The tumor suppressor gene p53 : methods for determination and their implications

The tumor suppressor p53 plays a central part in the intracellular defense against malignant transformation and tumor progression. This, and the fact that mutations in the p53 gene are the most common genetic abnormalities so far described in human cancer, illustrates the requirement for accurate and reliable techniques for p53 status determinations.

This thesis describes the development and comparison of mutation detection strategies as well as evaluation of the clinical value of p53 mutational status determinations in breast cancer.

A method was developed for sequence determination of the p53 status in primary breast cancers using cDNA obtained from frozen tumors. This method was applied to a population-derived breast cancer cohort of 315 patients. The results showed that the p53 gene had a mutation frequency of 22%, and that mutations in certain regions of the p53 gene had a detrimental impact on the survival expectations. Protein-based analyses displayed an inability to achieve the same high resolution with respect to prognostic value when compared with the cDNA sequencing results. This lack of resolution was mainly due to an inability to detect deletions, insertions and other protein truncating mutations such as non-sense mutations. This was true for both immunohistochemical analysis of p53 on tissue sections as well as p53 protein levels measurements in cytosol preparations using the luminometric immunoassay (LIA).

The concordance and benefits of basing the analysis on p53 cDNA or on p53 genomic DNA as templates for sequence determinations were evaluated in a double-blind study of 100 breast tumors. The results showed that the more labor-intensive method of using genomic DNA from microdissected tissue sections had a slightly higher sensitivity in detecting p53 mutations than the cDNA-based method. The genomic DNA-based method however, was in this study only applied across exons 4-9, where the cDNA-based method covered the whole coding region of p53 (exons 2- 11).

The Enzymatic Mutation Detection (EMD) technique was examined whether it could function as a reliable screening tool for detection of p53 mutations. We analyzed 203 archival cDNA samples with EMD in a double-blind study and compared the results with data derived from cDNA-based sequencing. Our data rather strongly indicated that EMD did not meet the requirements of high specificity, high sensitivity, high throughput and cost effectiveness when used on archival cDNA.

The p53 mutation status in primary breast cancers and their corresponding recurring tumors was studied by cDNA-based and genomic DNA-based sequencing, respectively. We have demonstrated that p53 mutations increase in frequency during tumor progression, suggesting that the p53 gene may potentially be involved in this process.

In conclusion, sequence analysis is the most accurate method for determination of the mutational status of the p53 gene. The more labor-intensive method of using DNA from microdissected tissue sections is likely to generate a more complete picture of the p53 mutational status, especially in combination with LOH analysis. cDNA-based sequencing of the complete coding region of p53 generates prognostic information in breast cancer patients, where protein-based methods have so far shown divergent results.

List of scientific papers

I. Bergh J, Norberg T, Sjogren S, Lindgren A, Holmberg L (1995). "Complete sequencing of the p53 gene provides prognostic information in breast cancer patients, particularly in relation to adjuvant systemic therapy and radiotherapy" Nat Med 1(10): 1029-1034
https://pubmed.ncbi.nlm.nih.gov/7489358

II. Sjogren S, Inganas M, Norberg T, Lindgren A, Nordgren H, Holmberg L, Bergh J (1996). "The p53 gene in breast cancer: prognostic value of complementary DNA sequencing versus immunohistochemistry." J Natl Cancer Inst 88(3-4): 173-182
https://pubmed.ncbi.nlm.nih.gov/8632491

III. Williams C, Norberg T, Ahmadian A, Ponten F, Bergh J, Inganas M, Lundeberg J, Uhlen M. (1998). "Assessment of sequence-based p53 gene analysis in human breast cancer: messenger RNA in comparison with genomic DNA targets." Clin Chem 44(3): 455-462
https://pubmed.ncbi.nlm.nih.gov/9510848

IV. Norberg T, Lennerstrand J, Inganas M, Bergh J. (1998). "Comparison between p53 protein measurements using the luminometric immunoassay and immunohistochemistry with detection of p53 gene mutations using cDNA sequencing in human breast tumors." Int J Cancer 79(4): 376-383
https://pubmed.ncbi.nlm.nih.gov/9699530

V. Norberg T, Klaar S, Lindqvist L, Lindahl T, Ahlgren J, Bergh J (2000). "Evaluation of the enzymatic mutation detection method (EMDTM)for detection of p53 mutations in a clinical breast cancer material." (Submitted)

VI. Norberg T, Klaar S, Kärf G, Nordgren H, Bergh J (2000). "Increased p53 mutation frequency during tumor progression -results from a breast cancer cohort." (Submitted)