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

BACKGROUND: In 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. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: JPLT-2 protocol achieved satisfactory survival among children with non-metastatic hepatoblastoma. New approaches are needed for patients with metastatic diseases.

Surgical intervention for patent ductus venosus.

Patent ductus venosus (PDV) is a rare condition, which usually presents secondary to hepatic atrophy and hepatic failure. We have treated eight cases of PDV, all with hypergalactosemia and hyperbilirubinemia. Ultrasonography and three-dimensional computed tomography demonstrated communication between the portal vein and the inferior vena cava. Of the eight PDV cases, three from the older age group (ages 9, 11, and 14 years) had high-density lesions in their brain nucleus, and one case (age 19 years) had undergone prior Kasai portoenterostomy for biliary atresia. Six PDV patients underwent ligation of PDV and the remaining two cases underwent partial banding of PDV with intraoperative monitoring to maintain portal vein pressure (PVP) under 30 cm H(2)O. Improvement of the intrahepatic portal vein flow was achieved by ligation or banding of PDV. Postoperatively, serum galactose and bilirubin fell to normal ranges, but portal thrombus occurred postoperatively in the first case. We subsequently administered postoperative anticoagulation in the remaining cases and experienced no major complications. These results suggest that PDV ligation and banding are effective surgical approaches for patients with PDV. Close postoperative monitoring to avoid portal thrombus is imperative in these cases.

Detection of CpG island hypermethylation of caspase-8 in neuroblastoma using an oligonucleotide array.

BACKGROUND: The caspase-8 gene (CASP8) is frequently inactivated in unfavorable neuroblastomas through DNA methylation. The present study utilized oligoarrays to evaluate the methylation status of a CpG island located between exons 2 and 3 of caspase 8 in neuroblastomas. PROCEDURE: DNA derived from 70 neuroblastomas was amplified by PCR after bisulfate modification and subjected to analysis on a self-made oligoarray that utilized a polycarbodiimide-coated slide to detect methylation of six intragenic CpG islands of caspase 8. In 30 cases, the methylation status was also analyzed by sequencing. In six cases, the PCR product was cloned into a vector and analyzed. RESULTS: Among the 70 tumor-derived DNAs, methylation was not detected in 18 cases, one methylated CpG was found in 12 cases, two in 18 cases, three in 3 cases, four in 8 cases, five in 1 case and six in 10 cases. All methylated CpG loci detected by sequencing were detected by oligoarray, but some methylated CpGs in three loci were detected by oligoarray alone. In these discrepant loci, methylation was detected in some clones after subcloning, indicating that the oligoarray might be more sensitive than sequencing. The CASP8 expression level was depressed in the tumors having two distinct CpG doublets. These results were significantly correlated with MYCN amplification and with clinical outcomes. CONCLUSIONS: A significant difference in the methylation status within the CpG island of CASP8 was shown between favorable and unfavorable subtypes, and CASP8 methylation detected by oligoarray may be useful in the clinical evaluation of neuroblastomas.

Evaluation of genes identified by microarray analysis in favorable neuroblastoma.

PURPOSE: The biological heterogeneity of neuroblastoma results in a varied outcome ranging from spontaneous regression to fatal tumor progression. Microarray expression profiling and genetic polymorphism arrays may help identify key genes that differ in aggressive neuroblastomas from those observed in tumors associated with a favorable outcome. METHODS: Total RNA was extracted from 16 neuroblastomas obtained from patients who subsequently died of the disease and from 16 favorable neuroblastomas and analyzed using a human whole genome oligomicroarray (55K CodeLink). Genes overexpressed in favorable tumors were subsequently analyzed in 121 neuroblastoma tumors obtained before chemotherapy using real-time RT-PCR. And among these cases, expression levels of these genes were also analyzed in 20 tumors obtained after chemotherapy. RESULTS: Oligomicroarray analysis revealed the overexpression of 283 genes in favorable tumors that were associated with either regressing or maturing tumors. Three candidate genes, including DHRS3, NROB1, and CYP26A1, were selected that were significantly overexpressed in favorable tumors by quantitative real-time RT-PCR (P < 0.01). No cases with overexpression of all three genes showed poor outcomes. In 20 post-chemotherapeutic tumors, the expression levels of these genes increased in the cases where patients survived but decreased in the fatal cases. CONCLUSIONS: Using microarray expression profiling, we identified genes that exhibit altered gene expression in neuroblastoma tumors associated with a favorable outcome. These candidates warrant further study for use in risk assessment and/or as therapeutic targets in neuroblastoma.

