Keiko Yamamoto

Spontaneous regression of localized neuroblastoma detected by mass screening.

PURPOSE To clarify whether and when neuroblastomas identified through screening do regress, and to ascertain how to treat them appropriately, we observed screened patients who had localized tumors, without any therapeutic intervention. PATIENTS AND METHODS The criteria for the observation program were as follows: disease stage I or II; tumor less than 5 cm in diameter; no invasion to the intraspinal canal or growth to the great vessels; urinary vanillylmandelic acid (VMA) and homovanillic acid (HVA) less than 50 microg/mg creatinine; and informed consent. Of 25 patients identified through screening for 6-month-old infants in Saitama Prefecture, Japan between April 1994 and March 1996, 11 patients who met the criteria and one other patient with stage III tumor were enrolled onto the program. They were examined by abdominal ultrasonography (US) and their urinary VMA and HVA levels were assessed approximately once per month. The observation periods ranged from 4 to 27 months. RESULTS The 11 tumors decreased in size, although one of these 11 tumors initially enlarged until the patient was 12 months of age and decreased in size thereafter. One other tumor slightly increased in size. Urinary VMA levels decreased in all patients. None of the tumors had completely disappeared by the last observation day. CONCLUSION Our results suggest that regression of screened neuroblastoma is not a rare phenomenon. At present, it seems reasonable to adopt a wait-and-see strategy, with careful observation, for selected stage I or II tumors identified in infants screened at 6 months of age.

Cytogenetic findings and prognosis in neuroblastoma with emphasis on marker chromosome 1

The relationship between cytogenetic findings and prognosis in 51 pediatric patients with neuroblastoma is described. Patients were classified into the following four groups based on karyotypic findings: (1) near diploidy, 42 to 47 chromosomes (n = 11); (2) hyperdiploidy, 50 to 56 chromosomes (n = 4); (3) near triploidy, 60 to 77 chromosomes (n = 33); and (4) hypotetraploidy, 80 to 83 chromosomes (n = 3). Patients with near diploid or hypotetraploid karyotypes also had several structural abnormalities including marker chromosome 1, with or without double minutes (DM) or homogeneously staining regions (HSR). Most of these patients were 1 year of age or older and had advanced tumors. The patients who were in the hyperdiploid or near triploid category had few structural abnormalities; all of them, except one, were younger than 1 year of age, had localized tumors, and are long‐term, disease‐free survivors. Kaplan‐Meier analysis of survival rates disclosed a significant difference favoring the latter group (P < 0.001). N‐myc gene amplification was found in five patients of the former group but in no patients of the latter group. The presence or absence of DM or HSR in the former group had no statistically demonstrable effect on survival. However, the presence of marker chromosome 1 appears to indicate a poor prognosis. Five patients with Stage IV‐S disease had near triploid abnormalities similar to findings in patients with localized tumors. We propose that localized and Stage IV‐S neuroblastomas can be classified as one disease category, and that patients with near diploid or hypotetraploid karyotypes are clinically distinct from those having hyperdiploid or near triploid karyotypes. We consider that chromosomal pattern is a more influential prognostic factor than age, disease stage, or N‐myc gene amplification.

MASS SCREENING FOR NEUROBLASTOMA IN INFANTS IN JAPAN: Interim Report of a Mass Screening Study Group

A mass screening system for the early detection by means of a vanillylmandelic acid test of neuroblastoma in 6-month-old infants in Japan has been developed in eight districts. 16 of the 281 939 infants screened by this test had neuroblastoma, equivalent to a very high incidence of 1 in 17 621. 15 of the 16 children with neuroblastoma are alive; the other child died 1 month after surgery. This mass screening system for neuroblastoma used in infancy can help to improve prognosis in infants with this malignant disorder.

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.

