Ryota Souzaki

Integrated genetic and epigenetic analysis defines novel molecular subgroups in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood. Here we studied 60 RMSs using whole-exome/-transcriptome sequencing, copy number (CN) and DNA methylome analyses to unravel the genetic/epigenetic basis of RMS. On the basis of methylation patterns, RMS is clustered into four distinct subtypes, which exhibits remarkable correlation with mutation/CN profiles, histological phenotypes and clinical behaviours. A1 and A2 subtypes, especially A1, largely correspond to alveolar histology with frequent PAX3/7 fusions and alterations in cell cycle regulators. In contrast, mostly showing embryonal histology, both E1 and E2 subtypes are characterized by high frequency of CN alterations and/or allelic imbalances, FGFR4/RAS/AKT pathway mutations and PTEN mutations/methylation and in E2, also by p53 inactivation. Despite the better prognosis of embryonal RMS, patients in the E2 are likely to have a poor prognosis. Our results highlight the close relationships of the methylation status and gene mutations with the biological behaviour in RMS.

EGFR Tyrosine Kinase Inhibition Worsens Acute Lung Injury in Mice with Repairing Airway Epithelium

RATIONALE Epidermal growth factor receptor (EGFR) and its ligands play important roles in the regeneration of damaged epithelium and proliferation of various epithelial tumors. Although the EGFR-tyrosine kinase inhibitor gefitinib is effective against advanced non-small cell lung cancer with EGFR mutations, some patients treated with this agent develop severe acute interstitial pneumonia. Characteristics of patients who develop interstitial pneumonia include older age, smoking history, and preexisting interstitial pneumonia suggesting a connection between airway injury and alveolar dysfunction. OBJECTIVES The purpose of this study was to investigate the effects of gefitinib on airway repair after injury. METHODS C57BL/6J mice received intraperitoneally naphthalene at Day 0. Gefitinib (20, 90, or 200 mg/kg) was given daily at Days--1 to 13 after naphthalene administration. Bronchoalveolar lavage fluid and lung tissue were obtained at Days 7 and 14. Terminal bronchial epithelial cells from Days 7 and 14 were retrieved with laser capture microdissection, and gene expression analyzed using microarray. MEASUREMENTS AND MAIN RESULTS Gefitinib treatment after naphthalene prolonged neutrophil sequestration and worsened acute lung injury. We found 17 genes with more than a threefold increase in bronchiolar epithelial cells from mice treated with 200 mg/kg of gefitinib after naphthalene at Day 14 compared with those treated with naphthalene alone. Up-regulated genes included S100A8, S100A6, and StefinA3. These genes are known to participate in neutrophil sequestration, acute inflammation, and airway remodeling. CONCLUSIONS EGFR inhibition in repairing airway epithelial cells modulated significant expression of genes involved in the airway microenvironment, prolonged inflammation, and potentiated acute lung injury.

Three-dimensional liver model based on preoperative CT images as a tool to assist in surgical planning for hepatoblastoma in a child

The patient is a 3-year-old female diagnosed with PRETEXT IV hepatoblastoma (HB). Although the tumor was decreased after the neoadjuvant chemotherapy, HB still located at the porta hepatis. The patient underwent extended left lobectomy successfully after surgical simulation using three-dimensional (3D) printing liver model based on preoperative CT.

An augmented reality navigation system for pediatric oncologic surgery based on preoperative CT and MRI images.

