Keiichiro Kawasaki

Quantitative assessment of PTPN11 or RAS mutations at the neonatal period and during the clinical course in patients with juvenile myelomonocytic leukaemia.

To evaluate minimal residual disease (MRD) after chemotherapy and haematopoietic stem cell transplantation in juvenile myelomonocytic leukaemia (JMML), a locked nucleic acid‐allele specific quantitative polymerase chain reaction (LNA‐AS‐qPCR) was developed for 13 patients (four types of PTPN11 mutation and four types of RAS mutation). The post‐transplant MRD detected by LNA‐AS‐qPCR analysis was well correlated with chimerism assessed by short tandem repeat PCR analysis. Non‐intensive chemotherapy exerted no substantial reduction of the tumour burden in three patients. There was no significant difference in the quantity of RAS mutant DNA after spontaneous haematological improvement in 4 patients with NRAS or KRAS 34G > A during a 2‐ to 5‐year follow‐up. PTPN11, NRAS, or KRAS mutant DNA was detected from Guthrie card dried blood in five of seven patients (who were aged <2 years at diagnosis) at a level of 1·0–6·5 × 10−1 of the values at diagnosis. Accordingly, these five patients might have already reached a subclinical status at birth. Considering the negative correlation between mutant DNA level in neonatal blood spots and age at diagnosis, JMML patients with a larger tumour burden at birth appeared to show earlier onset.

Rab15 expression correlates with retinoic acid-induced differentiation of neuroblastoma cells

Neuroblastoma is the most common extracranial solid tumor in children and accounts for 15% of pediatric cancer deaths. Although retinoic acid (RA) is currently used to treat high-risk neuroblastoma patients in the clinic, RA-responsiveness is variable and unpredictable. Since no alterations in the RA-signaling pathway have been found in neuroblastoma cells, molecules correlated with RA-induced differentiation will provide predictive markers of RA-responsiveness for clinical use. The Rab family of small G proteins are key regulators of membrane traffic and play a critical role in cell differentiation and cancer progression. Although an increasing number of cancer-associated alternative splicing events have been identified, alternative splicing of Rab proteins remains to be characterized in neuroblastoma. In the present study, we focused on Rab15 that was originally identified as a brain-specific Rab protein and regulates the endocytic recycling pathway. We identified alternatively spliced Rab15 isoforms designated as Rab15CN and Rab15AN in neuroblastoma cells. Rab15CN was composed of 7 exons encoding 212 amino acids and showed brain-specific expression. Alternative splicing of exon 4 generated Rab15AN that was predicted to encode 208 amino acids and was predominantly expressed in testis. RA induced neuronal differentiation of neuroblastoma BE(2)-C cells and specifically up-regulated Rab15CN expression. Reciprocally, RA-induced differentiation was observed in Rab15CN-expressing BE(2)-C cells in preference to Rab15AN-expressing BE(2)-C cells. Furthermore, Rab15CN expression was also specifically up-regulated during RA-induced differentiation of newly established neuroblastoma cells from high-risk patients. These results suggest that Rab15 expression correlates with RA-induced differentiation of neuroblastoma cells.

Rab15 alternative splicing is altered in spheres of neuroblastoma cells

Neuroblastoma is an aggressive pediatric tumor that accounts for 15% of cancer-related deaths in children. More than half of high-risk neuroblastoma patients develop tumor relapse that is lethal in most cases. A small population of tumor-initiating cells (TICs), recently identified from high-risk neuroblastoma patients as spheres, is believed to be responsible for tumor relapse. Rab family small G proteins are essential in controlling membrane traffic and their misregulation results in several cancers. Rab15 was originally isolated as a brain-specific Rab protein regulating the endocytic recycling pathway and was recently identified as a downstream target of the neural transcription factor Atoh1. Previously, we identified two alternatively spliced Rab15 isoforms in neuroblastoma cells and showed a significant correlation between Rab15 expression and neuronal differentiation. As aberrant alternative splicing is intimately associated with an increasing number of cancers, its use as a new diagnostic and/or prognostic biomarker has attracted considerable attention. In the present study, we explored cancer-associated changes of Rab15 alternative splicing in neuroblastoma TICs. We found that Rab15 alternative splicing generated two novel isoforms designated as Rab15(AN2) and Rab15(AN3) in addition to two known isoforms designated as Rab15(CN) and Rab15(AN1). Although both Rab15(AN2) and Rab15(AN3) contained premature termination codons, they were detected in not only neuroblastoma cells but also in normal human tissues. One isoform was predominantly expressed in the brain and testis, while the other isoform was more specifically expressed in the brain. In neuroblastoma, Rab15 isoform balance measured by the Rab15(CN)/Rab15(AN1+AN2+AN3) ratio was significantly decreased in spheres compared to parental cells. These results suggest that Rab15 alternative splicing may serve as a biomarker to discriminate TICs from non-TICs in neuroblastoma.

Persistent detection of a novel MLL-SACM1L rearrangement in the absence of leukemia

Most chromosomal rearrangements including the mixed lineage leukemia (MLL) gene are manifested as leukemia and predict a poor prognosis. Although more than 50 MLL-rearrangement partners are characterized, MLL-related leukemogenesis remains to be understood. Here we report a case of a 3-year old boy bearing a novel MLL-rearrangement with the suppressor of actin mutations 1-like (SACM1L) gene in the absence of leukemia. Bone marrow cells harboring the MLL-SACM1L rearrangement appeared during chemotherapy for acute lymphoblastic leukemia with hyperdiploidy and were continuously detected over 7 years without clonal expansion.