Allelic imbalance on chromosome 2q and alterations of the caspase 8 gene in neuroblastoma

We previously reported a high incidence of loss of heterozygosity (LOH) on chromosome 2q33 in neuroblastoma (NB), observed in various types of human cancers including lung cancer, head and neck cancer and follicular thyroid carcinoma. To better elucidate the role of chromosome 2q aberrations in NB, we examined common allelic imbalance (AI) regions on chromosome 2q in 82 NB patients using 10 polymorphic microsatellite markers. AI on 2q was detected in 26 (32%) of 82 NB cases. There was a distinct common AI region between the D2S115 and D2S307 markers on 2q33. The distance between these markers was about 2.0 cM. Recently, the caspase 8 and caspase 10 genes, both of which encode cystein protease, were mapped to chromosome 2q33. Since the common AI region on 2q33 includes the caspase 8 and caspase 10 genes, the alterations of these genes were examined further. Absent or reduced expression of caspase 8 and caspase 10 were found in 19 (70%) of 27 and two (7%) of 27 NB cell lines by reverse transcription-polymerase chain reaction, respectively. A missense mutation was detected at codon 96, GCT (Alanine) to GTT (Valine), of the caspase 8 gene in one of the NB cell lines lacking caspase 8 expression. Thirteen (68%) of 19 cell lines lacking caspase 8 expression displayed methylation of the CpG island of the caspase 8 gene, whereas only one (13%) of eight cell lines with caspase 8 expression showed caspase 8 methylation (P=0.031). Furthermore, there was a significant association between AI at 2q33 and loss of caspase 8 expression (P=0.026). These results indicated that there was a tumor suppressor gene in the common AI region on chromosome 2q33 involved in the pathogenesis of a subset of NB. It is possible that the caspase 8 gene is one of the candidate tumor suppressor genes for NB and inactivation of this gene plays an important role in the tumorigenesis of NB through mainly its methylation.

Mass screening and age-specific incidence of neuroblastoma in Saitama Prefecture, Japan.

PURPOSE To provide the population-based incidence rate of neuroblastoma and to determine the effect of mass screening on the annual age-specific incidence of the tumor in Saitama Prefecture, Japan, from 1981 to 1992. METHODS Data on screened infants and patients detected by the screening were obtained from the records of the Prefectural Screening Center. Data on neuroblastomas in this area were obtained from the Childrens Cancer Registry of the Saitama Prefectural Government (Prefectural Registry) and from the Japan Childrens Cancer Registry (National Registry). Population data were obtained from the Prefectural Census. Mass screening for 6-month-old infants was performed by qualitative assessment of urinary vanillylmandelic acid (VMA) from June 1981 to September 1989 and by quantitative measurement of VMA/creatinine (Cre) and homovanillic acid (HVA)/Cre from October 1989 to December 1992. RESULTS Between 1981 and 1992, 199 cases of neuroblastoma, which include 74 cases detected by mass screening, were identified in Saitama Prefecture. The incidence rate for children under 15 years of age increased from 6.4/10(6) to 20.1/10(6), that for children 0 to 4 years of age increased from 17.0/10(6) to 64.1/10(6), and that for infants under 1 year of age increased from 27.9/10(6) to 260.4/10(6) during these 12 years. No significant reduction in the incidence rate was observed for children over 1 year of age. CONCLUSION The incidence rate for children under 15 years of age increased with mass screening. The rate for infants was sharply increased, with no corresponding decrease in the rate for children at older ages. These data suggest that there is a subset of neuroblastoma that can be detected by mass screening at 6 months of age but would not be diagnosed later clinically.

Gene expression profiling and identification of novel prognostic marker genes in neuroblastoma.

To investigate the various genetic characteristics of and differences between early‐ and advanced‐stage neuroblastoma (NB) and to identify candidate genes involved in NB progression, we performed DNA microarray analysis on 20 primary tumors. Two‐way clustering analysis based on the expression pattern of approximately 500 of 1,700 genes revealed genetic subgroups in these NB tumors. Although 9 of the 13 early‐stage tumors (69%) and 4 of the 6 advanced‐stage tumors (67%) were classified as being in the same cluster, the remaining tumors showed different expression profiles. This indicates that both the early‐ and advanced‐stage tumors were heterogeneous. Based on the microarray data, we identified the BIRC, CDKN2D, and SMARCD3 genes as those that are predominantly expressed in either the early or the advanced stage of NB. These genes have been reported to be associated with apoptosis, cell cycles, and the transcriptional activator, respectively. To better assess the prognostic value of the expression of these genes in NB, real‐time polymerase chain reaction was carried out on 50 primary tumors. The expression of both the BIRC3 and CDKN2D genes was significantly higher in the early‐stage group than in the advanced‐stage group (P = 0.002 and 0.003, respectively), whereas the expression of the SMARCD3 gene was significantly reduced in the early‐stage group (P = 0.02). Therefore, the BIRC, CDKN2D, and SMARCD3 genes are possible candidates for being novel prognostic markers for NB.