PURPOSE In pediatric endoscopic surgery, a limited view and lack of tactile sensation restrict the surgeons abilities. Moreover, in pediatric oncology, it is sometimes difficult to detect and resect tumors due to the adhesion and degeneration of tumors treated with multimodality therapies. We developed an augmented reality (AR) navigation system based on preoperative CT and MRI imaging for use in endoscopic surgery for pediatric tumors. METHODS The patients preoperatively underwent either CT or MRI with body surface markers. We used an optical tracking system to register the reconstructed 3D images obtained from the CT and MRI data and body surface markers during surgery. AR visualization was superimposed with the 3D images projected onto captured live images. Six patients underwent surgery using this system. RESULTS The median age of the patients was 3.5 years. Two of the six patients underwent laparoscopic surgery, two patients underwent thoracoscopic surgery, and two patients underwent laparotomy using this system. The indications for surgery were local recurrence of a Wilms tumor in one case, metastasis of rhabdomyosarcoma in one case, undifferentiated sarcoma in one case, bronchogenic cysts in two cases, and hepatoblastoma in one case. The average tumor size was 22.0±14.2 mm. Four patients were treated with chemotherapy, three patients were treated with radiotherapy before surgery, and four patients underwent reoperation. All six tumors were detected using the AR navigation system and successfully resected without any complications. CONCLUSIONS The AR navigation system is very useful for detecting the tumor location during pediatric surgery, especially for endoscopic surgery.

Comprehensive analyses of imprinted differentially methylated regions reveal epigenetic and genetic characteristics in hepatoblastoma

BackgroundAberrant methylation at imprinted differentially methylated regions (DMRs) in human 11p15.5 has been reported in many tumors including hepatoblastoma. However, the methylation status of imprinted DMRs in imprinted loci scattered through the human genome has not been analyzed yet in any tumors.MethodsThe methylation statuses of 33 imprinted DMRs were analyzed in 12 hepatoblastomas and adjacent normal liver tissue by MALDI-TOF MS and pyrosequencing. Uniparental disomy (UPD) and copy number abnormalities were investigated with DNA polymorphisms.ResultsAmong 33 DMRs analyzed, 18 showed aberrant methylation in at least 1 tumor. There was large deviation in the incidence of aberrant methylation among the DMRs. KvDMR1 and IGF2-DMR0 were the most frequently hypomethylated DMRs. INPP5Fv2-DMR and RB1-DMR were hypermethylated with high frequencies. Hypomethylation was observed at certain DMRs not only in tumors but also in a small number of adjacent histologically normal liver tissue, whereas hypermethylation was observed only in tumor samples. The methylation levels of long interspersed nuclear element-1 (LINE-1) did not show large differences between tumor tissue and normal liver controls. Chromosomal abnormalities were also found in some tumors. 11p15.5 and 20q13.3 loci showed the frequent occurrence of both genetic and epigenetic alterations.ConclusionsOur analyses revealed tumor-specific aberrant hypermethylation at some imprinted DMRs in 12 hepatoblastomas with additional suggestion for the possibility of hypomethylation prior to tumor development. Some loci showed both genetic and epigenetic alterations with high frequencies. These findings will aid in understanding the development of hepatoblastoma.

High EGFR mRNA expression is a prognostic factor for reduced survival in pancreatic cancer after gemcitabine-based adjuvant chemotherapy

Pancreatic ductal adenocarcinoma (PDAC) still presents a major therapeutic challenge and a phase III clinical trial has revealed that the combination of gemcitabine and a human epidermal growth factor receptor type I (HER1/EGFR) targeting agent presented a significant benefit compared to treatment with gemcitabine alone. The aim of this study was to investigate EGFR mRNA expression in resected PDAC tissues and its correlation with patient prognosis. We obtained formalin-fixed paraffin-embedded (FFPE) tissue samples from 88 patients with PDAC who underwent pancreatectomy, and measured EGFR mRNA levels by quantitative real-time reverse transcription-polymerase chain reaction. The high-level EGFR group had significantly shorter disease-free-survival (p=0.029) and overall-survival (p=0.014) as shown by univariate analyses, although these did not reach statistical significance, as shown by multivariate analyses. However, we found that high EGFR expression was an independent prognostic factor in patients receiving gemcitabine-based adjuvant chemotherapy (p=0.023). Furthermore, we measured EGFR mRNA levels in 20 endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytological specimens. Altered EGFR levels were distinguishable in microdissected neoplastic cells from EUS-FNA cytological specimens compared to those in whole cell pellets. In conclusion, quantitative analysis of EGFR mRNA expression using FFPE tissue samples and microdissected neoplastic cells from EUS-FNA cytological specimens could be useful in predicting prognosis and sensitivity to gemcitabine in PDAC patients.

