Genetic locus copy number variation level and minimally invasive assessment of radiation therapy efficiency in rectal cancer patients

Aim. The screening of molecular genetic markers for a minimally invasive assessment of the radiation therapy effectiveness for rectum malignant tumors.

Materials and methods. The study was carried out in 4 stages: 1) bioinformatic analysis of TCGA (The Cancer Ge- nome Atlas) databases using the GISTIC algorithm; 2) validation of bioinformatics analysis data in a model experiment on cell culture; 3) study of genes copy number features validated in a model experiment in patients with different responses to radiation therapy; 4) determination of the gene copy number in cell-free DNA in patientswith different responses to radiation therapy. 100 patients with rectum adenocarcinoma (G1–2), as well as 30 apparently healthy individuals, took part in the work. Radiotherapy was carried out according to the standard protocol (single focal dose 2.4 Gy, total focal dose 54 Gy) on a Novalis TX linear accelerator. The relative copy number of genetic loci was determined by real-time quantitative polymerase chain reaction.

Results. Bioinformatic analysis revealed 32 candidate genetic loci. Validation of these markers on irradiated HT-29 cells showed that the copy number of BRCA2, H2AX, CASP9 and RBBP8 genes was increased, while the copy number of BCL2 gene was reduced relative to intact cells. In 74 patients with a partial response to radiation therapy, an increase in the copy number of BRCA2, H2AX, RBBP8 and BCL2 was found, which positively correlated with the copy number of these genes in blood plasma cell-free DNA.

Conclusion. The application of an integrated approach based on TCGA database bioinformatic analysis, radiation therapy modeling in cell culture and validation of the identified markers on tissue and blood samples of patients with rectal adenocarcinoma revealed RBBP8, BRCA2, H2AX and BCL2 genes copy number association with the preoperative radiation therapy effectiveness.

1. Kutilin D.S., Gusareva M.A., Kosheleva N.G. et al. Genetic and epigenetic predictors of rectal tumors radiotherapy effectiveness. Sovremenniye problemy nauki i obrazovaniya = Modern problems of science and education 2021;4. Available at: https://science-education.ru/ru/article/view?id=30963 (In Russ.). DOI: 10.17513/spno.30963

2. Romano G.M., Bianco F., De Franciscis S., Belli A. The management of recurrent rectal cancer: a European perspective. In: Comprehensive Rectal Cancer Care. Ed. by M. Kwaan, A. Zbar. Springer, Cham: 2019. P. 521–536. DOI: 10.1007/978-3-319-98902-0_27

3. Cammà C., Giunta M., Fiorica F. et al. Preoperative radiotherapy for resectable rectal cancer: a meta-analysis. JAMA 2000;284(8):1008–15. DOI: 10.1001/jama.284.8.1008

4. Hearn N., Atwell D., Cahill K. et al. Neoadjuvant radiotherapy dose escalation in locally advanced rectal cancer: a systematic review and meta-analysis of modern treatment approaches and outcomes. Clin Oncol (R Coll Radiol) 2021;33(1):e1–e14. DOI: 10.1016/j.clon.2020.06.008

5. Sun W., Li G., Wan J. et al. Circulating tumor cells: a promising marker of predicting tumor response in rectal cancer patients receiving neoadjuvant chemo-radiation therapy. Oncotarget 2016;7(43):69507–17. DOI: 10.18632/oncotarget. 10875

6. Kutilin D.S., Kosheleva N.G., Gusareva M.A. et al. Gene copy number variation as a factor of radioresistance of colon adenocarcinoma cells of the line HT-29. Sovremenniye problemy nauki i obrazovaniya = Modern problems of science and education 2019;5. Available at: https://science-education.ru/ru/article/view?id=29224. (In Russ.). DOI: 10.17513/spno.29224

7. Kutilin D.S., Tsandekova M.R., Porkhanova N.V. Features of the copy number variation of certain genes in tumor cells in patients with serous ovarian adenocarcinoma. Bull Exp Biol Med 2021;170(3):332–9. DOI: 10.1007/s10517-021-05062-1

