Tomoko Yanai

Automatic detection of immature platelets for decision making regarding platelet transfusion indications for pediatric patients

Immature or reticulated platelets are known as a clinical marker of thrombopoiesis. Recently, an automatic method was established to detect reticulated platelets as immature platelet fraction (IPF) by means of hematology analyzer XE-2100. We assessed the effects of IPF detection after chemotherapy for various pediatric malignant disorders of 16 patients. Our results indicate that IPF should be considered a useful marker of imminent platelet recovery so that unnecessary platelet transfusion can be avoided.

The first report of adolescent TAFRO syndrome, a unique clinicopathologic variant of multicentric Castleman’s disease

BackgroundTAFRO syndrome is a unique clinicopathologic variant of multicentric Castleman’s disease that has recently been identified in Japan. It is characterized by a constellation of symptoms: Thrombocytopenia, Anasarca, reticulin Fibrosis of the bone marrow, Renal dysfunction and Organomegaly (TAFRO). Previous reports have shown that affected patients usually respond to immunosuppressive therapy, but the disease sometimes has a fatal course. TAFRO syndrome occurs in the middle-aged and elderly and there are no prior reports of the disease in adolescents. Here we report the first adolescent case, successfully treated with anti-IL-6 receptor antibody (tocilizumab, TCZ) and monitored with serial cytokine profiles.Case presentationA 15-year-old Japanese boy was referred to us with fever of unknown origin. Whole body computed tomography demonstrated systemic lymphadenopathy, organomegaly and anasarca. Laboratory tests showed elevated C-reactive protein and hypoproteinemia. Bone marrow biopsy revealed a hyperplastic marrow with megakaryocytic hyperplasia and mild reticulin fibrosis. Despite methylprednisolone pulse therapy, the disease progressed markedly to respiratory distress, acute renal failure, anemia and thrombocytopenia. Serum and plasma levels of cytokines, including IL-6, vascular endothelial growth factor, neopterin and soluble tumor necrosis factor receptors I and II, were markedly elevated. Repeated weekly TCZ administration dramatically improved the patient’s symptoms and laboratory tests showed decreasing cytokine levels.ConclusionTo our knowledge, this is the first report of TAFRO syndrome in a young patient, suggesting that this disease can occur even in adolescence. The patient was successfully treated with TCZ. During our patient’s clinical course, monitoring cytokine profiles was useful to assess the disease activity of TAFRO syndrome.

ASSOCIATION OF GENETIC POLYMORPHISMS WITH HEPATOTOXICITY IN PATIENTS WITH CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA OR LYMPHOMA

The objective of this study was to identify novel pharmacogenetic determinants of treatment-related hepatotoxicity during the maintenance phase in children with acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL). Although the authors first determined whether genotypes of drug-metabolizing enzymes and transporters—glutathione S-transferase (GST) genes, GSTM1 positive/null, GSTT1 positive/null and GSTP1 A313G, methylenetetrahydrofolate reductase (MTHFR) C677T, reduced folate carrier 1 (RFC1) G80A, and breast cancer resistant protein (BCRP) C421A—were associated with hepatotoxicity for 24 patients, no significant difference was detected for genotype and allelic frequencies between the patients with and those without severe treatment-related hepatotoxicity. Therefore, the authors explored potential candidate polymorphisms associated with hepatotoxicity using the Illumina Infinium HumanHap300, encompassing more than 318,000 tag single-nucleotide polymorphisms (SNPs), for 8 of 24 patients with or without severe hepatotoxicity. Genome-wide genotyping uncovered a total of 28 candidate SNPs. rs1966862, in Rho GTPase-activating protein 24 (ARHGAP24), was the most significant of the candidates, and the genotypes of rs13424027 (PARD3B), rs1156304 (KCNIP4), rs10255262 (SLC13A1), rs7403531 (RASGRP1), and rs381423 (unidentified gene) were also significantly associated with severe hepatotoxicity. This study suggested rs1966862 (ARHGAP24) and the other SNPs to be predictive factors for drug-induced hepatotoxicity during the maintenance phase in pediatric patients with ALL or LBL.

High Level of Ezrin mrna Expression in an Osteosarcoma Biopsy Sample With Lung Metastasis

Osteosarcoma (OS) remains a life-threatening malignancy and its molecular character is not fully understood. Ezrin is a cytoskeleton linker protein involved in regulating the growth and metastatic capacity of cancer cells. However, the correlation between ezrin mRNA expression and clinical severity has not yet been examined in OS biopsy samples. Furthermore, recent evidence has demonstrated that the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression is increased in human cancers of various origins, but this has not yet been examined in OS cells. To clarify the correlation between the clinical severity and the levels of ezrin and GAPDH mRNA expression, we quantified these mRNA levels in 4 pediatric OS biopsy samples using real-time quantitative polymerase chain reaction. Among these 4 samples, ezrin mRNA expression was approximately 5-fold higher in a case with lung metastasis compared with the other cases without metastasis, suggesting an association between the ezrin mRNA expression level and metastasis. On the other hand, the GAPDH mRNA expression level was not related to the clinical severity. This is the first report to demonstrate a high level of ezrin mRNA expression in an OS biopsy sample with lung metastasis.

Collaboration of cancer-associated fibroblasts and tumour-associated macrophages for neuroblastoma development.

