Shigeto Kawauchi

Analysis of DNA copy number aberrations in hepatitis C virus-associated hepatocellular carcinomas by conventional CGH and array CGH

To clarify the genetic aberrations involved in the development and progression of hepatitis C virus-associated hepatocellular carcinoma (HCV-HCC), we investigated DNA copy number aberrations (DCNAs) in 19 surgically resected HCCs by conventional CGH and array CGH. Conventional CGH revealed that increases of DNA copy number were frequent at 1q (79% of the cases), 8q (37%), 6p (32%), and 10p (32%) and that decreases were frequent at 17p (79%), 16q (58%), 4q (53%), 13q (42%), 10q (37%), 1p (32%), and 8p (32%). In general, genes that showed DCNAs by array CGH were usually located in chromosomal regions with DCNAs detected by conventional CGH analysis. Increases in copy numbers of the LAMC2, TGFB2, and AKT3 genes (located on 1q) and decreases in copy numbers of FGR/SRC2 and CYLD (located on 1p and 16q, respectively) were observed in more than 30% of tumors, including small, well-differentiated carcinomas. These findings suggest that these genes are associated with the development of HCV-HCC. Increases of MOS, MYC, EXT1, and PTK2 (located on 8q) were detected exclusively in moderately and poorly differentiated tumors, suggesting that these alterations contribute to tumor progression. In conclusion, chromosomal and array CGH technologies allow identification of genes involved in the development and progression of HCV-HCC.

Overexpression of Polo-Like Kinase 1 (PLK1) and Chromosomal Instability in Bladder Cancer

Polo-like kinase 1 (PLK1) participates in bipolar spindle formation and entry into mitosis. Chromosomal instability (CIN) is caused by abnormalities in spindle formation and chromosome segregation. In this study, we investigated the relationship of PLK1 overexpression to CIN, and compared the PLK1 status with clinicopathological parameters in 101 human urothelial carcinomas of the urinary bladder. Expression of PLK1 and the number of centrosomes were assessed by immunohistochemistry. Numerical aberrations of chromosomes 7, 9 and 17 spots that allowed estimation of CIN were evaluated by fluorescence in situhybridization, and DNA ploidy was assessed by laser scanning cytometry. Cancers with a large intercellular variation in centromere copy number were defined as CIN cancers.Tumors with PLK1 overexpression were associated more frequently with CIN (p < 0.0001), DNA aneuploidy (p = 0.0007) and centrosome amplification (p = 0.0013) than those without. Overexpression of PLK1 was significantly related to higher pathological grade (p = 0.0024), multiple tumors (p = 0.0241) and positive urine cytology (p = 0.0192). These data suggest that a high level expression of PLK1 confers tumor progression advantages to urothelial cancer cells, although other factors are also involved.

Centrosome Hyperamplification Predicts Progression and Tumor Recurrence in Bladder Cancer

Purpose: Recent studies have reported that centrosome hyperamplification (CH) is closely related to chromosomal instability in bladder cancer. In this study, we investigated whether CH could be used as a prognostic biomarker for patients with bladder cancer. Experimental Design: CH was evaluated by immunohistochemistry in 50 bladder cancers (≤pT1: 43; ≥pT2: 7). In addition, numerical aberrations of chromosomes 7, 9, and 17 and gain of 20q13, on which the Aurora-A gene is located, were evaluated by fluorescence in situ hybridization, and DNA ploidy was assessed. Preliminary experiments on eight bladder cancer cell lines found that six had over 5% of CH cells associated with a gain of 20q13 and overexpression of Aurora-A; therefore, CH-positive cases (CH+) were defined as those having over 5% of cells with ≥3 centrosomes per cell. Results: CH+, 20q13 gain, chromosomal instability, and DNA aneuploidy were detected in 30 (60%), 18 (36%), 22 (44%), and 19 (38%) patients, respectively. There were significant differences in tumor number, grade, recurrence, and progression between the CH+ and CH− groups. The later had significantly higher recurrence-free and progression-free survivals than the former (P = 0.0028 and P = 0.0070, respectively, log-rank test). Multivariate analysis revealed that CH+ was the strongest predictor for tumor recurrence in nonmuscle invasive (pTa and pT1) bladder cancer (hazard ratio, 1.882; 95% confidence interval, 1.161–3.325; P = 0.0094). Conclusions: Detection of CH may provide crucial prognostic information about tumor recurrence in bladder cancer.

