Recurrent coamplification of cytoskeleton-associated genes EMS1 and SHANK2 with CCND1 in oral squamous cell carcinoma

Chromosomal band 11q13 is frequently amplified in oral squamous cell carcinoma (OSCC) and assumed to be critically involved in tumor initiation and progression by proto-oncogene activation. Though cyclin D1 (CCND1) is supposed to be the most relevant oncogene, several additional putative candidate genes are inside this chromosomal region, for which their actual role in tumorigenesis still needs to be elucidated. To characterize the 11q13 amplicon in detail, 40 OSCCs were analyzed by comparative genomic hybridization to DNA microarrays (matrix-CGH) containing BAC clones derived from chromosomal band 11q13. This high-resolution approach revealed a consistent amplicon about 1.7 Mb in size including the CCND1 oncogene. Seven BAC clones covering FGF3, EMS1, and SHANK2 were shown to be frequently coamplified inside the CCND1 amplicon. Subsequent analysis of tissue microarrays by FISH revealed amplification frequencies of 36.8% (88/239) for CCND1, 34.3% (60/175) for FGF3, 37.4% (68/182) for EMS1, and 36.3% (61/168) for SHANK2. Finally, quantitative mRNA expression analysis demonstrated consistent overexpression of CCND1 in all tumors and of EMS1 and SHANK2 in a subset of specimens with 11q13 amplification, but no expression of FGF3 in any of the cases. Our study underlines the critical role of CCND1 in OSCC development and additionally points to the functionally related genes EMS1 and SHANK2, both encoding for cytoskeleton-associated proteins, which are frequently coamplified with CCND1 and therefore could cooperatively contribute to OSCC pathogenesis.     

A consistent pattern of RIN1 rearrangements in oral squamous cell carcinoma cell lines supports a breakage-fusion-bridge cycle model for 11q13 amplification

Gene amplification is a common feature of tumors. Overexpression of some amplified genes plays a role in tumor progression. Gene amplification can occur either extrachromosomally as double-minute chromosomes (dmin) or intrachromosomally in the form of homogeneously staining regions (hsrs). Approximately one-half of our oral squamous cell carcinomas (OSCCs) are characterized by amplification of band 11q13, usually as an hsr located entopically (occurring or situated at the normal chromosomal site, as opposed to ectopically). Using chromosomal fluorescence in situ hybridization (FISH), we confirmed the amplification of the cyclin D1 (CCND1/PRAD1) and fibroblast growth factor types 3 and 4 (FGF3/INT2 and FGF4/HSTF1) genes within the 11q13 amplicon in our series of primary OSCCs and derived cell lines. The human RIN1 gene was isolated as an RAS interaction/interference protein in a genetic selection in yeast and has been described as a putative effector of both the RAS and ABL oncogenes. We mapped RIN1 to 11q13.2. FISH analysis of 10 11q13-amplified OSCC cell lines revealed high-level RIN1 amplification in two cell lines. Three additional cell lines have what appear to be duplications and/or low-level amplification of RIN1, visible in both interphase and metaphase cells. The hybridization pattern of RIN1 on the metaphase chromosomes is particularly revealing; RIN1 signals flank the 11q13 hsr, possibly as a result of an inverted duplication. The gene amplification model of Coquelle et al. (1997) predicted that gene amplification occurs by breakage-fusion-bridge (BFB) cycles involving fragile sites. Our data suggest that the pattern of gene amplification at 11q13 in OSCC cell lines is consistent with a BFB model. RIN1 appears to be a valuable probe for investigating the process of gene amplification in general and, specifically, 11q13 amplification in oral cancer.     

