Genomic changes defining the progression of human colorectal and cervical tumors

Defining changes during the carcinogenesis and progression of tumors is a major way to obtain a better understanding of the mechanisms of cancer development. We therefore investigated the carcinogenesis process in the colon-rectum and in the uterine cervix by different cell chemical, immunohistochemical and cytogenetic methods. Cell proliferation, assessed by immunohistochemical detection of the Ki-67 antigen (MIB 1 antibody), DNA ploify, determined by image cytometry, expression of the tumor suppressor gene p53 (antibody DO1) and the cycline-dependent kinase inhibitor p21/WAF1 (antibody EA10), detected immunohistochemically, and chromosomal aberrations, determined by comparative genomic hybridization (CGH), were monitored in a series of normal mucosa, low and high grade ademonas and adenocarcinomas of the colon-rectum (mainly pT3-tumors) and in a series of normal epithelium, mild, moderate and severe dysplasias/CIS and early stage squamous cell carcinomas (stage I) of the uterine cervix. The cervix material was also tested for human papillomavirus (HPW) infection. To investigate the behaviour of this panel of markers during tumor progression we additionally examined a material of advanced cervical carcinomas (stage II-IV).

A comparison of the two carcinogenesis models showed that in both systems proliferation increases rapidly with increasing grade of atypia of the lesions. 60-90% of the tumor cells of the high grade adenomas, the severe dysplasias/CIS and the invasive carcinomas stained positively for MIB1. DNA ploidy measurements showed that aneuploidy in colorectal carcinogenesis often developed via triploid DNA distribution patterns while in cervical lesions a tetraploidisation of the cells preceded crude aneuploidy. Carcinomas of both the colon and the uterine cervix showed high percentages of aneuploidy (80-95%) Thirty-eight percent (6/16) of the colon carcinomas exhibited strong expression of p53 protein but were negative for p21/WAF1 expression which could possibly be interpreted as being due to a mutation of the p53 gene.

Almost all cervical carcinomas showed a weak p53 expression combined with a relatively strong p21-WAF1 expression. p53 PCR-SSCP did not reveal any mutations in 10 stage I cervix carcinomas (SSCP data not published) indicating that the immunohistochemically detected p53 expression is due to wild type p53. All of the severe cervical dysplasias, 90% of the stage I cervix carcinomas and 83% of the advanced cervix carcinomas (stage IIb-IV) were positive for high risk HPW infection. High risk HPW E6 region is known to be able to degrade p53 wild type protein which could explain why there is no need to block the p53-p21/WAF1 cell cycle control pathway by mutations of the p53 gene. On the other hand it is not clear whether the strong expression of p21/WAF1 indicates that the inactivation of p53 via the E6-proteins is incomplete, or whether p21/WAF1 expression is up-regulated via alternative regulatory pathways.

The screening for chromosomal aberrations showed that the striking major CGH event in cervical cancer was a gain of the long arm of chromosome 3 which was detected in 1 of 13 severe dysplasias, 9 of 10 stage I carcinomas and 23 of 30 advanced cervical carcinomas, wild colorectal carcinomas exhibited a large number of different changes with gains on 20q (12/16 cases), 13q (8/16 cases), 8q (8/16) and 7p (8/16) and losses on 8p (7/16) and 18q (6/16) as the most frequent ones. In advanced cervical carcinomas acquisition of genetic material also occurred frequently on chromosomes 1q (14/30) and 5p (9/30) and recurrent losses were mapped to chromosomes 3p (15/30), 2q (10/30), 6q (8/30) and 13q (8/30). The average number of chromosomal aberrations (ANCA) was 3,8 for the stage I cervical carcinomas, 5.6 for the colorectal adenocarcinomas (mainly pT3-tumors) and 8.2 for the advanced cervical carcinomas stage IIb-IV indicating that ANCA is tumor stage-correlated.

The investigation of anal carcinomas as an example of another HPV-related tumor revealed a very similar picture to the results obtained for the uterine cervix concerning proliferation, DNA ploidy and p21/WAF1 expression. One exceptional case showed a strong p53 expression, p21/WAF1 negativity and a high level copy number increase on 2p23-24. 56% of the cases which could be evaluated by HPV-analysis tested positively. Non-random copy number increases of chromosomes 17 (9/23) and 19 (14/23) and chromsome arm 3q (7/23) and consistent losses on 4p (7/23), 11q (9/23), 13q (6/23) and 18q (8/23) could be observed. The average number of chromosomal aberrations (ANCA) was 4.8.

Major differences in the carcinogenesis of colorectal cancer and the HPV-related cancers of the uterine cervix and the anus concerning DNA ploidy, p53-p21/WAF1 expression pattern and chromosomal aberrations can be observed. A recurrent pattern of chromosomal aberrations defines each of the carcinoma types. The most common chromosomal aberration in cervical carcinogenesis is a gain on chromosome 3q which becomes visible at the transition from severe dysplasia/CIS to early stage carcinomas. 3q gain also plays a major role in HPV-infected anal carcinomas. Progression of cervical carcinomas is correlated to an increased frequency of specific chromosomal aberrations with 3q gain still playing the predominant role.