Naomi Ohnuma

Expression profiling and differential screening between hepatoblastomas and the corresponding normal livers: identification of high expression of the PLK1 oncogene as a poor-prognostic indicator of hepatoblastomas

Hepatoblastoma is one of the most common malignant liver tumors in young children. Recent evidences have suggested that the abnormalities in Wnt signaling pathway, as seen in frequent mutation of the β-catenin gene, may play a role in the genesis of hepatoblastoma. However, the precise mechanism to cause the tumor has been elusive. To identify novel hepatoblastoma-related genes for unveiling the molecular mechanism of the tumorigenesis, a large-scale cloning of cDNAs and differential screening of their expression between hepatoblastomas and the corresponding normal livers were performed. We constructed four full-length-enriched cDNA libraries using an oligo-capping method from the primary tissues which included two hepatoblastomas with high levels of alpha-fetoprotein (AFP), a hepatoblastoma without production of AFP, and a normal liver tissue corresponded to the tumor. Among the 10 431 cDNAs randomly picked up and successfully sequenced, 847 (8.1%) were the genes with unknown function. Of interest, the expression profile among the two subsets of hepatoblastoma and a normal liver was extremely different. A semiquantitative RT–PCR analysis showed that 86 out of 1188 genes tested were differentially expressed between hepatoblastomas and the corresponding normal livers, but that only 11 of those were expressed at high levels in the tumors. Notably, PLK1 oncogene was expressed at very high levels in hepatoblastomas as compared to the normal infants livers. Quantitative real-time RT–PCR analysis for the PLK1 mRNA levels in 74 primary hepatoblastomas and 29 corresponding nontumorous livers indicated that the patients with hepatoblastoma with high expression of PLK1 represented significantly poorer outcome than those with its low expression (5-year survival rate: 55.9 vs 87.0%, respectively, p=0.042), suggesting that the level of PLK1 expression is a novel marker to predict the prognosis of hepatoblastoma. Thus, the differentially expressed genes we have identified may become a useful tool to develop new diagnostic as well as therapeutic strategies of hepatoblastoma.

Intensified chemotherapy increases the survival rates in patients with stage 4 neuroblastoma with MYCN amplification.

Purpose Patients with high-risk neuroblastoma who have multiple copies of MYCN fare much worse than do those without MYCN amplification; however, it has not been clarified whether intensified chemotherapy with or without blood stem cell transplantation can alter the extremely poor prognosis of patients with amplified MYCN. Methods and Results Between 1985 and 1999, 301 patients older than age 12 months with stage 4 neuroblastoma were treated. From January 1985 to February 1991, 80 patients with stage 4 neuroblastoma with and without MYCN amplification uniformly received induction chemotherapy with regimen A1 (cyclophosphamide 1,200 mg/m2 and vincristine 1.5 mg/m2 on day 1, tetra-hydropyranyl [THP]-Adriamycin 40 mg/m2 on day 3, and cisplatin 90 mg/m2 on day 5). Among 22 patients with MYCN amplification, nine (40.9%) achieved a complete remission and seven (31.8%) underwent stem cell transplantation. Of 58 patients without MYCN amplification, 43 (74.1%) achieved a complete remission and 14 (24.1%) underwent stem cell transplantation. The 5-year relapse-free survival rates were 23.2% for stage 4 patients with MYCN amplification and 33.3% for those without MYCN amplification (P = 0.029); the 5-year overall survival rates were 32.8% for stage 4 patients with MYCN amplification and 42.8% for those without MYCN amplification (P > 0.05). From March 1991 to June 1998, patients with stage 4 neuroblastoma who had 10 or more copies of MYCN were treated with regimen A3 (cyclophosphamide 1,200 mg/m2 per day on days 1 and 2, THP-Adriamycin 40 mg/m2 on day 3, etoposide 100 mg/m2 per day on days 1 to 5, and cisplatin 25 mg/m2 per day on days 1 to 5); those with fewer than 10 copies of MYCN received regimen new A1 (cyclophosphamide 1,200 mg/m2 on day 1, THP-Adriamycin 40 mg/m2 on day 3, etoposide 100 mg/m2 per day on days 1 to 5, and cisplatin 90 mg/m2 on day 5), which is similar in intensity to regimen A1. Among 88 patients with MYCN amplification, 63 (71.6%) achieved a complete remission and 63 (71.68%) underwent stem cell transplantation. Of 133 patients without MYCN amplification, 93 (69.9%) achieved a complete remission and 71 (53.4%) underwent stem cell transplantation. The 5-year relapse-free survival rates were 36.0% for stage 4 patients with MYCN amplification and 32.2% for those without MYCN amplification (P > 0.05), the 5-year overall survival rates were 34.0% for stage 4 patients with MYCN amplification and 38.9% for those without MYCN amplification (P > 0.05). The difference in relapse-free survival rates was significantly different (P = 0.003) between patients with MYCN-amplified tumor treated before (regimen A1) versus after 1991 (regimen A3). Conclusions With the use of the more intensive induction regimen A3 plus blood stem cell transplantation for MYCN-amplified patients, survival curves for those with or without MYCN amplification now appear similar. Higher doses of chemotherapy may ameliorate the effect of MYCN amplification in patients with high-risk neuroblastoma.