Neuroblastoma is the most common extracranial solid tumour in children and is histologically classified by its Schwannian stromal cells. Although having fewer Schwannian stromal cells is generally associated with more aggressive phenotypes, the exact roles of other stromal cells (mainly macrophages and fibroblasts) are unclear. Here, we examined 41 cases of neuroblastoma using immunohistochemistry for the tumour‐associated macrophage (TAM) markers CD68, CD163, and CD204, and a cancer‐associated fibroblast (CAF) marker, alpha smooth muscle actin (αSMA). Each case was assigned to low/high groups on the basis of the number of TAMs or three groups on the basis of the αSMA‐staining area for CAFs. Both the number of TAMs and the area of CAFs were significantly correlated with clinical stage, MYCN amplification, bone marrow metastasis, histological classification, histological type, and risk classification. Furthermore, TAM settled in the vicinity of the CAF area, suggesting their close interaction within the tumour microenvironment. We next determined the effects of conditioned medium of a neuroblastoma cell line (NBCM) on bone marrow‐derived mesenchymal stem cells (BM‐MSCs) and peripheral blood mononuclear cell (PBMC)‐derived macrophages in vitro. The TAM markers CD163 and CD204 were significantly up‐regulated in PBMC‐derived macrophages treated with NBCM. The expression of αSMA by BM‐MSCs was increased in NBCM‐treated cells. Co‐culturing with CAF‐like BM‐MSCs did not enhance the invasive ability but supported the proliferation of tumour cells, whereas tumour cells co‐cultured with TAM‐like macrophages had the opposite effect. Intriguingly, TAM‐like macrophages enhanced not only the invasive abilities of tumour cells and BM‐MSCs but also the proliferation of BM‐MSCs. CXCL2 secreted from TAM‐like macrophages plays an important role in tumour invasiveness. Taken together, these results indicate that PBMC‐derived macrophages and BM‐MSCs are recruited to a tumour site and activated into TAMs and CAFs, respectively, followed by the formation of favourable environments for neuroblastoma progression.

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.

Sterility Testing of Stem Cell Products by Broad-Range Bacterial 16S Ribosomal DNA Polymerase Chain Reaction.

OBJECTIVE To evaluate broad-range 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) as a rapid screening tool to detect bacterial contamination of stem-cell products. METHODS We performed the evaluation using whole blood spiked with serially diluted bacterial-type strains. Detection sensitivity was defined as the bacterial concentration for which all replicates were positive at each concentration (100% detection). We tested the sterility of 29 bags of autologous peripheral blood stem cell (PBSC) products harvested at our facility using the 16S rDNA PCR method. RESULTS The detection sensitivity of 16S rDNA PCR in spiked whole blood was 10¹ to 10² colony-forming units (CFU) per mL, depending on the bacterial strain. We detected no amplified 16S rDNA among the PBSCs we used in this study. The BacT/ALERT automated bacterial culture system that we used also showed no positive signals in any of the PBSCs tested. CONCLUSIONS Our data indicate that bacterial 16S rDNA PCR is a useful alternative for rapid sterility testing, not only for blood products used in transfusion medicine but also for stem-cell products used in regenerative medicine.

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.

The emergence of CD20-/CD19- tumor cells after rituximab therapy for Epstein-Barr virus-associated post-transplant lymphoproliferative disorder complicated with hemophagocytic lymphohistiocytosis.

Post-transplant lymphoproliferative disorder (PTLD) is a well-recognized aggressive disease commonly associated with Epstein–Barr virus (EBV) infection after hematopoietic stem cell transplantation (HSCT). Although rituximab (RTX) is incorporated into the first-line therapy for EBV-PTLD patients, the outcome of the clinically overt disease is still not optimal mainly due to the regrowth of tumor cells. The proliferation of CD20−/CD19+ tumor cells is increasingly reported in RTX-treated EBV-PTLD patients, whereas the emergence of CD20−/CD19− tumor cells is barely recognized. Here, we report a fatal case of an 18-year-old patient who developed EBV-PTLD after allogeneic HSCT for anaplastic large-cell lymphoma. On day 60 after HSCT, the patient developed abdominal pain, watery diarrhea, and low-grade fever. Colon biopsy revealed the proliferation of CD20+/CD19+/EBV-encoded RNA (EBER)+ tumor cells, and an increase of EBV DNA was detected in peripheral blood (PB). He was treated with RTX for EBV-PTLD and was cleared of EBV DNA in PB. However, he manifested high-grade fever, pancytopenia, and elevated soluble interleukin-2 receptor with a prominent hemophagocytosis in bone marrow aspirates and was treated with etoposide for hemophagocytic lymphohistiocytosis (HLH) complication. He then developed EBV DNA positivity in PB and finally died of Bacteroides fragilis sepsis subsequent to bloody stool and ileus on day 163. Autopsy revealed erosion and bleeding in the whole colon with the proliferation of CD20−/CD19−/EBER+ tumor cells. Immunohistochemical analysis uncovered the CD3−/CD56−/CD79a+/CD79b+ B-cell origin of tumor cells. This case clinically demonstrates the removal of both CD20 and CD19 antigens from EBER+ B cells in an RTX-treated EBV-PTLD patient with HLH complication.