8. Tsandekova M.R., Porkhanova N.V., Kit O.I., Kutilin D.S. Minimally invasive molecular diagnostics of serous ovarian adenocarcinoma of high and low malignancy. Onkoginekologiya = Oncogynecology 2021;4(40):35–49. (In Russ.). DOI: 10.52313/22278710_2021_4_35

9. Kutilin D. Genetic and epigenetic bases of prostate tumor cell radioresistance. Klin Onkol 2021;34(3):220–34. DOI: 10.48095/ccko2021220

10. Kutilin D.S., Kosheleva N.G., Maksimov A.Yu. et al. Effects of various radiotherapy doses on survival of colon adenocarcinoma cells line HT-29. Sovremennye problemy nauki i obrazovaniya = Modern problems of science and education 2019;3. Available at: https://science-education.ru/ru/article/view?id=28918 (In Russ.). DOI: 10.17513/spno.28918

11. Tamkovich S.N., Bryzgunova O.E. Circulating NK in the blood of patients with stomach and colon cancer. Klinicheskie issledovaniya = Clinical studies 2005;51(3):321–8. (In Russ.).

12. Frydrych L.M., Ulintz P., Bankhead A. et al. Rectal cancer subclones respond differentially to neoadjuvant therapy. Neoplasia 2019;21(10):1051–62. DOI: 10.1016/j.neo.2019.08.004

13. Kim S.C., Shin Y.K., Kim Y.A. et al. Identification of genes inducing resistance to ionizing radiation in human rectal cancer cell lines: re-sensitization of radio-resistant rectal cancer cells through down regulating NDRG1. BMC Cancer 2018;18(1):594. DOI: 10.1186/s12885-018-4514-3

14. Ghadimi B.M., Grade M., Difilippantonio M.J. et al. Effectiveness of gene expression profiling for response prediction of rectal adenocarcinomas to preoperative chemoradiotherapy. J Clin Oncol 2005,23(9):1826–38. DOI: 10.1200/JCO.2005.00.406

15. Mandard A.M., Dalibard F., Mandard J.C. et al. Pathologic assessment of tumor regression after preoperative chemoradiotherapy of esophageal carcinoma. Clinicopathologic correlations. Cancer 1994;73(11):2680–6. DOI: 10.1002/1097-0142(19940601)73:11<2680::aid-cncr2820731105>3.0.co;2-c

16. Camps J., Grade M., Nguyen Q.T. et al. Chromosomal breakpoints in primary colon cancer cluster at sites of structural variants in the genome. Cancer Res 2008;68(5):1284–95. DOI: 10.1158/0008-5472.CAN-07-2864

17. Grade M., Gaedcke J., Wangsa D. et al. Chromosomal copy number changes of locally advanced rectal cancers treated with preoperative chemoradiotherapy. Cancer Genet Cytogenet 2009;193(1):19–28. DOI: 10.1016/j.cancergencyto. 2009.03.016

18. Diep C.B., Kleivi K., Ribeiro F.R. et al. The order of genetic events associated with colorectal cancer progression inferred from meta-analysis of copy number changes. Genes Chromosomes Cancer 2006;45(1):31–41. DOI: 10.1002/gcc.20261

19. Molinari C., Ballardini M., Teodorani N. et al. Genomic alterations in rectal tumors and response to neoadjuvant chemoradiotherapy: an exploratory study. Radiat Oncol 2011;6:161. DOI: 10.1186/1748-717X-6-161

20. Ashktorab H., Schäffer A.A., Daremipouran M. et al. Distinct genetic alterations in colorectal cancer. PLoS One 2010;5(1):e8879. DOI: 10.1371/journal.pone.0008879

21. Douglas J.K., Callahan R.E., Hothem Z.A. et al. Genomic variation as a marker of response to neoadjuvant therapy in locally advanced rectal cancer. Mol Cell Oncol 2020;7(3):1716618. DOI: 10.1080/23723556.2020.1716618

22. Kozlov V.A. Free extracellular DNA is normal and pathological. Meditsinskaya immunologiya = Medical Immunology 2013;15(5):399–412. (In Russ.). DOI: 10.15789/1563-0625-2013-5-399-412