We have chosen tumors of the uterine cervix as a model system to identify chromosomal aberrations that occur during carcinogenesis. A phenotype/genotype correlation was established in defined regions of archived, formalinfixed, and hematoxylin/eosin-stained tissue sections that were dissected from normal cervical epithelium (n = 3), from mild (n = 4), moderate (n = 6), and severe dysplasias/carcinomas in situ (CIS) (n = 13), and from invasive carcinomas (n = 10) and investigated by comparative genomic hybridization. The same tissues were analyzed for DNA ploidy, proliferative activity, and the presence of human papillomavirus (HPV) of the carcinomas and was also found to have undergone a high-level copy-number increase (amplification). We therefore conclude that the gain of chromosome 3q that occurs in HPV16-infected, aneuploid cells represents a pivotal genetic aberration at the transition from severe dysplasia/CIS to invasive cervical carcinoma.The multistep nature of carcinogenesis is firmly established (1-4). The sequence of genetic aberrations can be studied best in organs in which a histomorphological phenotype is defined for certain stages of tumor progression-e.g., colon cancer (5) and cancer of the uterine cervix. Regarding carcinomas of the cervix, infection with human papillomavirus (HPV) is known to play a crucial role in the immortalization of epithelial cells (6, 7). However, the persistence of HPV infection in women that do not develop dysplasias or carcinomas (8) and the long latency of the transition from severe dysplasia/carcinoma in situ (CIS) to carcinoma strongly suggest that factors in addition to HPV infection are required for the malignant transformation of epithelial cells (9). In analogy to colon carcinogenesis, mutations affecting tumor-suppressor genes or cellular oncogenes are likely candidates for additional "hits." Cytogenetically, those mutations are often present as specific chromosomal aberrations. However, relatively little is known about tumor-specific recurrent chromosomal aberrations in dysplastic lesions and primary invasive carcinomas of the uterine cervix-the second most frequent carcinoma in women worldwide-despite the fact that the importance of chromosomal aberrations in cervical carcinogenesis was recognized some 25 years ago (10). However, to date no landmark aberrations have been identified in cervical carcinomas (11, 12).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.The scarcity of data on recurrent chromosomal aberrations that occur during the initiation and progression of cervical tumors prompted us to screen tissue from defined stages of cervical tumorigenesis by a molecular cytogenetic approach termed comparative genomic hybridization (CGH) (13,14). CGH serves as a screening test for DNA copy-number changes in tumor genomes. CGH is based on a two-color fluorescence in situ hybridization, whe...
Comparative genomic hybridization was used to screen the DNA extracted from histologically defined tissue sections from consecutive stages of colorectal carcinogenesis for chromosomal aberrations. No aberrations were detected in normal epithelium (n = 14). Gain of chromosome 7 occurred as a single event in low‐grade adenomas (n = 14). In high‐grade adenomas (n = 12), an overrepresentation of chromosomes 7 and 20 was present in 30% of the cases analyzed. The transition to colon carcinomas (n = 16) was characterized by the emergence of multiple chromosomal aberrations. Chromosomes 1, 13, and 20 and chromosome arms 7p and 8q were frequently gained, whereas chromosome 4 and chromosome arms 8p and 18q were recurrently underrepresented. The same tissue sections that were used for CGH were analyzed by means of DNA‐ploidy measurements and immunohistochemical staining to quantify proliferative activity and p21/WAF‐I and TP53 expression. We observed that crude aneuploidy and increased proliferative activity are early events in colorectal carcinogenesis, followed by TP53 overexpression and the acquisition of recurrent chromosomal gains and losses during the progression from high‐grade adenomas to invasive carcinomas. Genes Chromosom Cancer (1996). © 1996 Wiley‐Liss, Inc.
Comparative genomic hybridization was used to screen the DNA extracted from histologically defined tissue sections from consecutive stages of colorectal carcinogenesis for chromosomal aberrations. No aberrations were detected in normal epithelium (n = 14). Gain of chromosome 7 occurred as a single event in low-grade adenomas (n = 14). In high-grade adenomas (n = 12), and overrepresentation of chromosomes 7 and 20 was present in 30% of the cases analyzed. The transition to colon carcinomas (n = 16) was characterized by the emergence of multiple chromosomal aberrations. Chromosomes 1, 13, and 20 and chromosome arms 7p and 8q were frequently gained, whereas chromosome 4 and chromosome arms 8p and 18q were recurrently underrepresented. The same tissue sections that were used for CGH were analyzed by means of DNA-ploidy measurements and immunohistochemical staining to quantify proliferative activity and p21/WAF-1 and TP53 expression. We observed that crude aneuploidy and increased proliferative activity are early events in colorectal carcinogenesis, followed by TP53 overexpression and the acquisition of recurrent chromosomal gains and losses during the progression from high-grade adenomas to invasive carcinomas.