Genetic differences detected by comparative genomic hybridization in head and neck squamous cell carcinomas from different tumor sites: construction of oncogenetic trees for tumor progression

For a better understanding of genetic alterations in head and neck squamous cell carcinoma (HNSCC), we applied comparative genomic hybridization (CGH) in the analysis of 75 HNSCCs, comprised of 18 pharyngeal squamous cell carcinomas (PSCCs), 23 laryngeal squamous cell carcinomas (LSCCs), and 34 oral squamous cell carcinomas (OSCCs). The three subgroups of HNSCC showed significant differences in genetic alteration patterns. Overall, PSCC and LSCC had more copy number aberrations (CNAs) per tumor than did OSCC. Apparent differing patterns of high-level amplification were also observed. The smallest recurrent chromosomal regions of high-level amplification (> or = 15% of cases) were 7q22, 8q24.1, and 11q12-13 in PSCC and 3q26.1-29 in OSCC. According to single frequency and combined frequencies of CNAs, we concluded that the most important chromosomal events for progression of head and neck cancer were +3q, +5p, +8q, and -3p for all subgroups of HNSCC; additionally, +7q, +17q, -9p, and -13q for PSCC; +7p, +9q, +11q12-13, +14q, and +17q for LSCC; and +1p and +11q12-13 for OSCC. To identify further important genetic alterations and the relationships among the alterations, we constructed oncogenetic tree models for tumor progression of HNSCC from CGH data using branching and distance-based tree models. The tree models predicted that: (1) +3q21-29 was the most important early chromosomal event, and -3p, which occurred after +3q21-29, was also an important chromosomal event for all subsites of HNSCC; (2) +8q is the second most important early chromosomal event; (3) there may be at least three subgroups of HNSCC: one characterized by -3p, -9p, +7p, and -13q; another by +5p, +9qter, and +17p; and the other by +8q and +18p. These results suggest that different chromosomal aberrations may play a role in the initiation and/or progression of different subgroups of HNSCC.     

Comprehensive genome and transcriptome analysis of the 11q13 amplicon in human oral cancer and synteny to the 7F5 amplicon in murine oral carcinoma

11q13 amplification occurs in a wide variety of tumors, including almost half of oral squamous cell carcinomas (OSCC) where it has been correlated with a poor outcome. In this study, we compiled 3.6 Mb of DNA sequence in the 11q13 amplicon core and refined the physical map of the amplicon. In the process, we determined the genomic structure and normal tissue expression patterns of two recently identified genes, TAOS2/TMEM16A and MRGF, which reside in the amplicon core. We then quantified DNA copy number and mRNA expression of all genes in the 11q13 amplicon in cell lines and primary tumors from OSCC. With the exception of FGF3, FGF4, FGF19, and MRGF, all genes were overexpressed in most tumors with genomic amplification. Furthermore, we found that the expression of genes in the amplicon appeared to be highly coordinated, making it difficult to determine which gene or genes are driving amplification. However, in nonamplified primary tumors, three genes, TAOS2/TMEM16A, OCIM, and TPCN2, are frequently overexpressed, whereas CCND1 and EMS1 are not. These results suggest that in addition to CCND1 and EMS1, other important genes also may be target genes driving 11q13 amplification. We hypothesize that 11q13 amplification may be driven by a cassette of genes that provide growth or metastatic advantage to cancer cells. This is supported by the finding that the human 11q13 amplicon core is syntenic to mouse chromosomal band 7F5, which is frequently amplified in chemically induced murine OSCC. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat     

Clinical significance of genome-wide minimally deleted regions in oral squamous cell carcinomas

Oral squamous cell carcinoma (OSCC) has the highest rate of increase among male cancers in Taiwan. An understanding of the molecular pathogenesis of this disease as well as the development of prognostic markers for the clinical management of this disease is very important. Thus, a systematic loss of heterozygosity (LOH) analysis was performed to define minimally deleted regions (MDRs) in 63 male OSCCs using 400 polymorphic microsatellite markers. For increasing reliability, genomic DNA was extracted from >90% tumor cells that had been purified by LCM, and only when a microsatellite marker provided LOH information in >30% of the OSCCs was there considered to be successful allelotyping. A correlation of the various MDRs with clinicopathological parameters and prognosis was carried out. In total, 32 MDRs were identified and ten were noted as novel. In addition, six MDRs were found to be associated with cigarette smoking. Among these markers, a loss of MDR c7r2 (7q32.2-q35) was significantly associated with poor disease-free survival (DFS) and ten MDRs were associated with allelic imbalance (AI) in tumors. Among the latter, a loss of MDR c14r1 (14q24.2-q32.12) and c11r1 (11q13.4-q25) had a synergistic effect on poor DFS and were able to reduce further the DFS rate in patients with MDR c7r2 loss. Taken together, the results generated in this study provide new insights that help with exploring the molecular mechanisms associated with OSCC tumorigenesis and cigarette smoking. They also should aid the development of potential prognostic markers for the clinical management of OSCC.     

