Molecular changes in the tumour suppressor genes p53 and CDKN2A/ARF in human urinary bladder cancer

Urinary bladder neoplasm is the ninth most common cancer in Europe and the United States. The incidence in Sweden is approximately 32 for men and 9 for women per 105 subjects and year and almost all cases are transitional cell carcinoma (TCC). Many genetic alterations have been identified in TCC that include activation of proto-oncogenes, inactivation of tumour suppressor genes and identification of multiple sites of non-random chromosomal deletion for example on chromosome 9 and 17. Through a unique collaboration between the hospitals in Stockholm during 1995 and 1996, nearly 600 bladder cancer samples were collected, representing almost all new cases within the area at that time. This thesis focuses on molecular changes in the tumour suppressor genes CDKN2A/ARF (chromosome 9p2 1) and p53 (17p 13), analysed in nearly 200 of the collected samples.

For analysis of p53 mutations in exons 5-8, a fluorescent multiplex-PCR based SSCP method amplifying four fragments simultaneously was developed. In addition, we used two microsatellite markers to determine loss of heterozygosity (LOH). We found p53 mutations at a rate of 14% and LOH in 30% of the samples. In 21% of informative samples, LOH was found but without accompanying mutation, indicating other mechanisms inactivating p53 than mutations. A strong correlation was found between both mutations and LOH and tumours of high stage and grade, suggesting p53 inactivation as a late event in tumourigenesis. We found a clear distinction between G2a and G2b neoplasms concerning the occurrence of p53 genetic events, supporting the proposal that moderately differentiated (G2) urinary bladder neoplasms are genetically heterogeneous and that they should not be grouped together but instead, for example, be categorised as either lowly or highly malignant. A linked polymorphism in p53 intron 7 was studied but found to have no bearing on the occurrence of urinary bladder cancer, however it showed large ethnic variations in distributed frequencies.

A second method was developed using real-time quantitative PCR (QPCR) to study deletions and duplications in the CDKN2A/ARF gene. We found 14% homozygous deletions but also hemizygous deletions and duplications. CDKN2A/ARF aberrations were found in both superficial papillary tumours and in muscle invasive high-grade tumours implying an early event in bladder tumourigenesis. However, all multiple duplications (n=3) were found in muscle invasive (>hT2), G3 tumours, indicating a late event in tumour progression. Comparing the results from real time QPCR analysis with results from microsatellite analysis show that CDKN2A and ARF can be separately targeted. This suggests the presence of alternate and specific inactivating pathways in addition to molecular mechanisms regulating larger deletions. We also found three samples that showed specific inactivation of both p53 and ARF although they are known to function in the same tumour suppressive pathway.

List of scientific papers

I. Berggren P, Steineck G, Hemminki K (2000). A rapid fluorescence based multiplex polymerase chain reaction--single-strand conformation polymorphism method for p53 mutation detection. Electrophoresis. 21(12): 2335-42.
https://pubmed.ncbi.nlm.nih.gov/10939443

II. Berggren P, Steineck G, Adolfsson J, Hansson J, Jansson O, Larsson P, Sandstedt B, Wijkstrom H, Hemminki K (2001). p53 mutations in urinary bladder cancer. Br J Cancer. 84(11): 1505-11.
https://pubmed.ncbi.nlm.nih.gov/11384101

III. Berggren P, Hemminki K, Steineck G (2000). p53 intron 7 polymorphisms in urinary bladder cancer patients and controls. Stockholm Bladder Cancer Group. Mutagenesis. 15(1): 57-60.
https://pubmed.ncbi.nlm.nih.gov/10640531

IV. Berggren P, Kumar R, Steineck G, Ichiba M, Hemminki K (2001). Ethnic variation in genotype frequencies of a p53 intron 7 polymorphism. Mutagenesis. 16(6): 475-8.
https://pubmed.ncbi.nlm.nih.gov/11682637

V. Berggren P, Kumar R, Sakano S, Hemminki L, Wada T, Steineck G, Adolfsson J, Larsson P, Norming U, Wijkstrom H, Hemminki K (2002). Detecting deletions and duplications in the CDKN2A/ARF gene in urinary bladder cancer, using a novel real-time PCR application. [Submitted]