Quantitative Measurement of HER2/neu Oncogene Amplification and p53 Tumor Suppressor Gene Deletion by RT-PCR in Breast Cancer

Abstract

Objective: The aim of the study was to quantitatively evaluate HER2/neu oncogene amplification and p53 tumor suppressor gene deletion in breast cancer with real-time polymerase chain reaction (RT-PCR). Materials and Methods: Sections obtained from the tumor tissues of 50 patients were paraffinized on slides, and DNA extraction was performed. HER2/neu amplification and p53 deletion were analyzed using RT-PCR with respect to immunohistochemistry (IHC). Results: For 25 patients with breast cancer, we compared IHC and RT-PCR results for the quantitative measurement of HER2/neu expression. We found a significant correlation between the results obtained using IHC and RT-PCR (p < 0.05). Taking the results of IHC as reference, the sensitivity and specificity of the RT-PCR method were 57% and 83%, respectively. HER2/neu amplification and p53 deletion did not have a significant correlation with tumor size, histological grade, lymph node invasion, and status of estrogen receptor and progesterone receptor (p>0.05, for all). Conclusion: RT-PCR measured gene levels reliably and accurately with high sensitivity and specificity, making it superior to IHC, which is subjective.

Keywords

• Breast cancer
• p53
• HER2/neu
• RT-PCR

References

1. 1. Ferlay J, Colombet M, Soerjomataram I, Parkin DM, Pineros M, Znaor A, et al. Cancer statistics for the year 2020: an overview. Int J Cancer 2021; 149: 778-9. google scholar
2. 2. Yedjou CG, Sims JN, Miele L, Noubissi F, Lowe L, Fonseca DD, et al. Health and racial disparity in breast cancer. Adv Exp Med Biol 2019; 1152: 31-49. google scholar
3. 3. Tanani ME, Al Khatib AO, Al-Najjar BO , Shakya AK, El-Tanani Y , Lee YF et al. Cellular and molecular basis of therapeutic approaches to breast cancer. Cellular Signalling 2023; 101: 1-12. google scholar
4. 4. Königshoff M, Wilhelm J, Bohle RM, Pingoud A, Hahn M. Her-2/ neu Gene copy number quantified by real-time PCR: comparison of gene amplification, heterozigoty, and immunohistochemical status in breast cancer tissue. Clin Chem 2003; 49: 219-9. google scholar
5. 5. Tsutsui S, Ohno S, Murakami S, Kataoka A, Kinoshita J, Hachitanda Y. Prognostic significance of the coexpression of p53 protein and c-erbB2 in breast cancer. Am J Surg 2003; 185: 165-7. google scholar
6. 6. Dimitrakakis C, Konstadoulakis M, Messaris E, Kymionis G, Karayannis M, Panoussopoulos D, et al. Molecular markers in breast cancer: Can we use c-erbB-2, p53, bcl-2 and bax gene expression as prognostic factors? Breast 2002; 11: 279-5. google scholar
7. 7. KKaltenboeck B, Wang C. Advances in real-time PCR: application toclinical laboratorydiagnostics.AdvCIin Chem 2005;40:219-59. google scholar
8. 8. Behl T, Kumar A, Vishakha, Sehgal A , Singh S , Sharma N , et al. Understanding the mechanistic pathways and clinical aspects associated with protein and gene based biomarkers in breast cancer. Int J Biol Macromol 2023; 253(Pt 1): 126595. google scholar

9. 9. Mocellin S, Rossi CR, Pilati P, Nitti D, Marincola FM. Quantitative real-time PCR: a powerful ally in cancer research. Trends Mol Med 2003; 9: 189-5. google scholar
10. 10. Viani GA, Afonso SL, Stefano EJ, De Fendi LI, Soares FV. Adjuvant trastuzumab in the treatment of her-2-positive early breast cancer: a metaanalysis of published randomized trials. BMC Cancer 2007; 7: 153. google scholar
11. 11. Murad NA, Razak ZA, Hussain RM, Hussain SA, Huat CK, Ali SA, et al. Quantification of Her-2/Neu gene in breast cancer patients using real time-polymerase chain reaction (Q-PCR) and correlation with immunohistochemistry findings. Asian Pacific J Cancer Prev 2013; 14: 1655-59. google scholar
12. 12. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ulrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu onkogene. Science 1987; 235: 177-2. google scholar
13. 13. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the HER-2/neu proto-onkogene in human breast and overian cancer. Science 1989; 244: 707-2. google scholar
14. 14. Borg A, Baldetotp B, Fernö M, Killander D, Olsson H, Ryden S, et al. ERBB2 amplification is associated with tamoxifen resistance in steroid -receptor positive breast cancer. Cancer Lett 1994; 81: 137-4. google scholar
15. 15. Battifora H, Gaffey M, Estaban J, Mehta P, Biley A, Faucett C, et al. Immunohistochemical assay of neu/ cerb2 onkogene product in parafin-embedded tissues in early breast cancer: retrospective follow-up study of 245 stage I and II cases. Modern Pathol 1991; 4: 466-4. google scholar
16. 16. Gentile M, Jungestrom MB, Olsen KE, Soderkvist P. p53 and survival in early onset breast cancer: analysis of gene mutations, loss of heterozygosity and protein Accumulation. Eur J Cancer 1999; 35: 1202-07. google scholar
17. 17. Fedorova O, Daks A , Shuvalov O, Kizenko A , Petukhov A, Gnennaya Y, et al.Attenuation of p53 mutant as an approach for treatment Her2-positive cancer. Cell Death Discovery 2020; 6: 100-8. google scholar
18. 18. Sangrajrang S, Arpornwirat W, Cheirsilpa A, Thisuphakorn P, Kalalak A, Sornprom A, et al. Serum p53 antibodies in correlation to other biological parameters of breast cancer. Cancer Detec Prev 2003; 27: 182-6. google scholar
19. 19. Keohavonga P, Gaoa W, Mady HH, Kanbour-Shakirb A, Melhemb MF. Analysis of p53 mutations in cells taken from paraffin-embedded tissue sections of ductal carcinoma in situ and atypical ductal hyperplasia of the breast. Cancer Lett 2004; 212: 121-30. google scholar
20. 20. Sadia H, Bhinder MA, Irshad A, Zahid B, Ahmed R, Ashiq S, et al. Determination of expression profile of p53 gene in different grades of breast cancer tissues by real time PCR. Afr Health Sci 2020; 20: 1273-82. google scholar