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.     

Comparative genomic hybridization analysis reveals 3q gain resulting in genetic alteration in 3q in advanced oral squamous cell carcinoma.

We analyzed DNA sequence copy number aberrations (DSCNAs) in 17 primary oral squamous cell carcinomas (OSCCs) by comparative genomic hybridization. DSCNAs were detected frequently at 3q25-qter (7/17), Xp21 (5/17), and Xq12-q23 and 8q23-q24 (4/17), and losses were detected frequently at 13q21-q22 (5/17), 3p21-pter, 4p15-pter and 17p13 (4/17), and 8p22-pter and 9p21-pter (3/17). Four tumors showed amplifications of seven loci: 3q11-qter, 3q13, 3q26, 7q21-q22, 8q23-qter, 9p22-pter, and 12p11. The total number of DSCNAs was significantly greater in stage III and stage IV tumors than in stage I and stage II tumors (P=.008). Furthermore, 3q gain was detected preferentially in stage III and stage IV tumors (6/8) rather than in stage I and stage II tumors (1/9, P=.013). In our study, all tumors with gain of 3q also contained one or more loss(es) in common regions. On the other hand, all tumors with gain of 9p did not contain 3q gains. These observations indicate that gain of 3q and accumulation of DSCNAs are strongly associated with tumor progression in OSCC. Furthermore, 3q gain and loss of one or more additional loci in common aberration regions appears to be a group of DSCNs associated with dominant genetic pathways of leading to advanced OSCCs.     

Recurrent Chromosomal Imbalances Detected in Biopsy Material from Oral Premalignant and Malignant Lesions by Combined Tissue Microdissection, Universal DNA Amplification, and Comparative Genomic Hybridization

Biopsies routinely performed for the histopathological diagnosis of oral epithelial lesions before treatment were screened for chromosomal imbalances by comparative genomic hybridization. Comparative genomic hybridization was performed on 12 oral premalignant lesions (OPLs; dysplasias and carcinomas in situ) and 14 oral squamous cell carcinomas (OSCCs). Eight biopsies displayed areas of different histopathological appearance, so that OPLs and OSCCs from the same patient were analyzed. To avoid contamination with nonneoplastic cells, defined cell populations were isolated by micromanipulation with a glass needle. Before comparative genomic hybridization analysis, universal DNA amplification was performed using the DOP-polymerase chain reaction protocol. In the 14 OSCCs examined, the average number of chromosomal imbalances was significantly higher than in the 12 OPLs (mean ± SEM: 11.9 ± 1.9 versus 3.2 ± 1.2; P = 0.003). The DNA copy number changes identified in more than one OPL were gains on 8q (3 of 12) and 16p (2 of 12), as well as losses on 3p (5 of 12); 5q (4 of 12); 13q (3 of 12); and 4q, 8p, and 9p (2 of 12 each). In more than 30% of OSCCs, gains of chromosomal material were identified on 20q (8 of 14); 8q, 11q, 22q (7 of 14 each); 3q, 15q, and 17p (6 of 14 each); and 14q, 17q, and 20p (5 of 14 each), and losses were identified on 3p and 4q (9 of 14 each), 5q (7 of 14), 13q (6 of 14), and 2q and 9p (5 of 14 each). These results were validated by positive and negative control comparative genomic hybridization experiments and microsatellite analysis for the detection of allelic loss. The vast majority of genomic alterations found in OPLs were again identified in OSCCs from the same biopsy, supporting the hypothesis that multiple lesions in the same patient are clonally related. In summary, we show that comprehensive information on the genomic alterations in oral epithelial lesions can be obtained from small biopsies. Such data may identify prognostic indicators that could eventually assist in designing therapeutic strategies.     

