasal mucosa was first studied using both a photo-oscillographic apparatus and by bullfrog palate clearance. Neither modification of cilialy activity nor change in bullfrog palate clearance was observed in the early phase after the administration of LPS.

The influence of lipopolysaccharide (LPS) of Haemophilus influenzae on the ciliary activity of human nasal mucosa was first studied using both a photo-oscillographic apparatus and by bullfrog palate clearance. Neither modification of cilialy activity nor change in bullfrog palate clearance was observed in the early phase after the administration of LPS.

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-MYH11contributes 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 GATA2on 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 GATA2contributes to leukemogenesis by CBFB-MYH11in the initiation phase; 2) GATA2deficiency contributes to the relapse of CBF-AML.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Transient abnormal myelopoiesis (TAM) is a clonal pre-leukemic disorder in neonates of Down syndrome. Although self-limiting in a majority of cases, TAM may evolve as non-self-limiting acute megakaryoblastic leukemia (DS-AMKL) after spontaneous remission. As such, the study of TAM/DS-AMKL can provide insight into the mechanisms of leukemic evolution. Our recent study has shown that DS-AMKL from TAM is shaped by acquisition of new mutations and clonal selection, and genomic mutations in epigenetic regulators frequently preceded other driver mutations. In this study, we address biological significance of the mutations in a progression from TAM to DS-AMKL using a newly established xenograft model of malignant conversion of the leukemia.

No relevant conflicts of interest to declare.

Abstract 2577

Acute lymphoblastic leukemia (ALL) is the most common type of childhood hematologic malignancy. Although improvements in treatment regimen have raised the 5-year survival rate as high as 80% for pediatric ALL patients, a minority of patients with various risk factors, including central nervous system (CNS) infiltration continue to have poor prognosis. Recently, bone marrow (BM) microenvironments which support leukemic stem cells have become noticed as an important element which can influence treatment response and relapse of the disease. Although leukemic cells appear to be completely eradicated through treatment, they are thought to survive within bone marrow and/or extramedullary microenvironments, such as CNS, causing disease recurrence. However, little is known about the CNS microenvironment for leukemic cells because of the lack of appropriate animal model. Even though several investigators have tried to establish a CNS infiltrated model of leukemia, major limitation with these studies are the use of leukemic cell lines and the preconditioning of recipient mice, which did not represent CNS leukemia observed in patients. Here we report the establishment of a novel xenograft model for primary human ALL using NOD/SCID/γc null (NOG) mouse. Without irradiation, this model recapitulates CNS as well as extramedullary leukemic infiltration (hereby referred to as the h-leukemic NOG model).Primary bone marrow samples were collected from 9 children with ALL at the time of diagnosis with informed consent. The leukemic cells (1×106cells) were injected into the tail veins of non-irradiated 8- to 10-week old NOG mice. Primary samples from 8 out of 9 patients were successfully engrafted. Engrafted leukemic cells could be serially transplanted into secondary, tertiary and quaternary recipients. Morphological and FACS analyses revealed as high as 95% BM chimerism and showed that blast phenotypes were conserved through serial transplantations. Of note, extramedullary organs including the CNS, liver, spleen, and kidneys showed the leukemic invasion consistent with those of the donor ALL patients. Liver pathology in the h-leukemic NOG model is identical to that seen in the ALL patients. We also showed the existence of a functional niche in the liver mediated by SDF-1/CXCR4 axis. In terms of the CNS involvement, we observed the progressive infiltration of leukemic cells into the Virchow-Robin space that is consistent with the pathology of human ALL patients. Using this model, we examined the mechanism of dissemination and harboring of leukemic cells in the CNS niche.NOG mice model for engraftment of human leukemic cells provides useful insights into the biology of ALL and allows us to answer various questions concerning the mechanism of extramedullary invasion and expansion. We have reported that NOG mice have significantly better human hematopoietic cell engraftment in the BM and extramedullary organs than other immunodeficient mice (Hiramatsu H. Blood. 2003), and is capable of supporting the growth of human neoplastic cells (Kato M. Nature. 2009). Here we report that this non-preconditioned mouse xenograft model reproduces leukemic extramedullary involvement, including the CNS, in sustaining leukemic cells. This approach provides a more sophisticated and physiological model suitable for the evaluation of molecular interactions between patient leukemic cells and host niche. Our h-leukemic NOG model will provide a powerful tool to analyze the CNS niche that harbors leukemia initiating cells. Moreover, this model would be a useful platform for developing novel anti-leukemic therapies that target CNS extramedullary niche.No relevant conflicts of interest to declare.

Abstract 3248

Acute lymphoblastic leukemia (ALL) is the most common type of childhood hematologic malignancy. Although the accumulated progresses in treatment regimen have raised the 5-year survival rate as high as 80% for whole pediatric patients, only poor prognosis, an overall survival rate of 30%, can be still now expected for the patients with relapsed diseases. Widespread extramedullary involvement such as liver, spleen, lymph nodes and central nervous system invasion is a well-known characteristic of ALL related to poor prognosis. Recently, bone marrow (BM) microenvironments supporting leukemic cells have been widely noticed as an important element which influences on treatment response and relapse of disease. Although the mechanism of extramedullary dissemination has been the most crucial issues in the study of leukemia, it still remains incompletely understood. In this study, we established a novel murine model of human ALL with NOD/SCID/γc null (NOG) mouse. Using this model, we examined the involvement of SDF-1-CXCR4 signaling axis in hepatomegary development in ALL.Primary bone marrow samples were collected from 13 children with ALL at the time of diagnosis with informed consent. The leukemic cells (1×106cells) were injected into the tail veins of non-irradiated 8- to 10-week old NOD/SCID/γc null (NOG) mice, a transgenic mouse with severe combined immunodeficiency and IL-2 receptor chain allelic mutation showing high potential to reconstitute the normal human hematopoietic system. Primary samples from 10 out of 13 patients were successfully engrafted into mice without any conditioning such as prior irradiation and DNA-damaging agents medication, and those engrafted leukemic cells were able to be serially transplanted into secondary, tertiary and quaternary recipients. Morphological and FACS analyses revealed as high as >80% blood chimerism and conserved blast phenotypes through serial transplantations. Moreover, extramedullary organs including liver, spleen and kidneys showed the leukemic invasion consistent with donor ALL disease. In contrast, no normal human hematopoiesis was observed in our xenotransplantation system without conditioning.CXCR4 is a known regulator of lymphocyte migration and has been suggested to be important for proliferation of normal leucocytes and leukemic cells. CXCR4 expression and function of leukemic cells in NOG mice were confirmed by flow cytometry and in vitro chemotaxis assays towards its known chemokine ligand SDF-1. Immunohistorical analysis of liver reveals that SDF-1 was detectable only in biliary duct endotherial cells. Finally, we demonstrated directly the effect of SDF-1-CXCR4 axis in our model by using the CXCR4 inhibitor AMD3100 in vivo and in vitro.NOG mice model for engraftment of human leukemic cells provides significant insights into the biology of ALL and allows us to answer various questions concerning the molecular mechanism of extramedullaly invasion. This non-conditioning approach may prevent possible damage to the host microenvironment, thereby providing a more natural model for growth of human leukemic cells in mice. Our present study on the involvement of SDF-1-CXCR4 axis in ALL dissemination could rink to the novel therapies in future which target the extramedullary sites in order to perfectly overcome ALL.No relevant conflicts of interest to declare.

Abstract 981