Outcome of hepatoblastomas treated using the Japanese Study Group for Pediatric Liver Tumor (JPLT) protocol-2: report from the JPLT

BackgroundIn the recent years, surgical resection with pre- and/or postoperative chemotherapy has markedly improved the survival rate of hepatoblastoma patients. We herein report the results of patients treated with the current protocol of the Japanese Study Group for Pediatric Liver Tumor, JPLT-2.MethodsA total of 279 patients with malignant liver tumor were enrolled in JPLT-2. Data from 212 hepatoblastoma cases were analyzed. PRETEXT I patients were treated with primary resection followed by low doses of cisplatin–pirarubicin (tetrahydropyranyl-adriamycin). Otherwise, patients received preoperative cisplatin–pirarubicin (CITA), followed by surgery and postoperative chemotherapy. Ifosfamide, pirarubicin, etoposide, and carboplatin (ITEC) were given as a salvage treatment. High-dose chemotherapy with hematopoietic stem cell transplantation (SCT) was reserved for patients with metastatic diseases.ResultsThe 5-year overall survival rate (OS) in non-metastatic cases was 100% for PRETEXT I, 87.1% for PRETEXT II, 89.7% for PRETEXT III, and 78.3% for PRETEXT IV. The 5-year OS in metastatic cases was 43.9%. The outcome in non-metastatic PRETEXT IV cases was markedly improved, while the results of metastatic tumors remained poor.ConclusionsJPLT-2 protocol achieved satisfactory survival among children with non-metastatic hepatoblastoma. New approaches are needed for patients with metastatic diseases.

Treatment results of advanced neuroblastoma with the First Japanese Study Group protocol

PURPOSE To elucidate the efficacy of intensive induction and consolidation chemotherapy regimens (Study Group of Japan for Advanced Neuroblastoma [JANB] 85) for patients with advanced neuroblastoma aged 1 year or older. PATIENT AND METHODS One hundred fifty-seven patients with newly diagnosed advanced neuroblastoma were entered into this study between January 1985 and December 1990. Eligible patients were 12 months old or older with stage III or IV disease. The patients first received six cyclic courses of intensive induction chemotherapy (designated regimen A1) consisting of cyclophosphamide (1,200 mg/m2), vincristine (1.5 mg/m2), tetrahydro-pyranyl Adriamycin (pirarubicin; 40 mg/m2), and cisplatin (90 mg/m2). The patients were further treated with three different consolidation protocols: 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosour ea, dacarbazine, and bone marrow transplantation. RESULTS Overall survival rates for patients with stage III disease without reference to the consolidation protocols were 80.8%, 76.9%, and 66.3% at 2, 5, and 10 years, respectively. The overall survival rates for patients with stage IV disease were 58.8%, 34.4%, and 28.9% at 2, 5, and 10 years, respectively. There were no statistically significant differences between the three consolidation treatment groups. Patients who did not achieve complete remission (CR) with induction chemotherapy and surgery all died, suggesting that CR is essential for the cure of advanced neuroblastoma. The overall 5-year survival rate of the 24 patients with N-myc amplified stage III and IV disease was 33.3%, and the longest survival time of a relapse-free patient was 103 months. CONCLUSION The intensive induction chemotherapy regimen used in this study may be of significant value in increasing the CR rate and survival for patients with N-myc amplified and nonamplified advanced neuroblastoma.