p53 immunoexpression in non-malignant oral mucosa adjacent to oral squamous cell carcinoma: potential consequences for clinical management

p53 is a tumour suppressor gene encoding a protein whose function is impaired in a very large proportion of human cancers. The objectives of this study were to determine the natural history of p53 alterations during stages of oral carcinogenesis, by comparing p53 immunoexpression in oral squamous cell carcinomas (OSCCs), their non-malignant adjacent mucosa, and respective metastases; and to define the potential practical consequences for clinical management of p53 staining in the non-malignant adjacent mucosa. Forty-two samples of non-malignant mucosa adjacent to OSCCs, the respective carcinomas, and six lymph node metastases derived from six of the OSCCs were investigated for p53 protein expression by immunohistochemistry. Seven out of 42 (17%) non-malignant mucosal samples immediately adjacent to OSCC showed suprabasal p53 staining and this was significantly associated with moderate/severe dysplasia (p=0.02). In six of these cases (86%), the respective carcinoma showed p53 immunoexpression in more than 50% of the neoplastic cells and in the remaining case, p53 immunoexpression was found in more than 25% of the neoplastic cells. In all p53-negative carcinomas that showed p53 immunoexpression in the non-malignant adjacent mucosa, p53 staining was never detected above the basal cell layer. Lymph node metastases showed the same patterns of p53 immunoexpression as the carcinomas from which they were derived. When suprabasal p53 staining is present in non-malignant mucosa immediately adjacent to OSCCs, this suggests stable p53 alterations which are maintained upon progression to overt malignancy. The immunostaining in non-malignant mucosa of the resection margins of OSCCs might be a valuable predictor for local recurrences and may therefore have implications for the management of patients who have received surgical treatment for OSCC.     

High-resolution genomic analysis of the 11q13 amplicon in breast cancers identifies synergy with 8p12 amplification, involving the mTOR targets S6K2 and 4EBP1

The chromosomal region 11q13 is amplified in 15-20% of breast cancers; an event not only associated with estrogen receptor (ER) expression but also implicated in resistance to endocrine therapy. Coamplifications of the 11q13 and 8p12 regions are common, suggesting synergy between the amplicons. The aim was to identify candidate oncogenes in the 11q13 region based on recurrent amplification patterns and correlations to mRNA expression levels. Furthermore, the 11q13/8p12 coamplification and its prognostic value, was evaluated at the DNA and the mRNA levels. Affymetrix 250K NspI arrays were used for whole-genome screening of DNA copy number changes in 29 breast tumors. To identify amplicon cores at 11q13 and 8p12, genomic identification of significant targets in cancer (GISTIC) was applied. The mRNA expression levels of candidate oncogenes in the amplicons [RAD9A, RPS6KB2 (S6K2), CCND1, FGF19, FGF4, FGF3, PAK1, GAB2 (11q13); EIF4EBP1 (4EBP1), PPAPDC1B, and FGFR1 (8p12)] were evaluated using real-time PCR. Resulting data revealed three main amplification cores at 11q13. ER expression was associated with the central 11q13 amplification core, encompassing CCND1, whereas 8p12 amplification/gene expression correlated to S6K2 in a proximal 11q13 core. Amplification of 8p12 and high expression of 4EBP1 or FGFR1 was associated with a poor outcome in the group. In conclusion, single nucleotide polymorphism arrays have enabled mapping of the 11q13 amplicon in breast tumors with high resolution. A proximal 11q13 core including S6K2 was identified as involved in the coamplification/coexpression with 8p12, suggesting synergy between the mTOR targets S6K2 and 4EBP1 in breast cancer development and progression.     

miR-23a-3p suppresses cell proliferation in oral squamous cell carcinomas by targeting FGF2 and correlates with a better prognosis: miR-23a-3p inhibits OSCC growth by targeting FGF2

