Sachiyo Suita

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.

Changing profile of abdominal wall defects in Japan: Results of a national survey

BACKGROUND/PURPOSE The incidence of gastroschisis has increased over the past 3 decades in a number of countries. To elucidate the Japanese status of anterior abdominal wall defects, the Japanese Society of Pediatric Surgeons conducted a national survey in Japan. METHODS Information was obtained by sending out a questionnaire to 192 University Hospitals, Childrens hospitals, and general hospitals that each had more than 200 beds. The characteristics of the patients including the birth date, birth weight, gestations, rate of associated anomalies, rate of antenatal diagnosis and prognosis, maternal age, gravidity, history of smoking, and drug use were analyzed. RESULTS The authors obtained answers from 149 institutions, including 1,785 cases of omphalocele and 970 cases of gastroschisis, which were treated between 1975 to 1997. There was a significant increase in the incidence of gastroschisis, from 0.131 in 1975 to 1980, 0.269 in 1981 to 1985, 0.337 in 1986 to 1990, 0.461 in 1991 to 1995 to 0.467 per 10,000 births in 1996 to 1997. The incidence of omphalocele was 0.322 in 1975 to 1980, 0.567 in 1981 to 1985, 0.657 in 1986 to 1990, 0.741 in 1991 to 1995 to 0.626 per 10,000 births in 1996 to 1997, respectively. In the omphalocele group, 43.1% of the mothers were between 25 to 29 years of age, whereas in the gastroschisis group 42.6% of the mothers were 20 to 24 years of age. In the gastroschisis group, the number of primipara mothers was larger than that of multipara mothers. In the omphalocele group, approximately 10% of the mothers smoked during each period, whereas in the gastroschisis group, the percentage of smoking mothers increased chronologically from 12.9% in 1981 to 1985, 18.7% in 1986 to 1990, 23.5% in 1991 to 1995 and 29.3% in 1996 to 1997. A history of drug use by the mother was approximately 10% for both the omphalocele and gastroschisis groups. In the omphalocele group, 55.9% had associated anomalies against 21.8% in the gastroschisis group. Approximately 10% in the omphalocele group and less than 3% in the gastroschisis group showed chromosomal abnormalities. From 1986, a significant number of cases detected antenatally has been observed. CONCLUSIONS There have been substantial changes in the incidence of anterior abdominal wall defects, particularly regarding gastroschisis in Japan. The reasons for such changes are most likely multifactorial, further epidemiological monitoring is thus called for.

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.

Prenatal phthalate causes cryptorchidism postnatally by inducing transabdominal ascent of the testis in fetal rats

Phathalate esters, which are commonly used as plasticizers for polyvinyl chloride, are also well known to disturb Sertoli cells. This study aims to show the effect of prenatally administered phthalate on testicular descent in pre- and postnatal rats. Pregnant rats were exposed to mono-n-butyl phthalate (MBP) by gavage from the 15th to the 18th gestational days. Rats administered with solvent only were used as controls. In 20-day-old fetuses (n = 15), the degree of transabdominal testicular ascent in relation to the bladder neck was thus found to be significantly higher in MBP-treated rats than that of the controls (n = 19). In addition, in MBP-treated male offspring (n = 26), 22 rats showed either bilateral or unilateral cryptorchidism at the age of 30 to 40 days old, and the occurrence of cryptorchidism was 84.6%. By contrast, the occurrence of cryptorchidism was 0% in the control rats (n = 15, P < .001). It is therefore suggested that prenatal exposure to MBP may disturb the Sertoli cells and elevate the fetal testes relative to the bladder neck while also inducing cryptorchidism postnatally. Sertoli cells may thus play an important role in the transabdominal descent of the testis by secreting Müllerian-inhibiting substance (MIS), which is known to act as a putative mediator of the transabdominal phase.

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.

Altered expression and molecular abnormalities of cell-cycle-regulatory proteins in rhabdomyosarcoma