Stratification of treatment of stage 4 neuroblastoma patients based on N-myc amplification status

BACKGROUND It has been shown that children aged more than 12 months with stage 3 and 4 neuroblastoma with N-myc amplification do worse than those without amplification. Development of an innovative chemotherapeutic protocol for patients in such an extremely poor-risk group was the purpose of this study. PROCEDURE Since March 1991 a new protocol for the treatment of advanced neuroblastoma was started. When N-myc was amplified more than 10-fold, patients received regimen A3, in which cyclophosphamide 1,200 mg/m2 was given on days 1 and 2; hence the dose of cytotoxic drugs was doubled. Patients with fewer than 10 copies of N-myc received regimen new A1, which is very similar to regimen A1 that had been used until March 1991 for all patients with advanced neuroblastoma with/without N-myc amplification. The efficacy of regimen A3 was evaluated. RESULTS The relapse-free survival rate at 1 and 2 years for stage 4 patients older than 12 months of age with N-myc amplification of more than 10-fold was 43% and 29%, respectively, with regimen A1 and that for the same subgroup of patients treated with regimen A3 since March 1991 was 79% and 49%, respectively; the difference is statistically significant. On the other hand, there were no differences in the relapse-free survival rate at 1 year and 2 years for stage 4 patients with fewer than 10 copies of N-myc between those treated with regimen A1 and those treated with new A1 since March 1991. CONCLUSIONS Stratification based on N-myc amplification into new A1 and A3 treatment protocols is of significant clinical importance. Regimen A3 was well tolerated and showed an improvement in clinical results in stage 4 patients with N-myc amplified more than 10-fold.

The role of ERCP in biliary atresia

BACKGROUND It is not easy to discriminate between infantile hepatitis and biliary atresia in spite of several diagnostic tests including laboratory analyses, ultrasound, and hepatobiliary scans. ERCP is the most useful procedure for visualization of the extrahepatic biliary system, but ERCP is still an uncommon procedure in children. METHODS ERCP examination was performed in 52 infants with biliary atresia (10 with infantile hepatitis, 5 with congenital biliary dilatation, 3 with paucity of intrahepatic bile duct, 2 with duodenal atresia, and 1 with postoperative jaundice of hepatoblastoma) aged from 8 days to 300 days (mean, 71 days). RESULTS ERCP was successful in 47 with biliary atresia, in 9 with infantile hepatitis, and 10 with another disease. Liver biopsy was performed in 1 infant with hepatitis in whom the cannulation failed; in 9 with hepatitis in whom the cannulation was successful, exploratory laparotomy could be avoided. The ERCP findings in 46 patients with biliary atresia (excluding 1 in whom evaluation could not be performed because of poor x-ray quality) were classified into four patterns. CONCLUSIONS A success rate of ERCP examinations in infants was 88%, so ERCP is recommended to make a correct decision regarding the need for surgery in cholestatic disorders.

Nonoperative management of blunt pancreatic injury in childhood

PURPOSE Nonoperative management for blunt pancreatic injury in children was performed between 1977 and 1998. The efficiency and safety of nonoperative management was examined. METHODS Pancreatic injury was diagnosed in 20 children. The surgical indication was determined by hemodynamic instability and the management of associated injuries. Children without surgical indications were treated initially by nonoperative management. RESULTS Nineteen of 20 children were treated initially nonoperatively, and 18 of the 19 survived. Surgical exploration was performed in only 1 child with perforation of the duodenum and bile duct. One child died of complications of total parenteral nutrition. Ultrasound scan and computed tomography scan showed pancreatic contusion in 9, laceration in 6, and injury of the main pancreatic duct (MPD) in 5. Pseudocysts were detected in 10 (5 laceration and 5 MPD injury). Pseudocysts smaller than 10 cm disappeared after nonoperative management, and those larger than 10 cm required operative management. Rupture of pseudocysts occurred in 2 children by rotating the upper torso. CONCLUSIONS Nonoperative management of pancreatic injuries is effective in children, although careful management is required to avoid complications. Pseudocysts smaller than 10 cm were treated successfully by nonoperative management, and those larger than 10 cm required surgical management.