Oral squamous cell carcinomas (OSCCs) are one of the most ubiquitous malignancies the world over, and are accompanied by a high mortality. microRNAs (miRNAs) have increasingly garnered attention with regards to the roles they play in initiation and progression of various kinds of cancers, including OSCC. It has been reported, that miR-23a-3p promotes the development of tumors for prostate cancer, gastric cancer and gliomas. The functions of miR-23a-3p in OSCC however, remain unclear. In this study, fibroblast growth factor 2 (FGF2) is revealed as a direct target of miR-23a-3p, based on luciferase assays and immunoblotting. The expression of miR-23a-3p and FGF2 were found to be significantly downregulated and upregulated in OSCC tissues respectively. This indicates a reverse correlation between miR-23a-3p and FGF2 levels. Using in vitro approaches we ascertained that miR-23a-3p might contribute to the inhibition of growth and inhibition through increasing apoptosis in OSCC cells; while an inhibitor of miR-23a-3p could reverse this effect. Examination of a clinical cohort of OSCC patients suggested that reduced expression of miR-23a-3p is correlated with more advanced cancerous stage and poorer differentiation of OSCC cell. Additionally, a survival analysis and the Cox-hazard regression model showed that higher levels of miR-23a-3p can be used reliably for prognosis of OSCC patients. This study indicates that miR-23a-3p might suppress tumor proliferation, invasion and promote apoptosis of OSCC by targeting FGF2. miR-23a-3p has the potential to be used as prognostic indicator, and could be exploited as a therapeutic reagent for OSCC in the future.     

Specific p53 immunostaining patterns are associated with smoking habits in patients with oral squamous cell carcinomas

AIMS: To identify immunostaining patterns that are predictive for p53 mutations and to investigate whether p53 mutations are associated with established risk factors for oral squamous cell carcinoma (OSCC). METHODS: Fifty five OSCCs were investigated for p53 protein expression by immunohistochemistry (IHC). Ten of these cases, including five p53 immunopositive and five p53 immunonegative cases, were subjected to microdissection of representative tumour areas followed by sequence analysis for the detection of TP53 mutations. RESULTS: Paired IHC and sequence analysis revealed that p53 immunoexpression in more than 25% of tumour cells was indicative of TP53 mutations, whereas p53 immunonegativity was not informative. Therefore, for p53 immunohistochemical interpretation, p53 immunonegative cases were excluded from the analysis and the cut off value for p53 immunoexpression was set at 25%. Of the OSCCs showing any p53 immunoexpression, 64% revealed staining in more than 25% of the tumour cells. p53 immunoexpression in more than 25% of the neoplastic cells was significantly associated with smoking but not with alcohol consumption. No significant association with smoking habits was found when OSCCs were dichotomised into p53 immunonegative and p53 immunopositive. CONCLUSIONS: In OSCCs the following conclusions can be made: (1) p53 immunonegativity is not informative for TP53 mutations; (2) 25% p53 immunopositive cells appears to be a good cut off value to predict TP53 mutations; (3) p53 immunostaining patterns that appeared to be predictive for TP53 mutations were associated with the smoking habits of the patients.     

Molecular cytogenetic analysis of oral squamous cell carcinomas by comparative genomic hybridization, spectral karyotyping, and fluorescence in situ hybridization

We investigated relationships between DNA copy number aberrations and chromosomal structural rearrangements in 11 different cell lines derived from oral squamous cell carcinoma (OSCC) by comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). CGH frequently showed recurrent chromosomal gains of 5p, 20q12, 8q23 approximately qter, 20p11 approximately p12, 7p15, 11p13 approximately p14, and 14q21, as well as losses of 4q, 18q, 4p11 approximately p15, 19p13, 8p21 approximately pter, and 16p11 approximately p12. SKY identified the following recurrent chromosomal abnormalities: i(5)(p10), i(5)(q10), i(8)(q10), der(X;1)(q10;p10), der(3;5)(p10;p10), and der(3;18)(q10;p10). In addition, breakpoints detected by SKY were clustered in 11q13 and around centromeric regions, including 5p10/q10, 3p10/q10, 8p10/q10 14q10, 1p10/1q10, and 16p10/16q10. Cell lines with i(5)(p10) and i(8)(q10) showed gains of the entire chromosome arms of 5p and 8q by CGH. Moreover, breakages near the centromeres of chromosomes 5 and 8 may be associated with 5p gain, 8q gain, and 8p loss in OSCC. FISH with a DNA probe from a BAC clone mapping to 5p15 showed a significant correlation between the average numbers of i(5)(p10) and 5p15 (R(2) = 0.8693, P< 0.01) in these cell lines, indicating that DNA copy number of 5p depends upon isochromosome formation in OSCC.     

