Tomoko Furuya

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.

Tumour lineage-homing cell-penetrating peptides as anticancer molecular delivery systems

Cell-penetrating peptides have gained attention owing to their promise in noninvasive delivery systems. Among the identified cell-penetrating peptides, the TAT peptide has been preferentially used for transduction into cells of diverse origins. However, this activity is nonselective between neoplastic and non-neoplastic cells. Here we describe artificial cell-penetrating peptides that are selectively and efficiently incorporated into human tumour cells, according to their lineage. Ten representative tumour lineage-homing cell-penetrating peptides were obtained by screening of a random peptide library constructed using messenger RNA display technology, and some of the isolates were further modified by amino-acid substitution. Their advantageous tumour cell-targeting ability is corroborated in an in vivo mouse model for imaging and growth suppression of metastatic xenoplant tumours. These cell-penetrating peptides are potentially useful for the efficient targeting of human neoplasms in a tumour origin-dependent manner, and provide a framework for the development of peptide-based anti-tumour technologies.

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.

Gain of 5p15.33 Is Associated with Progression of Bladder Cancer

Objective: To search for a biological marker to distinguish low-risk from high-risk bladder cancer indicating disease progression. Methods: The whole genome-wide copy numbers were screened in 18 patients with bladder cancer using array comparative genomic hybridization (CGH) consisting of 4,030 bacterial artificial chromosome clones. Results: Gain of 5p15.33, including TPPP (tubulin polymerization-promoting protein)and ZDHHC11 (zinc finger DHHC domain-containing protein 11) genes, was detected in 5 of 9 (55.6%) high-grade bladder cancers and no (0%; n = 9) low-grade bladder cancer. To confirm the preliminary data, 5p15.33 gain was studied by fluorescence in situhybridization (FISH) in 100 patients, and the results were compared with biological characteristics. In FISH analysis, gain of 5p15.33 was significantly correlated with higher histological grade (p < 0.0001) and advanced pathological stage (p = 0.0284). Tumors with a gain of 5p15.33 had a significantly higher progression-free survival rate than those without (p = 0.0006, log-rank test). Multivariate analysis revealed that gain of 5p15.33 was a predictor for disease progression in bladder cancer (hazard ratio: 1.887, 95% confidence interval: 1.215–2.968, p = 0.0050). Conclusion: These data suggest that gain of 5p15.33 (TPPP and ZDHHC11) may become a potential biomarker identifying high-risk patients with disease progression in bladder cancer.

Examination of oncogene amplification by genomic DNA microarray in hepatocellular carcinomas: comparison with comparative genomic hybridization analysis

To identify amplified oncogenes involved in hepatocellular carcinomas (HCC), we applied a genomic DNA microarray spotted with 57 oncogenes to 20 HCCs. Aberrations in DNA copy number also were analyzed by comparative genomic hybridization (CGH) using an aliquot of DNA samples. In 5 of 20 HCCs, only 6 oncogenes (CCND1, FGF3/FGF4, SAS/CDK4, TERC, MET, and MYC) were amplified, whereas in the remaining 15 tumors no oncogenes were amplified. A comparison of DNA microarray and conventional CGH analyses showed that, although 5 of 6 amplified oncogenes shown by microarray were located in chromosomal regions shown by CGH to have increased DNA copy numbers, not all genes located in such chromosomal regions were affected. One of the amplified oncogenes (SAS/CDK4) was found in a chromosomal region that was undetected by CGH. We, therefore, conclude that amplification of the oncogenes examined in this series is not directly implicated in hepatocellular carcinogenesis.

A comparison of DNA copy number changes detected by comparative genomic hybridization in malignancies of the liver, biliary tract and pancreas.

Tumors arising from the liver, biliary tract and pancreas, which originate in the foregut and are in close anatomical proximity to each other, sometimes show similar histological features. No studies have focused on genetic similarities and differences between tumors of these organs. To elucidate the similarities and differences in DNA copy number alterations between tumors of these organs, we applied comparative genomic hybridization (CGH) to cancers of the liver (31 cases), biliary tract (42 cases) and pancreas (27 cases). Some alterations were common to tumors of all three organs, and some were preferential in certain types of tumor. Gains of 1q and 8q and losses of 8p and 17p were common to all tumors. In contrast, 13q14 and 16q losses were detected exclusively in hepatocellular carcinomas (HCCs; p < 0.01). The incidence of 17q21 gain and 5q loss was higher in biliary tract cancers than in the other two types (p < 0.05). Pancreatic cancers exhibited higher incidence of 5q14-q23 gain and 19p loss than tumors of other organs (p < 0.01). Gains of 7p, 7q, 12p and 20q and losses of 3p, 6q, 9p and 18q were frequent in both biliary tract and pancreatic cancers but rare in HCCs (p < 0.05). The present results suggest that although genes located at 1q, 8p, 8q and 17p are frequently involved in HCC, biliary tract and pancreatic cancer, at least some of the genes implicated in carcinogenesis are different between these three types. It is also suggested that CGH analysis is useful as a potential adjunct for the diagnosis and management of these tumors of organs that are anatomically close to one another.

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

Detection of amplified oncogenes by genome DNA microarrays in human primary esophageal squamous cell carcinoma: comparison with conventional comparative genomic hybridization analysis

Oncogene amplification in 20 surgically resected esophageal squamous cell carcinomas (ESCC) was examined with DNA microarrays that detected 57 oncogenes and two reference DNA. Alterations in DNA copy numbers detected by microarrays were compared to those obtained by conventional comparative genomic hybridization (CGH). Amplification of eight oncogenes (CCND1, FGF3/FGF4, EMS1, SAS, ERBB2, PDGFRA, MYC, and BCL2) was detected by DNA microarrays in 9 of 20 tumors. Although ERBB2 was 23.2 times higher than the control level in one case, the average magnitude of gene amplification was approximately two to four times that of the control level. EMS1, CCND1, and FGF3/FGF4, which are all located on 11q13, were amplified in 7, 5, and 4 of 20 ESCC, respectively, and they were coamplified in 3 tumors. A comparison of genome DNA microarrays and CGH data revealed that although most amplified oncogenes were included in chromosomal regions for which DNA copy number gains were detected by conventional CGH, not all amplified genes on microarrays showed concomitant DNA copy number gains on CGH. In conclusion, microarrays of oncogenes are useful for the comprehensive identification of amplified oncogenes and for analysis of areas of specific amplification within chromosomal regions with DNA copy number increases detected by CGH analysis.