The methylation status of RASSF1A promoter predicts responsiveness to chemotherapy and eventual cure in hepatoblastoma patients

Despite the progress of therapy, outcomes of advanced hepatoblastoma patients who are refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the mortality rate, novel prognostic markers are needed for better therapy planning. We examined the methylation status of 13 candidate tumor suppressor genes in 20 hepatoblastoma tumors by conventional methylation‐specific PCR (MSP) and found hypermethylation in 3 of the 13 genes. We analyzed the methylation status of these 3 genes (RASSF1A, SOCS1 and CASP8) in 97 tumors and found hypermethylation in 30.9, 33.0 and 15.5%, respectively. Univariate analysis showed that only the methylation status of RASSF1A but not the other 2 genes predicted the outcome, and multivariate analysis showed a weak contribution of RASSF1A methylation to overall survival. Using quantitative MSP, we found RASSF1A methylation in 44.3% of the 97 tumors. CTNNB1 mutation was detected in 67.0% of the 97 tumors. While univariate analysis demonstrated RASSF1A methylation, CTNNB1 mutation and other clinicopathological variables as prognostic factors, multivariate analysis identified RASSF1A methylation (p = 0.043; relative risk 9.39) and the disease stage (p = 0.002; relative risk 7.67) but not CTNNB1 mutation as independent prognostic factors. In survival analysis of 33 patients in stage 3B or 4, patients with unmethylated tumor had better overall survival than those with methylated tumor (p = 0.035). RASSF1A methylation may be a promising molecular‐genetic marker to predict the treatment outcome and may be used to stratify patients when clinical trials are carried out.

Analysis of treatment outcome for children with recurrent or metastatic hepatoblastoma.

For better total survival rate of children with hepatoblastoma, the therapeutic strategy for recurrent or metastatic hepatoblastoma should be improved. From 1991 to 1999, 134 cases of hepatoblastoma were treated by surgery and combination chemotherapy of cisplatin (CDDP) and THP-Adriamycin (THP-ADR) based on the JPLT-1 protocol. In 114 non-metastatic cases, 90 primary liver tumors were resected completely by partial hepatectomy, but 12 recurrences were observed in the liver (4 cases) and the lungs (8 cases). Distant metastases on the diagnosis were observed in 20 cases. The treatment outcome of these 12 recurrent and 20 metastatic tumors was analyzed. In four recurrent liver tumors, surgical resection was performed in all four cases, and all the patients were alive and well. In eight recurrent lung tumors, surgical resection was performed completely in six cases with unilateral lung disease, and five of the six patients were alive and well. In stage IV tumors, the survival rate of the patients having primary tumors within two hepatic sections was significantly higher than that of the patients having primary tumors over three hepatic sections. Active surgical intervention to lung metastases and a more intensive chemotherapy to facilitate complete resection of primary hepatic tumor could improve survival rate of children with refractory hepatoblastoma.

High expression of telomerase is an independent prognostic indicator of poor outcome in hepatoblastoma

Telomerase, an enzyme related with cellular immortality, has been extensively studied in many kinds of malignant tumours for clinical diagnostic or prognostic utilities. Telomerase activity is mainly regulated by the expression of hTERT (human telomerase reverse transcriptase), which is a catalytic component of human telomerase. To evaluate whether the levels of hTERT mRNA provides a molecular marker of hepatoblastoma malignancy, we examined hTERT mRNA expression levels in the primary hepatoblastoma tissues by fluorescent RT–PCR using LightCycler technology and followed up the clinical outcomes in 63 patients listed in the Japanese Study Group of Pediatric Liver Tumor between 1991 and 2002. The hTERT mRNA expression was detected in 61 (96.8%) specimens and their expression levels ranged between 0.1/1000 and 745.1/1000 copies of PBGD gene that was used as an internal control. Among these cases, frozen 39 tumour samples and 14 adjacent noncancerous liver tissues were analysed for semiquantitative telomerase assay. In the 39 tumour samples, the levels of telomerase activity ranged between 0.11 and 2709 TPG and 12 (30.7%) had high telomerase activity (>100 TPG), whereas only nine of 14 noncancerous liver tissue samples showed telomerase activity which was less than 1.0 TPG. The levels of telomerase activity were significantly correlated with the levels of hTERT mRNA expression (P<0.001). The frequency of high hTERT mRNA expression and/or high telomerase activity did not significantly associate with the clinicopathological factors except for stage of disease. The prognosis of the patients with high hTERT mRNA expression was significantly worse than that of others (P<0.01), as was the patients with high telomerase activity (P<0.01). Multivariate analysis indicated that high levels of hTERT mRNA expression as well as telomerase activity are independent prognosis-predicting factors in patients with hepatoblastoma.