Fluorescence in situ hybridization techniques allow the visualization and localization of DNA target sequences on the chromosomal and cellular level and have evolved as exceedingly valuable tools in basic chromosome research and cytogenetic diagnostics. Recent advances in molecular cytogenetic approaches, namely comparative genomic hybridization and spectral karyotyping, now allow tumor genomes to be surveyed for chromosomal aberrations in a single experiment and permit identification of tumor-specific chromosomal aberrations with unprecedented accuracy. Comparative genomic hybridization utilizes the hybridization of differentially labeled tumor and reference DNA to generate a map of DNA copy number changes in tumor genomes. Comparative genomic hybridization is an ideal tool for analyzing chromosomal imbalances in archived tumor material and for examining possible correlations between these findings and tumor phenotypes. Spectral karyotyping is based on the simultaneous hybridization of differentially labeled chromosome painting probes (24 in human), followed by spectral imaging that allows the unique display of all human (and other species) chromosomes in different colors. Spectral karyotyping greatly facilitates the characterization of numerical and structural chromosomal aberrations, therefore improving karyotype analysis considerably. We review these new molecular cytogenetic concepts, describe applications of comparative genomic hybridization and spectral karyotyping for the visualization of chromosomal aberrations as they relate to human malignancies and animal models thereof, and provide evidence that fluorescence in situ hybridization has developed as a robust and reliable technique which justifies its translation to cytogenetic diagnostics.
W e have analyzed 30 cases of advanced-stage cervical squamous cell carcinoma (stages lib-IV) by comparative genomic hybridization (CGH). The most consistent chromosomal gain in the aneupioid tumors was mapped to chromosome arm 3q in 77% of the cases. Acquisition of genetic material also occurred frequently on Iq (47%), 5p (30%), 6p (27%), and 20 (23%). Recurrent losses were mapped on 2q (33%), 3p (50%), 4 (33%), 8p (23%), and 13q (27%). High-level copy number increases were mapped to chromosome 8, chromosome arms 3q, 5p, 8q, I2p, I4q, I7q, I9q, 20p, and 20q, and chromosomal bands 3q26-27, 9p23-24, I I q22-23, and 12p 13. In the majority of the cases, the presence of high-risk human papilloma virus genomes was detected. High proliferative activity was accompanied by crude aneuploidy. Increased p2l/W A F-l activity, but low or undetectable expression of TP53 were representative for the immunophenotype. This study confirms the Importance of a gain of chromosome arm 3q in cervical carcinogenesis and identifies additional, recurrent chromosomal aberrations that are required fo r progression from stage I tumors to advanced-stage carcinomas.
The expression level of stromelysin-3 (ST3) mRNA was analyzed by in situ hybridization of formalin-fixed, paraffin-embedded primary breast-tumor samples from 76 patients. Digital image analysis of the dark-field in situ hybridization signal was used to measure the maximal level of ST3 expression in each tumor. All 55 invasive ductal carcinomas and 9 of 10 invasive lobular carcinomas were positive for ST3. Invasive tumors had significantly higher levels of ST3 than in situ tumors. Furthermore, ST3 levels were higher in invasive ductal carcinomas than in invasive lobular carcinomas. The ST3 expression level was significantly correlated to fatal metastatic disease (mean follow-up 104 months). ST3 levels of < 2,500 units were associated with distant metastasis in 46% of patients, whereas levels of > 2,500 units were associated with metastasis in 79% of patients selected for study. ST3 mRNA levels did not correlate with tumor size, microvessel density, DNA ploidy or estrogen-receptor levels. Studies of ST3 expression may provide information valuable for the understanding of breast cancer biology and for prognosis.
Summary Squamous cell carcinomas of the anus are rare neoplasias that account for about 3% of large bowel tumours. Infections with human papillomaviruses are frequently detected in these cancers, suggesting that pathogenic pathways in anal carcinomas and in carcinomas of the uterine cervix are similar. Little is known regarding recurrent chromosomal aberrations in this subgroup of squamous cell carcinomas. We have applied comparative genomic hybridization to identify chromosomal gains and losses in 23 cases of anal carcinomas.A non-random copy number increase of chromosomes 17 and 19, and chromosome arm 3q was observed. Consistent losses were mapped to chromosome arms 4p, 11q, 13q and 18q. A majority of the tumours were aneuploid, and most of them showed increased proliferative activity as determined by staining for Ki-67 antigen. p53 expression was low or undetectable, and expression of p21/WAF-1 was increased in most tumours. Sixteen cancers were satisfactorily tested for the presence of HPV by consensus Li -primer polymerase chain reaction; nine were HPV positive, of which eight were positive for HPV 16.
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