Malignant melanoma in a 12-year-old boy 17 months after completing hepatoblastoma treatment.

BACKGROUND: Melanoma is rare as a secondary malignant neoplasm among childhood cancer survivors. CASE: We report a case of a 12-year-old boy who developed malignant melanoma with systemic metastases 17 months after completing treatment for hepatoblastoma. The diagnosis was made unexpectedly based on a bone marrow examination. The patient did not respond to immune checkpoint inhibitor therapy and died 6 weeks after being diagnosed with melanoma. Whole-exome sequencing to examine 103 genes associated with cancer predisposition did not identify any germ-line variants. CONCLUSION: This case study provides a unique example of melanoma in a childhood cancer survivor following hepatoblastoma treatment but does not identify any candidate variant to link hepatoblastoma and melanoma.

Effect of Japanese Kampo medicine, eppikajutsuto, in patients with lymphatic malformation: A retrospective observational study.

ABSTRACT: Lymphatic malformations (LMs) are congenital malformations of the lymphatic system that cause considerable cosmetic and functional complications. In this study, we present 8 children with LM who were treated with the Kampo medicine eppikajutsuto (EKJT).Between 2001 and 2020, 8 children (male: 4, female: 4) with LMs who underwent magnetic resonance imaging (MRI) evaluation both before and after treatment or observation were selected for investigating the effect of EKJT. Two patients were observed without any treatment for 24 and 60 months. EKJT was evaluated based on percentage reduction, defined as the percentage of total lesions that decreased in size, confirmed by radiological examination after initiating treatment with EKJT or determined by observation alone. Volumetric analysis of LMs on MRI was performed using the Digital Imaging and Communications in Medicine viewer.Six patients were treated with EKJT. The mean observational period was 13.2 months (range: 6-24 months). The mean reduction in LM volume on MRI was 73.0% in treated patients and -66.3% in observed patients. Two of the 6 lesions exhibited complete reduction, 2 exhibited marked (>90%) reduction, 1 exhibited moderate reduction, and 1 exhibited a small response. The treatment was well-tolerated, with no severe adverse events.This preliminary study demonstrated the beneficial effects of EKJT. Prospective evaluations of this promising therapeutic modality are warranted based on the results of this study.

Diagnosis and management of genital injuries in girls: 14-year experience.

BACKGROUND: Pediatric patients with genital injuries are often recommended to receive an examination under general anesthesia; however, detailed clinical data of such patients are rarely reported. METHODS: A single-center retrospective review was conducted in 45 girls less than 16 years of age with genital injuries between January 2005 and December 2018. RESULTS: The median patient age was 5.0 years. Forty-two patients were hospitalized, of whom 38 required an examination under general anesthesia and all consequently required surgical repair. The diagnosis obtained after a thorough examination under general anesthesia was inconsistent with the diagnosis obtained at the emergency room in five patients. In 20 patients, the source of bleeding was not clarified at the time of initial examination at the emergency room; four of these patients were later revealed to have vaginal or rectal injuries that had been overlooked during the examination at the emergency room. Injuries occurring in the bathroom were the most frequent and tended to be serious. Multiple injuries were found in 10 patients. The exterior of the labia minora was the most commonly injured site, found in 18 patients. CONCLUSIONS: We analyzed the clinical data of girls with genital injuries in detail, which allowed us to find a detailed classification of injured sites and the characteristics of serious cases, and to re-recognize the importance of a thorough examination under general anesthesia.

Forced expression of NR4A3 induced the differentiation of human neuroblastoma-derived NB1 cells.

Nuclear receptor subfamily 4, group A, member 3 (NR4A3) is a member of the NR4A subgroup of orphan nuclear receptors, implicated in the regulation of diverse biological functions, including metabolism, angiogenesis, inflammation, cell proliferation, and apoptosis. Although many reports have suggested the involvement of NR4A3 in the development and/or progression of tumors, its role varies among tumor types. Previously, we reported that DNA hypomethylation at NR4A3 exon 3 is associated with lower survival rate of neuroblastoma (NB) patients. As hypomethylation of this region results in reduced expression of NR4A3, our observations suggested that NR4A3 functions as a tumor suppressor in NB. However, the exact mechanisms underlying its functions have not been clarified. In the present study, we analyzed public databases and showed that reduced NR4A3 expression was associated with shorter survival period of NB in two out of three datasets. An in vitro study revealed that forced expression of NR4A3 in human NB-derived cell line NB1 resulted in elongation of neurites along with overexpression of GAP43, one of the differentiation markers of NB. On the other hand, siRNA-mediated knockdown of NR4A3 suppressed the expression level of GAP43. Interestingly, the forced expression of NR4A3 induced only the GAP43 but not the other molecules involved in NB cell differentiation, such as MYCN, TRKA, and PHOX2B. These results indicated that NR4A3 directly activates the expression of GAP43 and induces differentiated phenotypes of NB cells, without affecting the upstream signals regulating GAP43 expression and NB differentiation.