High-throughput tissue microarray analysis of 11q13 gene amplification (CCND1, FGF3, FGF4, EMS1) in urinary bladder cancer

Gene amplification is a common mechanism for oncogene overexpression. High-level amplifications at 11q13 have been repeatedly found in bladder cancer by comparative genomic hybridization (CGH) and other techniques. Putative candidate oncogenes located in this region are CCND1 (PRAD1, bcl-1), EMS1, FGF3 (Int-2), and FGF4 (hst1, hstf1). To evaluate the involvement of these genes in bladder cancer, a tissue microarray (TMA) containing 2317 samples was screened by fluorescence in situ hybridization (FISH). The frequency of gains and amplifications of all genes increased significantly from stage pTa to pT1-4 and from low to high grade. In addition, amplification was associated with patient survival and progression of pT1 tumours. Among 123 tumours with amplifications, 68.3% showed amplification of all four genes; 19.5% amplification of CCND1, FGF4, and FGF3; and 0.8% co-amplification of FGF4, FGF3, and EMS1. Amplification of CCND1 alone was found in 9% of the tumours, while EMS1 alone was amplified in 1.6% and FGF4 in 0.8%. Overall, the amplification frequency decreased with increasing genomic distance from CCND1, suggesting that, among the genes examined, CCND1 is the major target gene in the 11q13 amplicon in bladder cancer.     

FGF4 and INT2 oncogenes are amplified and expressed in Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a vascular tumor, the pathogenesis of which has been suggested to include human herpesvirus 8 (HHV-8) as well as various cytokines and growth factors. Very little is known about cytogenetic and molecular genetic changes in KS. We studied DNA copy number changes in KS and found a recurrent gain at 11q13. We then analyzed the amplification and expression status of two known oncogenes, FGF4 and INT2, residing at 11q13. Comparative genomic hybridization, interphase fluorescence in situ hybridization with yeast artificial chromosome probes containing FGF4 and INT2, and immunoperoxidase immunostaining with anti-FGF4 and -INT2 antibodies were used on 12 KS samples. All samples tested were shown by polymerase chain reaction to be HHV-8 positive. A recurrent gain at 11q13 was shown by comparative genomic hybridization in 4 of 10 cases studied. Of six cases studied by interphase fluorescence in situ hybridization, four showed a 3- to 4-fold amplification with the probes containing FGF4 and INT2. Expression of FGF4 and INT2 was found in nine and three cases, respectively, of nine studied. Amplification and expression of these genes is particularly interesting in the context of oncovirus involvement, because INT2 is a homolog of mouse int2 which causes mammary carcinoma in mice when activated by integration of retrovirus mouse mammary tumor virus. This raises the question of whether HHV-8 represents an integrating oncovirus that causes amplification and activation of genomic oncogenes in humans.     