Prevention of chemotherapy-induced vomiting in children receiving multiple-day cisplatin chemotherapy: A hospital-based, retrospective cohort study: Kishimoto et al.

Optimal prevention of chemotherapy‐induced vomiting (CIV) has not been established for patients receiving cisplatin in divided doses. The aim of this study was to describe the incidence and risk factors of CIV in children who received multiple‐day cisplatin chemotherapy.

Familial Hemophagocytic Lymphohistiocytosis Presenting as Hydrops Fetalis.

Background Familial hemophagocytic lymphohistiocytosis (FLH) is an autosomal recessive disorder of immune regulation that leads to a hyperinflammatory syndrome. Fetal onset FHL is extremely rare and is considered to be the most severe form of FHL. Case We report a preterm case of FHL that presented as hydrops fetalis. The infant was treated with a chemotherapy regimen based on the HLH-2004 protocol from the third day of life. However, he had persistent cytopenia and died on the 18th day of life due to bacteremia. The detection of defective perforin expression in the patients natural killer cells and mutations in the PRF1 gene resulted in a molecular diagnosis of FHL. Conclusion We suggest that early diagnosis and the development of an appropriate immunosuppressive strategy that can induce and maintain remission until hematopoietic stem cell transplantation can be performed are required to improve the outcomes of fetal onset FHL.

Fatal degeneration of specialized cardiac muscle associated with chronic active Epstein–Barr virus infection

Daiichiro Hasegawa, Michiko Kaji, Hiroki Takeda, Keiichiro Kawasaki, Hironobu Takahashi, Hiroshi Ochiai, Tomohiro Morio, Yasuhiro Omori, Hiroshi Yokozaki and Yoshiyuki Kosaka Department of Hematology and Oncology, Kobe Children’s Hospital, Division of Pathology, Kobe University Graduate School of Medicine, Kobe, Department of Pediatrics, Himeji Red Cross Hospital, Hyogo and Center for Cell Therapy, Tokyo Medical and Dental University, Tokyo, Japan

Successful Treatment of Transplantation-associated Atypical Hemolytic Uremic Syndrome With Eculizumab

We herein reported a 4-month-old boy with transplantation-associated atypical hemolytic uremic syndrome (TA-aHUS) who was successfully treated with eculizumab. The patient diagnosed with type 3 of familial hemophagocytic lymphohistiocytosis underwent cord blood transplantation. After transplantation, he developed TA-aHUS, but plasma exchanges were unsuccessful. We identified deletions in CFH-related gene 1 (del-CFHR1) by the multiplex ligation-dependent probe amplification testing procedure and CFH autoantibodies. Eculizumab has been administered to the patient, with a marked improvement being achieved in thrombocytopenia. He has been well except for the persistent microhematuria for a year after transplantation. Uncontrolled complement activation might be involved in the pathophysiology of TA-aHUS.

Reemergence of translocation t(11;19)(q23;p13.1) in the absence of clinically overt leukemia

We report the case of a 10-year-old female with acute myeloid leukemia (AML) FAB M0 carrying a novel t(11;19)(q23;p13.1) MLL–ELL variant, in which intron 8 of MLL is fused to exon 6 of ELL. Complete remission, judged by morphology and cytogenetic analysis, was achieved after the conventional chemotherapy. Eight months after completion of therapy, the level of WT-1 in peripheral blood and the number of cells with the MLL–ELL fusion transcript resurged. However, the patient remained overtly healthy and the morphology in the bone-marrow smear was innocuous, with no sign of relapse or secondary leukemia. Without any evidence of relapse, the patient has been closely observed without any therapeutic intervention. For approximately 2 years after the completion of therapy, despite clonal proliferation of pre-leukemic cells with an MLL–ELL fusion gene, she has maintained complete remission. In this case, the rare variant form of MLL–ELL fusion that has been identified may be related to diminished leukemogenic capacity, resulting in the persistence of pre-leukemic status; an additional genetic abnormality may thus be necessary for full transformation of pre-leukemic cells.

Congenital immature pure erythroid leukemia with E-cadherin expression

Congenital pure erythroid leukemia is exceedingly rare and poses a diagnostic challenge. We report an atypical case of congenital pure erythroid leukemia that did not express typical erythroid markers. The patient presented with a high white blood cell count with blastic cells at birth. Although flow cytometric analyses of peripheral blood and bone marrow showed a large CD45-negative cell population, we did not identify any evidence of monoclonality. While the circulating blasts decreased with only supportive care, hepatomegaly with multiple nodules was accompanied by liver failure, disseminated intravascular coagulation, and development of hemophagocytic lymphohistiocytosis. Pathological examination of the liver biopsy specimen revealed a small round cell tumor that was negative for nearly all hematopoietic cell markers, including classical erythroid cell markers, and positive for CD43, CD71, and E-cadherin, an early erythroid marker epithelial calcium-dependent adhesion protein, suggesting that these tumor cells originated from an immature erythroblast. We found high β-catenin and c-Myc protein expression, which were not previously described in pure erythroid leukemia. Cytosine arabinoside temporarily alleviated clinical symptoms; however, the patient died of progressive disease at 8 months of age. This case indicates that E-cadherin is useful for diagnosing pure erythroid leukemia, even in immature cases.