NRP1 knockdown promotes the migration and invasion of human neuroblastoma-derived SK­N­AS cells via the activation of ß1 integrin expression.

Neuropilin 1 (NRP1) is a transmembrane glycoprotein, which regulates many aspects of cellular function by functioning as co-receptor of various ligands. Recent studies have suggested that NRP1 promotes tumorigenesis, not only by activating the growth of tumor vessels, but also by activating the growth or migration of tumor cells themselves. The present study was performed to elucidate the roles of NRP1 in the development and/or progression of neuroblastoma (NB). In contrast to previous observations in various types of cancer, the analysis of public datasets indicated that lower levels of NRP1 expression were significantly associated with a shorter survival period of patients with NB. Consistent with this finding, wound-healing assay and Matrigel invasion assay revealed that NB cells in which NRP1 was knocked down exhibited increased migratory and invasive abilities. Further analyses indicated that ß1 integrin expression was markedly increased in NB cells in which NRP1 was knocked down, and NB cells in which ß1 integrin was knocked down exhibited decreased migratory and invasive abilities. The results presented herein indicate that NRP1 exerts tumor suppressive effects in NB, at least in part by regulating the expression of ß1 integrin.

ZAR1 knockdown promotes the differentiation of human neuroblastoma cells by suppression of MYCN expression.

Although DNA hypermethylation at non-promoter region of the Zygote arrest 1 (ZAR1) gene has been observed in many types of tumor, including neuroblastoma (NB), the role of this gene in tumor development and/or progression is unclear. One reason is that knowledge about the function of ZAR1 protein is limited. Although it has been reported that ZAR1 plays a crucial role in early embryogenesis and may act as a transcriptional repressor for some transcripts, the detailed mechanism is still elusive. In the present study, we analyzed public data of NB patients and found that higher expression levels of ZAR1 were significantly associated with a shorter survival period. Consistent with this result, ZAR1-depleted NB cells showed well-differentiated phenotypes with elongated neurites and upregulated expression of TRKA and RET, which are markers for differentiated NB. Moreover, the expression level of MYCN protein was markedly suppressed in ZAR1-depleted NB cells. MYCN-depleted cells showed similar phenotypes to ZAR1-depleted cells. The present findings indicate that ZAR1 has oncogenic effects in NB by suppressing cell differentiation via regulation of MYCN expression.

Depletion of TFAP2E attenuates adriamycin-mediated apoptosis in human neuroblastoma cells.

Neuroblastoma is a childhood malignancy originating from the sympathetic nervous system and accounts for approximately 15% of all pediatric cancer-related deaths. To newly identify gene(s) implicated in the progression of neuroblastoma, we investigated aberrantly methylated genomic regions in mouse skin tumors. Previously, we reported that TFAP2E, a member of activator protein-2 transcription factor family, is highly methylated within its intron and its expression is strongly suppressed in mouse skin tumors compared with the normal skin. In the present study, we analyzed public data of neuroblastoma patients and found that lower expression levels of TFAP2E are significantly associated with a shorter survival. The data indicate that TFAP2E acts as a tumor suppressor of neuroblastoma. Consistent with this notion, TFAP2E-depleted neuroblastoma NB1 and NB9 cells displayed a substantial resistance to DNA damage arising from adriamycin (ADR), cisplatin (CDDP) and ionizing radiation (IR). Silencing of TFAP2E caused a reduced ADR-induced proteolytic cleavage of caspase-3 and PARP. Of note, compared with the untransfected control cells, ADR-mediated stimulation of CDK inhibitor p21WAF1 was markedly upregulated in TFAP2E­knocked down cells. Therefore, our present findings strongly suggest that TFAP2E has a pivotal role in the regulation of DNA damage response in NB cells through the induction of p21WAF1.

Pyrrole-imidazole polyamide-mediated silencing of KCNQ1OT1 expression induces cell death in Wilms' tumor cells.