Rhabdomyosarcoma is the most commonly occurring soft-tissue sarcoma in children. Some reports have discussed the altered expression and molecular abnormalities of cell-cycle-regulatory proteins in rhabdomyosarcoma; however, variable frequencies of occurrence have been noted. In the current study, among 72 cases of rhabdomyosarcoma, the authors evaluated for the expression of p53, MDM2, p16, p21/WAF1, p27, cyclin D1, cyclin E, pRb and E2F-1 protein immunohistochemically and assessed for proliferative activities using MIB-1. We also analyzed the mutation of the p53 gene in 45 cases, the amplification of the MDM2 gene in 18 cases and the mutation of the H-ras gene in 29 cases, using formalin-fixed paraffin-embedded materials. Furthermore, we assessed the correlation between clinicopathologic factors and the results of both immunohistochemical and molecular analyses. Alveolar type affected older patients, and it had a significantly higher mitotic rate compared with the embryonal type (P=0.0226). p53 overexpression was detected in 22 (30.6%) of 72 cases, and 10 (22.2%) of 45 cases had p53 gene abnormalities. As for MDM2, its overexpression was found in nine (12.5%) of 72 cases, and three (16.7%) of 18 cases showed MDM2 amplification. A statistically significant association was observed between immunoreaction for MDM2 and p53 overexpression (P=0.0002), and p53 and MDM2 overexpression was significantly correlated with high MIB-1 labeling indices. E2F-1 labeling indices showed a significantly higher score in alveolar type compared with that seen in embryonal type (P=0.0334), but MIB-1 did not. In conclusion, our study suggests that p53 overexpression may be related to tumor progression because tumors with p53 overexpression have a high proliferative activity in the current study. Alveolar type had a significantly higher both mitotic rate and E2F-1 labeling indices when compared with the embryonal type. The current study is the first report of the correlation of E2F-1 with alveolar rhabdomyosarcoma.

Influence of age on the presentation and outcome of choledochal cyst

PURPOSE The aim of this study was to identify the most appropriate timing for surgery in newborns with choledochal cysts. METHODS The clinical and histological data of 8 newborn cases of choledochal cysts (newborn group) were compared with 45 cases that were diagnosed later than the newborn period (late group). RESULTS The mean and standard deviation of age at diagnosis was 0.4+/-0.4 months and 43.4+/-37.8 months in the newborn and late group, respectively. The age at operation was significantly earlier in the newborn group than in the late group (4.9+/-4.5 months v54.7+/-47.0 months). Although no significant difference in the time that elapsed between the operation and the onset of symptoms between the newborn and late groups (4.5+/-4.7 months v11.3+/-21.1 months), the serum bilirubin level (4.6+/-3.8 mg/dL v1.8+/-3.4 mg/dL) and the grade of liver fibrosis (2.0+/-0.8 v1.1+/-0.8) were significantly higher in the newborn group than in the late group. CONCLUSION The newborn group should be considered as a special group of patients with different clinical course and pathology than those of the late group, which necessitate the early surgical intervention to prevent progression of liver fibrosis.

Phase I study of irinotecan in pediatric patients with malignant solid tumors.

Purpose To determine the dose-limiting toxicity, maximum tolerated dose, and potential efficacy of irinotecan in children with refractory malignant solid tumors. Patients and Methods In the present phase I clinical trial, 28 patients received irinotecan 50 to 200 mg/m2 per day by intravenous 2-hour infusion over the course of 3 days, repeated once after an interval of 25 days. Fifty-one treatment courses were administered to these patients. Results Dose-limiting toxicities were observed at the dose of 200 mg/m2 per day for 3 days. Diarrhea and hematopoietic toxicities were the dose-limiting factors, and the former required support with intravenous fluid administration. The occurrence of vomiting was variable. Decreases in clinical tumor marker levels were observed in the majority of patients who received two cycles of irinotecan 80 mg/m2 per day to 200 mg/m2 per day over the course of 3 days, and partial response was attained in four patients who received irinotecan in two cycles of 140 mg/m2 per day to 200 mg/m2 per day over the course of 3 days. Pharmacokinetic studies showed that the plasma concentration of irinotecan and its active metabolite SN-38 ranged from 93 to 2,820 ng/mL and 5.2 to 34.8 ng/mL, respectively, during 3-day infusions of irinotecan 200 mg/m2 per day. The mean clearance of irinotecan was 14.54 L/h per m2 (range 8.45–20.83 L/h per m2). Conclusion The maximum tolerated dose was determined to be a dose of irinotecan between 160 mg/m2 per day and 180 mg/m2 per day administered over the course of 3 consecutive days on an inpatient basis, repeated once after 25 days off, and our results indicate that irinotecan is a promising anticancer agent that is worthy of phase II trials in pediatric solid tumors.

Management of ovarian cyst detected antenatally

Ovarian cyst was diagnosed in nine fetuses by antenatal ultrasonographic examination. All deliveries were uncomplicated; two were delivered by cesarean section. The clinical condition in all cases was good. Five of the neonates with a large cyst (more than 5 cm in diameter) were managed surgically, while four were merely followed with repeated ultrasonic examinations. In three of the latter four infants, the cysts disappeared without clinical complications during the subsequent 12 months, and one has been followed for only 2 weeks. The treatment of neonatal ovarian cyst is controversial. Since small ovarian cysts are not clinically significant and may involute, conservative treatment after birth is possible. Repeated ultrasonographic examination may be used to follow small cysts in asymptomatic patients. However, surgical excision in the newborn period has been suggested for larger lesions and/or those with solid components inside the cyst before life-threatening complications occur.