Allelic loss at BRCA1, BRCA2, and adjacent loci in relation to TP53 abnormality in breast cancer

Cells with abnormal TP53 lose cell cycle checkpoints, resulting in genomic instability and neoplastic transformation. However, the evidence linking the tumor-specific targets of genomic alteration to an abnormal TP53 is limited. The present study tested the hypothesis that TP53 abnormalities are correlated with an increased frequency of deletion of breast cancer susceptibility loci (17q and 13q) in breast carcinomas. Tumors from 90 patients were examined for TP53 abnormality and loss of heterozygosity (LOH) at 11 loci on 17q (17q11.2–21) and 13q (13q12–14), including the loci for BRCA1 and BRCA2. A higher frequency of LOH was consistently found at 17q or 13q loci in tumors with an abnormal TP53. The increased LOH in relation to TP53 abnormality was statistically significant at the BRCA1, D17S588, and D13S267 loci (P < 0.05) but not at the locus for BRCA2 (P = 0.64). These observations imply a possible link between an abnormal TP53 and specific genomic deletions of breast cancer susceptibility loci, which may provide clues to the role of TP53 during breast tumorigenesis. Genes Chromosomes Cancer 20:377–382, 1997. © 1997 Wiley-Liss, Inc.     

Combined array comparative genomic hybridization and tissue microarray analysis suggest PAK1 at 11q13.5-q14 as a critical oncogene target in ovarian carcinoma

Amplification of chromosomal regions leads to an increase of DNA copy numbers and expression of oncogenes in many human tumors. The identification of tumor-specific oncogene targets has potential diagnostic and therapeutic implications. To identify distinct spectra of oncogenic alterations in ovarian carcinoma, metaphase comparative genomic hybridization (mCGH), array CGH (aCGH), and ovarian tumor tissue microarrays were used in this study. Twenty-six primary ovarian carcinomas and three ovarian carcinoma cell lines were analyzed by mCGH. Frequent chromosomal overrepresentation was observed on 2q (31%), 3q (38%), 5p (38%), 8q (52%), 11q (21%), 12p (21%), 17q (21%), and 20q (52%). The role of oncogenes residing in gained chromosomal loci was determined by aCGH with 59 genetic loci commonly amplified in human tumors. DNA copy number gains were most frequently observed for PIK3CA on 3q (66%), PAK1 on 11q (59%), KRAS2 on 12p (55%), and STK15 on 20q (55%). The 11q13-q14 amplicon, represented by six oncogenes (CCND1, FGF4, FGF3, EMS1, GARP, and PAK1) revealed preferential gene copy number gains of PAK1, which is located at 11q13.5-q14. Amplification and protein expression status of both PAK1 and CCND1 were further examined by fluorescence in situ hybridization and immunohistochemistry using a tissue microarray consisting of 268 primary ovarian tumors. PAK1 copy number gains were observed in 30% of the ovarian carcinomas and PAK1 protein was expressed in 85% of the tumors. PAK1 gains were associated with high grade (P < 0.05). In contrast, CCND1 gene alterations and protein expression were less frequent (10.6% and 25%, respectively), suggesting that the critical oncogene target of amplicon 11q13-14 lies distal to CCND1. This study demonstrates that aCGH facilitates further characterization of oncogene candidates residing in amplicons defined by mCGH.     

Microsatellite alterations in hepatocellular carcinoma and intrahepatic cholangiocarcinoma

A series of 20 hepatocellular carcinomas and 8 intrahepatic cholangiocarcinomas was screened from the Korean population for microsatellite alterations, including a loss of heterozygosity and replication errors using nine microsatellite markers containing several genes. The microsatellite results and our previous comparative genomic hybridization results of two tumors were compared at each locus, and the correlations between these and clinicopathologic variables were examined. The most characteristic findings were found at 13q. Replication errors were prevalent at D13S160 (13q21.2 approximately q31) and D13S292(13q12). The incidence of loss of heterozygosity, however, was higher at D13S153 (13q14.1 approximately q14.3) and D13S265(13q31 approximately q32). In contrast, there were higher deletion frequencies observed in hepatocellular carcinoma (HCC) and higher amplification frequencies observed in intrahepatic cholangiocarcinoma at 13q in our previous comparative genomic hybridization (CGH) study. Higher frequencies of replication errors were observed at D16S408 (13q12 approximately q21) and D16S504(13q23 approximately q24) in the HCC. This study found that significant differences in the patterns of genetic instability of microsatellites were dependent on the chromosomal loci. It is believed that certain genes at altered CGH regions, which are relevant to the development and/or progression of these cancers, are activated by different mutation mechanisms.