Marginal Decrease in Mortality and Marked Increase in Incidence as a Result of Neuroblastoma Screening at 6 Months of Age: Cohort Study in Seven Prefectures in Japan

PURPOSE To determine the usefulness of 6-month screening for neuroblastoma. PATIENTS AND METHODS The cumulative incidence rates (IRs) and cumulative mortality rates (MRs) of neuroblastoma in children younger than 60 months of age were analyzed for control (n = 713,025), qualitative screening (Qual Screen, n = 1,142,519), and quantitative screening (Quan Screen, n = 550,331) cohorts, and for Screened and Unscreened subgroups within screening cohorts. RESULTS IRs (per 100,000) for infants aged 6 to 11 months were 1.12 in Control, 5.69 in Qual Screen (P <.0001), and 17.81 in Quan Screen (P <.0001); IRs for children aged 12 to 59 months were 7.29 in Control, 5.86 in Qual Screen (P =.28), and 6.36 in Quan Screen (P =.60). IRs for children aged 12 to 59 months in Unscreened or Screened subgroups remained at the same level. When patients diagnosed at younger than 6 months of age were excluded, the MR (per 100,000) under 60 months for Control was 4.21; those in Unscreened and Screened subgroups were 3.84 and 2.53 in Qual Screen (P =.30), and 3.20 and 1.97 in Quan Screen (P =.73), respectively; MRs between Control and Unscreened subgroups revealed no significant differences (P =.89 in Qual Screen, P =.85 in Quan Screen). CONCLUSION Six-month screening resulted in a marked increase in incidence for infants with no significant decrease in incidence for children older than 1 year of age. A decrease in mortality was observed, but it was not significant. The usefulness of screening is questionable, because the decrease of mortality should be balanced against the adverse effect of overdiagnosis and the psychological burden on parents and children.

Endosurgical procedures for pediatric solid tumors

The aim of this study was to evaluate the advantages and complications of endosurgical procedures for benign and malignant pediatric solid tumors. Endosurgical techniques of biopsy and excision were used for diagnosis and treatment of solid tumors, respectively. Since July 1997, a total of 24 biopsies and 24 excisions have been performed laparoscopically for neuroblastoma (n=24), ovarian solid tumors (n=10) and other tumors. Seventeen biopsies and six excisions were performed for abdominal neuroblastoma, while ten excisions were performed for ovarian tumor. In these patients, the length of the operation, blood loss, time to start postoperative feeding, time to start postoperative chemotherapy and length of hospital stay were evaluated and compared to the those of the open surgery group. Furthermore, intra- and postoperative complications were analyzed in all patients of both groups. The length of the hospital stay and time to start postoperative feeding were significantly shorter in the group of patients who underwent endosurgical procedures for either abdominal neuroblastoma or ovarian tumor when compared to the open procedure group. The time to start postoperative chemotherapy was shorter only in the abdominal neuroblastoma group. The procedure for two patients undergoing endosurgical tumor excision had to be converted to open surgery due to large tumor size. Two weeks after thoracoscopic excision of a dumb bell-type neurofibroma, one patient underwent open repair of the dura mater because of leakage of cerebrospinal fluid. There were no port-site recurrences in any tumor types. Endosurgical procedures for solid tumors are effective and minimally invasive. However, better indicators are needed for their implementation in order to prevent complications and subsequent conversions to open procedures.

Chromosome findings and prognosis in 15 patients with neuroblastoma found by VMA mass screening

We performed chromosome analysis of 15 neuroblastomas found by mass screening using a vanillymandelic acid spot test. We found near triploid chromosome abnormalities, ranging from 60 to 77 chromosomes, in the tumor cells from 14 patients, and hyperdiploidy with the mode of 50 in cells from one. A structural abnormality was observed in only one patient. We did not find a marker chromosome 1, homogeneously staining region, or double minutes, which have been previously reported in advanced neuroblastomas or in cell lines. All of our patients were completely free of disease 4 to 32 months after diagnosis. We consider that patients with hyperdiploidy or near triploidy are different from those with marker chromosome 1, homogeneously staining region, or double minutes and may constitute a subgroup with a good prognosis in childhood neuroblastoma.