Biological Characteristics in Bladder Cancer Depend on the Type of Genetic Instability

Purpose: Malignant tumors show an inherent genetic instability that can be classified as microsatellite instability (MSI) or chromosomal instability (CIN). To elucidate the differences in biological characteristics of bladder cancer between the two types of genetic instability, the expression of the mismatch repair (MMR) proteins, Aurora-A and p53 proteins, the number of centrosomes, numerical aberrations of chromosomes and 20q13, and DNA ploidy were examined in 100 human urothelial carcinomas of the bladder. Experimental Design: Expressions of the MLH1, MSH2, Aurora-A, and p53 proteins and the numbers of centrosomes were immunohistochemically assessed. Numerical aberrations of chromosomes 7, 9, 17, and 20q13 spots were evaluated by fluorescence in situ hybridization, and DNA ploidy was assessed by laser scanning cytometry. Results: The expression levels of the MMR related-proteins decreased in 9 of 100 tumors. Tumors with low MLH1 or MSH2 expression (designated as MSI cancers) were not linked with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, 20q13 gain, DNA aneuploidy, and disease progression. MSI cancers showed a favorable prognosis. CIN cancers (49 cases), defined as tumors with a large intercellular variation in centromere copy numbers, were associated more frequently with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, and 20q13 gain than the others (51 cases). Tumors with disease progression were included in the CIN cancer group. Conclusions: The present observations suggest that there are differences in the biological characteristics of the two types of genetic instability.

Comparative genomic hybridization reveals genetic progression of oral squamous cell carcinoma from dysplasia via two different tumourigenic pathways

To clarify the genetic pathway(s) involved in the development and progression of oral squamous cell carcinoma (OSCC), as well as the relationship between genetic aberrations and biological characteristics of OSCC tumours, comparative genomic hybridization was used to analyse genetic alterations in both primary OSCCs and adjacent dysplastic lesions of the same biopsy specimens from 35 patients. Gain of 8q22–23 was the most frequent alteration in both OSCC and mild dysplasia, and was considered the earliest event in the process of oral tumourigenesis. The average number of DNA sequence copy number aberrations (DSCNAs) increased with progression from mild dysplasia to invasive carcinoma (r = 0.737, n = 70, p < 0.001). OSCC samples were classified as having a large or small number of DSCNAs (OSCC‐L, 21.4 ± 4.7 DSCNAs or OSCC‐S, 10.0 ± 1.7 DSCNAs, respectively; p < 0.0001). Gains of 3q26‐qter, 8q, 11q13, 14q, and 20q and losses of 4q, 5q12–22, 6q, 8p, 13q, and 18q22‐qter were common to OSCC‐L and OSCC‐S. Gains of 5p15, 7p, 17q11–22, and 18p and losses of 3p14–21, 4p, and 9p were detected exclusively in OSCC‐L. The average number of DSCNAs depended on whether the samples showed OSCC‐ L or dysplasia plus OSCC‐L, or showed OSCC‐S or dysplasia plus OSCC‐S (p = 0.001). Gain of 5p15 and losses of 4p and 9p were detected even in dysplastic lesions adjacent to OSCC‐L samples. Loss of 4p was associated with node metastasis by multivariate analysis (p = 0.013). OSCC‐L tumours were more often T3–T4 stage tumours than T1–T2 stage tumours (p = 0.03). These findings suggest that two different types of OSCC, OSCC‐L associated with high‐stage cancer and OSCC‐S associated with low‐stage cancer, arise from different types of dysplasia via different genetic pathways. Copyright

Comparative genomic hybridization analysis for pancreatic cancer specimens obtained by endoscopic ultrasonography-guided fine-needle aspiration.

BackgroundComparative genomic hybridization (CGH) analysis of pancreatic cancer has been done exclusively for surgical and autopsy specimens, because of the difficulty of tissue sampling without surgery. To overcome this difficulty, we applied CGH technology to cells obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA).MethodsIn the present study, we performed EUS-FNA for 17 patients with pancreatic cancer before surgery. Tumor cells were selected by microdissection. DNA was extracted from the cells and amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). Then CGH was carried out.ResultsIn the 15 patients with tubular adenocarcinoma, the most common loci of gains (including amplification) were 5p, 8q, and 20q (60% of the patients); and 1q, 7p, and 12p (27%). The most frequent losses were 17p (73%); 9p, 18q, and 19p (47%); and 8p (33%). These findings were similar to our previously reported data. Both of the patients with acinar cell carcinoma showed gains of 2q and 5p, and losses of 1p, 9p, 9q, 11p, 11q, 14q, 17p, 17q, and 18q.ConclusionsThe results of this study suggest that comprehensive genetic analysis is possible for EUS-FNA biopsy specimens, with a combination of microdissection and DOP-PCR. This analytical strategy will enable us to evaluate the biological characteristics of pancreatic cancer before treatment.

New perspectives for tumor pathology provided by comparative genomic hybridization.

Abstractu2002Comparative genomic hybridization (CGH) allows rapid screening for DNA copy number gains and losses across the entire genome. CGH analyses have revealed a number of common aberrations and characteristics associated with specific tumor cells or pathogeneses. Recurrent aberrations suggest that tumor-related gene(s) may be located in such regions. Furthermore, some specific aberrations may serve as novel diagnostic features. Quantitative chromosomal analyses based on CGH have also provided stimulating information associated with chromosomal instability, genetic pathways, telomerase activity status, and DNA ploidy and have yielded valuable insights into tumor pathology. This review focuses on scientific advances facilitated by this technique.