Nobumoto Tomioka

Identification and characterization of a 500-kb homozygously deleted region at 1p36.2-p36.3 in a neuroblastoma cell line

Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s) might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have identified for the first time a homozygously deleted region at the marker D1S244 within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines, NB-1 and NB-C201 (MASS-NB-SCH1), although our genotyping has suggested the possibility that both lines are derived from the same origin. The 800-kb PAC contig covering the entire region of homozygous deletion was made and partially sequenced (about 60%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes within the region which include three known genes (DFF45, PGD, and CORT) as well as three other genes which have been reported during processing our present project for the last 3½ years (HDNB1/UFD2, KIAA0591F/KIF1B-β, and PEX14). They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-β, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor, although RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes.

Novel risk stratification of patients with neuroblastoma by genomic signature, which is independent of molecular signature

Human neuroblastoma remains enigmatic because it often shows spontaneous regression and aggressive growth. The prognosis of advanced stage of sporadic neuroblastomas is still poor. Here, we investigated whether genomic and molecular signatures could categorize new therapeutic risk groups in primary neuroblastomas. We conducted microarray-based comparative genomic hybridization (array-CGH) with a DNA chip carrying 2464 BAC clones to examine genomic aberrations of 236 neuroblastomas and used in-house cDNA microarrays for gene-expression profiling. Array-CGH demonstrated three major genomic groups of chromosomal aberrations: silent (GGS), partial gains and/or losses (GGP) and whole gains and/or losses (GGW), which well corresponded with the patterns of chromosome 17 abnormalities. They were further classified into subgroups with different outcomes. In 112 sporadic neuroblastomas, MYCN amplification was frequent in GGS (22%) and GGP (53%) and caused serious outcomes in patients. Sporadic tumors with a single copy of MYCN showed the 5-year cumulative survival rates of 89% in GGS, 53% in GGP and 85% in GGW. Molecular signatures also segregated patients into the favorable and unfavorable prognosis groups (P=0.001). Both univariate and multivariate analyses revealed that genomic and molecular signatures were mutually independent, powerful prognostic indicators. Thus, combined genomic and molecular signatures may categorize novel risk groups and confer new clues for allowing tailored or even individualized medicine to patients with neuroblastoma.

Array comparative genomic hybridization analysis revealed four genomic prognostic biomarkers for primary gastric cancers

Unlike the case with some other solid tumors, whole genome array screening has not revealed prognostic genetic aberrations in primary gastric cancer. Comparative genomic hybridization (CGH) using bacterial artificial chromosome (BAC) arrays for 56 primary gastric cancers resulted in identification of four prognostic loci in this study: 6q21 (harboring FOXO3A; previously FKHRL1), 9q32 (UGCG), 17q21.1 approximately q21.2 (CASC3), and 17q21.32 (HOXB3 through HOXB9). If any one of these four loci was deleted, the prognosis of the patient was significantly worse (P = 0.0019). This was true even for advanced tumors (stage IIIA, IIB, or IV, n = 39) (P = 0.0113), whereas only 1 of the 17 patients with less advanced tumors (stage IA, IB, or II; n = 17) died of disease after surgery. Multivariate analysis according to the status of four BACs or pathological stage based on the Japanese Classification of Gastric Carcinoma (stages IA, IB, and II vs. stages IIIA, IIIB, and IV) demonstrated that the BAC clone status was also an independent prognostic factor (P = 0.006). These findings may help predict which patients with malignant potential need more intensive therapy, or may point to new therapeutic approaches especially for advanced tumors. The parameter here termed the integrated genomic prognostic biomarker may therefore be of clinical utility as a prognostic biomarker.

Biology of neuroblastomas that were found by mass screening at 6 months of age in Japan

Mass screening (MS) of neuroblastoma has been carried out by measuring the urinary catecholamine metabolites in infants at the age of 6 months in Japan. We assessed the incidence of neuroblastoma that may be a target for MS by studying tumor biology.

Chromosomes that show partial loss or gain in near-diploid tumors coincide with chromosomes that show whole loss or gain in near-triploid tumors: evidence suggesting the involvement of the same genes in the tumorigenesis of high- and low-risk neuroblastomas.

We performed two‐color fluorescence in situ hybridization analysis to detect the numbers of chromosomes 1 and 17, 1p deletion, and 17q gain in 177 neuroblastomas, including 101 tumors that were found by a mass‐screening program for infants. Sixty‐eight tumors with disomy 1 or tetrasomy 1 were classified as the Dis1 group, and 109 tumors with trisomy 1, pentasomy 1, or a mixed population of cells with trisomy 1 and cells with tetrasomy 1 were classified as the Tris1 group. 17q gain was the most frequent genetic event, followed by 1p deletion, and MYCN amplification in both Dis1 and Tris1 tumors. However, the incidence of all the genetic events was higher in Dis1 tumors than in Tris1 tumors. These findings suggest that Tris1 tumors are more resistant to acquiring the genetic events than are Dis1 tumors. In addition, there was an accumulation of genetic events in more advanced stages, with the exception of a high incidence of 17q gain in the stage IVS Tris1 tumors. Comparative genomic hybridization analysis, which was performed in 59 of the 177 tumors, showed that chromosomes partially lost or gained in Dis1 tumors coincided with chromosomes totally lost or gained in Tris1 tumors. Dis1 and Tris1 tumors were considered to have near‐diploid/tetraploid and near‐triploid/pentaploid chromosome numbers, respectively. These findings suggest that the same tumor‐suppressor genes or oncogenes may be involved in the development and progression of both high‐ and low‐risk neuroblastomas, and that the ploidy state of the tumor plays a fundamental role in the heterogeneous behavior.

Safety and tolerance of dose-dense Epirubicin and Cyclophosphamide (EC) with Pegfilgrastim for Japanese patients with early breast cancer

With the approval of pegfilgrastim, the use of dose-dense epirubicin and cyclophosphamide (EC) for breast cancer has become acceptable in Japan. Thus, we aimed to evaluate its safety and tolerability in Japanese patients. Nine breast cancer patients with a high risk of preoperative or postoperative recurrence received EC therapy(epirubicin 90 mg/m(2) and cyclo- phosphamide 600 mg/m(2))for 4 cycles every 2 weeks in combination with a subcutaneous injection of pegfilgrastim (3.6 mg) on day 2 of each cycle. Treatment was discontinued in 1 and extended in 1 of the 9 patients, and the mean relative dose intensity(RDI)was good at 0.93. No serious adverse events were observed, indicating good tolerability. The regimen has potential for use in cases in which the treatment dose needs to be increased. grade 4 neutropenia was observed in all the 9 patients on day 8, with 6 patients developing febrile neutropenia. In Japan, data on changes in neutrophil count associated with pegfilgrastim administration under anthracycline-based chemotherapy are currently insufficient, and further study is required.