Association between the HER2 expression and histological differentiation in Wilms tumor

Human epidermal growth factor receptors (HER) play a critical role in the branching morphogenesis of renal tubules. In the current study, we analyzed the expression of HER2 in Wilms tumor and assessed the role of this gene in the tumorgenesis of Wilms tumor. During the period from 1960 to 2005, 40 patients with Wilms tumor were treated in our department. Twenty-four of those patients (except those with clear cell sarcoma of the kidney and malignant rhabdoid tumor of the kidney) were collected and assessed. The histological component of each Wilms tumor was divided into three categories (epithelial, blastemal, and mesenchymal) and the extent of HER2 protein expression was analyzed immunohistochemically. The normal kidney tissue accompanied with 12 cases of Wilms tumor was also examined. In the normal kidney, HER2 showed a strong immunoreactivity in the cell membranes of the collecting tubules and in the endothelial cells. Of 24 cases, 15 cases showed an epithelial component, while 24 cases had a blastemal component and 21 cases had a mesenchymal component, respectively. Among the 15 specimens with epithelial cell differentiation, eight (53.3%) showed HER2 immunoreactive epithelial cells. HER2 immunoreactive blastemal cells were present in 11 (45.8%) of 24 specimens with blastemal cells. On the other hand, only 3 (14.3%) of 21 specimens containing mesenchymal cells showed HER2 immunoreactivity. These results suggest that the extent of HER2 expression is associated with epithelial differentiation in Wilms tumor. These histological findings may therefore help to explain the development of Wilms tumor from the standpoint of histological differentiation.

Diagnostic value of lectin reactive alpha-fetoprotein for neoinfantile hepatic tumors and malignant germ cell tumors: Preliminary study

Background and Purpose The serum alpha-fetoprotein (AFP) level has been used as a tumor marker for hepatoblastoma, and malignant germ cell tumors in pediatric patients. The AFP has 3 isoforms (L1, L2, L3), and the usefulness of the L3 fraction as a diagnostic marker for the adult hepatocellular carcinoma is well known. However, there are few reports dealing with various pediatric malignant tumors. In the current study, we analyzed the diagnostic value of AFP fractions for pediatric diseases, in particular, those occurring in the neoinfantile period. Materials and Methods From 2003 to 2006, two cases of hepatoblastoma, and 5 cases of germ cell tumor, all of which were neoinfantile, were treated in our department. In our analytical system (LiBASys), the level of the L3 fraction contains the majority of the L2 fraction. The total AFP (ng/mL) level and the L3 fraction (%) were measured to assess the usefulness of the L3 fraction as a diagnostic marker. Results In all cases of hepatoblastoma and yolk sac tumor, both the total AFP and the L3 fraction were high, either before treatment or in the presence of malignant tumors. Most of the cases of neonatal immature teratoma showed a high total AFP level during the neoinfantile period, however, the L3 fraction was around 10%, and decreased after surgical treatment. Only 1 case of the immature teratoma demonstrated malignant transformation, when the patient was 8 months old. As the total AFP and the AFP-L3 fraction were proportionally elevated, the patient was treated with additional surgical resection and chemotherapy. In the case of neonatal mature teratoma, the L3 fraction was below 0.5%, even when the total AFP level was high. Discussion Our results indicated that the level of the L3 fraction accurately confirmed the existence, or the malignant potential of hepatic tumor or germ cell tumor. The L3 fraction is useful as a tumor marker during the neoinfantile period.