KvDMR (an intronic CpG island within the KCNQ1 gene) is one of the imprinting control regions on human chromosome 11p15.5. Since KvDMR exists within the promoter region of KCNQ1OT1 (antisense transcript of KCNQ1), it is likely that genomic alterations of this region including deletion, paternal uniparental disomy and de-methylation in maternal allele lead to aberrant overexpression of KCNQ1OT1. Indeed, de-methylation of KvDMR accompanied by uncontrolled overexpression of KCNQ1OT1 occurs frequently in Beckwith-Wiedemann syndrome (BWS), and around 10% of BWS patients developed embryonal tumors (Wilms' tumor or hepatoblastoma). These observations strongly suggest that silencing of KCNQ1OT1 expression might suppress its oncogenic potential. In the present study, we designed two pyrrole-imidazole (PI) polyamides, termed PI-a and PI-b, which might have the ability to bind to CCAAT boxes of the KCNQ1OT1 promoter region, and investigated their possible antitumor effect on Wilms' tumor-derived G401 cells. Gel retardation assay demonstrated that PI-a and PI-b specifically bind to their target sequences. Microscopic observations showed the efficient nuclear access of these PI polyamides. Quantitative real-time PCR analysis revealed that the expression level of KCNQ1OT1 was significantly decreased when treated with PI-a and PI-b simultaneously but not with either PI-a or PI-b single treatment. Consistent with these results, the combination of PI-a and PI-b resulted in a significant reduction in viability of G401 cells in a dose-dependent manner. Furthermore, FACS analysis demonstrated that combinatory treatment with PI-a and PI-b induces cell death as compared with control cells. Taken together, our present observations strongly suggest that the combinatory treatment with PI polyamides targeting KCNQ1OT1 might be a novel therapeutic strategy to cure patients with tumors over-expressing KCNQ1OT1.

Non-promoter DNA hypermethylation of Zygote Arrest 1 (ZAR1) in neuroblastomas.

BACKGROUND: The comprehensive methylation analysis of tumor-specific differently methylated regions in malignant melanomas and brain tumors has led to the identification of non-promoter hypermethylation of zygote arrest 1 (ZAR1). To search the non-promoter ZAR1 hypermethylation in neuroblastomas, we analyzed the levels of the methylation and transcript expression of ZAR1. METHODS: The MassARRAY® EpiTYPER (Sequenom Inc., San Diego, CA, USA) system was optimized to determine the quantitative methylation levels of ZAR1 for 12 neuroblastoma cell lines, 23 neuroblastoma samples and four adrenal samples. ZAR1 expression levels were evaluated through a quantitative, real-time reverse transcription-polymerase chain reaction. The quantitative methylation levels of ZAR1 were subjected to correlation studies with the established markers of progressive disease and outcome. RESULTS: Strikingly, the hypermethylation of ZAR1 regions and ZAR1 expression levels was observed in the neuroblastoma cell lines and neuroblastoma samples, compared to the adrenal samples. Somatic changes in ZAR1 methylation and ZAR1 expression were found in all three neuroblastoma patients. In the ZAR1 regions, poor-outcome tumors that were MYCN-amplified and/or Stage 3 or 4 and/or the age at diagnosis was≥18months, and/or showed an unfavorable histology were frequently hypermethylated. CONCLUSION: Our results indicate that the hypermethylation of ZAR1 regions is extremely frequent in neuroblastomas and correlates with established markers of progressive disease and outcome.

Identification of aberrant methylation regions in neuroblastoma by screening of tissue-specific differentially methylated regions.

BACKGROUND: The identification of tissue-specific differentially methylated regions (tDMRs) is key to our understanding of mammalian development. Research has indicated that tDMRs are aberrantly methylated in cancer and may affect the oncogenic process. PROCEDURE: We used the MassARRAY EpiTYPER system to determine the quantitative methylation levels of seven neuroblastomas (NBs) and two control adrenal medullas at 12 conserved tDMRs. A second sample set of 19 NBs was also analyzed. Statistical analysis was carried out to determine the relationship of the quantitative methylation levels to other prognostic factors in these sample sets. RESULTS: Screening of 12 tDMRs revealed 2 genomic regions (SLC16A5 and ZNF206) with frequent aberrant methylation patterns in NB. The methylation levels of SLC16A5 and ZNF206 were low compared to the control adrenal medullas. The SLC16A5 methylation level (cut-off point, 13.25%) was associated with age at diagnosis, disease stage, and Shimada classification but not with MYCN amplification. The ZNF206 methylation level (cut-off point, 68.80%) was associated with all of the prognostic factors analyzed. Although the methylation levels at these regions did not reach statistical significance in their association with prognosis in mono-variant analysis, patients with both hypomethylation of SLC16A5 and hypermethylation of ZNF206 had a significantly prolonged event-free survival, when these two variables were analyzed together. CONCLUSIONS: We demonstrated that two tDMRs frequently displayed altered methylation patterns in the NB genome, suggesting their distinct involvement in NB development/differentiation. The combined analysis of these two regions could serve as a diagnostic biomarker for poor clinical outcome.