Clinical feature of anaplastic lymphoma kinase-mutated neuroblastoma.

PURPOSE: Anaplastic lymphoma kinase (ALK) has recently been identified as a gene conferring a predisposition for neuroblastoma. We have analyzed tyrosine kinase domain mutations and amplification/expression of the ALK gene and focused on clinical features of neuroblastoma cases with ALK aberrations. METHODS: The frequency of ALK mutations, copy number gain, and expression were analyzed in 538 neuroblastoma tumors derived from 361 cases, including 161 cases detected by mass screening. These cases were analyzed according to clinicopathologic features including the International Neuroblastoma Staging System and patient outcomes. RESULTS: Three cases (0.8%) had ALK amplification, and 16 cases (5.2%) had missense mutations at positions F1174, F1245, D1249, and R1275. Among them, 7 cases were diagnosed at more than 14 months of age, and 11 cases were infants, including 9 cases detected by mass screening and 1 multiple neuroblastoma with a germline mutation. Of the 11 infants, 3 cases relapsed, and 1 case died of disease. Among cases detected by screening, activated ALK cases showed significantly worse prognosis (P = .002). Of 7 older cases, 5 had MYC amplifications, and 5 died of disease. The expression levels of ALK were up-regulated in cases with unfavorable outcomes. In cases with activated ALK neuroblastoma, survival rates of patients detected by screening were significantly better than those in the clinically detected group (P = .025). CONCLUSIONS: The results of the present study support the hypothesis that activated ALK tumors represent a specific subset of neuroblastomas. These tumors usually develop in infants and may have a high capacity for recurrence.

Wnt signaling and telomerase activation of hepatoblastoma: correlation with chemosensitivity and surgical resectability.

PURPOSE: Recently, it became apparent that telomerase directly modulated Wnt signaling as a cofactor in a ß-catenin transcriptional complex. In this study, we investigated Wnt/ß-catenin signaling and telomerase activation in hepatoblastoma (HBL). METHODS: Tumors derived from 56 HBL cases treated with the Japanese Study Group for Pediatric Liver Tumors (JPLT) Protocol-2 were analyzed for oncogenic mutations (missense mutations and interstitial deletions in the third exon) of the CTNNB1 gene-encoding ß-catenin and for the expression levels of telomerase reverse transcriptase (TERT). RESULTS: Oncogenic mutations of CTNNB1 were detected in 42 cases (75%). The expression levels of TERT were significantly higher in 14 cases without mutation (P < .05) and in 8 cases with metastasis (P < .01). Interestingly, Wnt/ß-catenin target genes were significantly activated in the tumors without mutations (P = .013). In cases with mutations, preoperative chemotherapy was more effective (P = .008), and complete resection rate was higher (P = .034). Consequently, 2 patients with mutations and 4 patients without mutations died of disease (P = .013). High expression of TERT was detected in all tumors of these dead patients. CONCLUSIONS: Wnt/ß-catenin signaling in the HBLs without CTNNB1 mutations was activated by high expression of TERT. The clinical courses in HBLs without CTNNB1 mutations seemed to be unfavorable because of chemoresistance and low rates of resectability.

Single nucleotide polymorphism array analysis to predict clinical outcome in neuroblastoma patients.

PURPOSE: Neuroblastoma (NB) is a heterogeneous tumor and demonstrates favorable or unfavorable outcomes. In Japan, a nationwide NB mass screening (MS) had been performed on 6-month-old infants for approximately 20 years, which might have detected almost all NB including regressing/maturing tumors. To clarify the heterogeneity of this tumor, we examined genetic alterations in the representative cases using genomewide microarrays. METHODS: Genomic DNA was extracted from 198 NB tissue samples and paired blood samples including 76 MS-detected cases and analyzed by single nucleotide polymorphism arrays. RESULTS: The single nucleotide polymorphism array classified the genetic aberrations into 4 types: whole gain/loss type, partial gain/loss type, MYCN-amplified type, and silent type. Most MS-detecting cases belonged to the whole gain/loss type, whereas unfavorable cases who died of disease showed partial gain/loss, MYCN-amplified, or silent types. CONCLUSIONS: Genomewide genetic analysis is useful to predict the outcome of patients. Although the cases whose tumors showed whole gain/loss may respond well to contemporary therapy, sparing intensive surgery, current therapeutic strategy may be insufficient for the subgroups with partial gain/loss, MYCN-amplified, or silent type. Validation of these results would provide new tools to predict clinical outcome of children with NB.