Hedgehog signaling pathway in neuroblastoma differentiation

PURPOSE The hedgehog (Hh) signaling pathway is activated in some adult cancers. On the other hand, the Hh signaling pathway plays an important role in the development of the neural crest in embryos. The aim of this study is to show the activation of Hh signaling pathway in neuroblastoma (NB), a pediatric malignancy arising from neural crest cells, and to reveal the meaning of the Hh signaling pathway in NB development. METHODS This study analyzed the expression of Sonic hedgehog (Shh), GLI1, and Patched 1 (Ptch1), transactivators of Hh signaling pathway, by immunohistochemistry in 82 NB and 10 ganglioneuroblastoma cases. All 92 cases were evaluated for the status of MYCN amplification. RESULTS Of the 92 cases, 67 (73%) were positive for Shh, 62 cases (67%) were positive for GLI1, and 73 cases (79%) were positive for Ptch1. Only 2 (10%) of the 20 cases with MYCN amplification were positive for Shh and GLI1, and 4 cases (20%) were positive for Ptch1 (MYCN amplification vs no MYCN amplification, P ≦ .01). The percentage of GLI1-positive cells in the cases with INSS stage 1 without MYCN amplification was significantly higher than that with INSS stage 4. Of 72 cases without MYCN amplification, 60 were GLI1-positive. Twelve cases were GLI1-negative, and the prognosis of the GLI1-positive cases was significantly better than that of the GLI1-negative cases (P = .015). CONCLUSIONS Most of NBs without MYCN amplification were positive for Shh, GLI1, and Ptch1. In the cases without MYCN amplification, the high expression of GLI1 was significantly associated with early clinical stage and a good prognosis of the patients. In contrast to adult cancers, the activation of the Hh signaling pathway in NB may be associated with the differentiation of the NB.

Glypican 3 Expression in Pediatric Malignant Solid Tumors

PURPOSE Glypican 3 (GPC3) is one of the cell surface heparan sulfate proteoglycans that binds to the cell membrane, and it is known as an oncofetal protein in adult malignant tumors. Clinical trials using a GPC3 peptide vaccine have already been started in Japan as a new immunotherapy for hepatocellular carcinoma in adult patients. To investigate the possibility of GPC3 immunotherapy for pediatric malignant tumors, we assessed the expression of GPC3 in pediatric malignant tumors. METHODS Immunohistochemically, the GPC3 expression was examined in 159 pediatric solid tumors, including 35 cases of neuroblastoma, 30 cases of Wilms tumor, 10 cases of hepatoblastoma, 25 cases of germ cell tumors, 56 cases of rhabdomyosarcoma, and 3 cases of other tumors. In addition, to clarify the physiological expression during the fetal to neoinfantile period, autopsy specimens of subjects without any neoplastic diseases were assessed in 9 fetal cases and 21 neoinfantile cases. The serum levels of GPC3 were also analyzed using specimens obtained from 53 subjects by the sandwich enzyme-linked immunosorbent assay method. RESULTS Histologically, a high rate of GPC3 expression was noted in 10 (90.9%) of the 11 subjects with yolk sac tumors and 6 (60.0%) of the 10 subjects with hepatoblastoma. In addition, 9 (30.0%) of the 30 subjects with Wilms tumors and 14 (25.0%) of the 56 subjects with rhabdomyosarcoma were positive for the expression of GPC3. Concerning autopsy specimens, most of the 23 subjects younger than 7 months showed positive findings in the liver (94.7%) and kidney (81.8%). Two subjects (100%) with yolk sac tumors and six (75.0%) of the eight subjects with hepatoblastoma serologically demonstrated a high rate of positive expression. Concerning the distribution of the serum GPC3 level according to age, 8 (80.0%) of the 10 subjects younger than 1 year showed a positive finding, while only 16 (37.3%) of the 43 subjects older than 1 year showed a positive finding. CONCLUSION Most cases of hepatoblastoma and yolk sac tumor, and some cases of other tumors were found to express GPC3 either histologically or serologically. On the other hand, GPC3 was physiologically expressed during the fetal and neoinfantile period under 1 year of age. Although, more preliminary data and experience are required, patients older than 1 year that show a positive finding for GPC3 are considered to be appropriate candidates to receive the new immunotherapy using GPC3 peptide vaccination.