Molecular Diagnostics of Oncological Disease: Prospects for the Development of a Reference Material for the HER2 gene Content

Cancer is the leading cause of death in the world. The development of oncopathology is closely related to various changes in the genetic material that occur in malignantly transformed cells. Medical decision-making requires a clear differentiation between normal and pathological indicators, which are, among other things, the results of application of quantitative methods in laboratory medicine. Studies of DNA isolated from a patient’s biological material, identification and measurement of the content of nucleotide sequences acting as oncopathology biomarkers allow to solve the problems of determining the genetic prerequisites for cancer, its early diagnosis, determining the treatment strategy, monitoring, and confirming the patient’s cure.

The purpose of this research is to develop the main approaches to the design of DNA reference materials (RMs) for metrological support of molecular diagnostics of oncopathology through the example of the RM for the HER2 gene sequence content in the human genome, with the value of «the number of copies of the DNA sequence» which is metrologically traceable to the natural SI unit «one».

In the course of the research, a technique for measuring the HER2 gene amplification (the number of copies of the gene sequence per genome) was developed based on the use of the digital PCR method (dPCR). Comparability of measurement results for the method developed by the authors, and the results obtained using a commercial kit by the MLPA method on samples of human biological material is shown.

Five permanent cell lines obtained from the CUC «Vertebrate Cell Culture Collection» were characterized in relation to the copy number ratios of HER2 gene sequence and CEP17 and RPPH1 genes sequences. A cell line with the HER2 gene amplification was identified. The results obtained will be used to create the RM for the copy number ratio of the HER2 gene sequences and the RPPH1 and CEP17 gene sequences. Creation of matrix DNA RMs based on human cell cultures certified using dPCR will allow transferring the unit of copy numbers of the DNA sequence to calibrators included in medical devices, thereby ensuring the required reliability and comparability of measurement results in the laboratory diagnostics of oncopathology, as well as the possibility of calibrating routine methods of DNA diagnostics and intralaboratory quality control.

1. Sung H., Ferlay J., Siegel R. L., Laversanne M., Soerjomataram I., Jemal A. et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 2021;71(3):209– 249. https://doi.org/10.3322/caac.21660

2. Loeb K. R., Loeb L. A. Significance of multiple mutations in cancer. Carcinogenesis. 2000;21(3):379–385. https://doi.org/10.1093/carcin/21.3.379

3. Kuchenbaecker K. B., Hopper J. L., Barnes D. R., Phillips K.-A., Mooij T. M., Roos-Blom M.-J. Risks et al. of breast, ovarian, and contralateral breast cancer for BRCA1 and BRCA2 mutation carriers. JAMA. 2017;317(23):2402–2416. https://doi:10.1001/jama.2017.7112

4. Antoniou A., Pharoah P. D. P., Narod S., Risch H. A., Eyfjord J. E., Hopper J. L. et al. Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: A combined analysis of 22 studies. American Journal of Human Genetics. 2003;72(5):1117–1130. https://doi:10.1086/375033

5. Chen S., Parmigiani G. Meta-analysis of BRCA1 and BRCA2 penetrance. Journal of Clinical Oncology. 2007;25(11):1329–1333. https://doi:10.1200/JCO.2006.09.1066

6. Li Sh., Silvestri V., Leslie G., Rebbeck T. R., Neuhausen S. L., Hopper J. L. et al. Cancer risks associated with BRCA1 and BRCA2 pathogenic variants. Journal of Clinical Oncology. 2022;40(14):1529–1541. https://doi.org/10.1200/JCO.21.02112

7. Berger M. F., Mardis E. R. The emerging clinical relevance of genomics in cancer medicine. Nature Reviews Clinical Oncology. 2018;15(6):353–365. https://doi.org/10.1038/s41571-018-0002-6

8. Haratani K., Hayashi H., Tanaka T., Kaneda H., Togashi Y., Sakai K. et al. Tumor immune microenvironment and nivolumab efficacy in EGFR mutation-positive non-small-cell lung cancer based on T790M status after disease progression during EGFR-TKI treatment. Annals of Oncology. 2017;28(7):1532–1539. https://doi.org/10.1093/annonc/mdx183

9. Cutsem E. V., Lenz H.-J., Köhne C.-H., Heinemann V., Tejpar S., Melezínek I. et al. Fluorouracil, leucovorin, and irinotecan plus cetuximab treatment and RAS mutations in colorectal cancer. Journal of Clinical Oncology. 2015;33(7):692–700. https://doi.org/10.1200/JCO.2014.59.4812

10. He H.-J., Almeida J. L., Lund S., Steffen C. R. Development of NIST standard reference material 2373: Genomic DNA standards for HER2 measurements. Biomolecular Detection and Quantification. 2016;8:1–8. https://doi.org/10.1016/j.bdq.2016.02.001

11. He H.-J., Das B., Cleveland M. H., Chen L., Camalier C. E., Liu L.-Ch. et al. Development and interlaboratory evaluation of a NIST reference material RM 8366 for EGFR and MET gene copy number measurements. Clinical Chemistry and Laboratory Medicine. 2019;57(8):1142–1152. https://doi.org/10.1515/cclm-2018–1306

12. Milavec M., Cleveland M. H., Bae Y.-K., Wielgosz R. I., Vonsky M., Huggett J. F. et al. Metrological framework to support accurate, reliable, and reproducible nucleic acid measurements. Analytical and Bioanalytical Chemistry. 2022;414(2):791–806. https://doi.org/10.1007/s00216–021–03712-x

13. Alkabban F. M., Ferguson T. Breast cancer. Treasure Island: StatPearls Publishing; 2022. Available at: https://www.ncbi.nlm.nih.gov/books/NBK482286 [Accessed 08 November 2022].

14. Bose R., Kavuri Sh. M., Searleman A. C., Shen W., Shen D., Koboldt D. C. et al. Activating HER2 mutations in HER2 gene amplification negative breast cancer. Cancer Discov. 2013;3(2):224–237. https://doi.org/10.1158/2159–8290.CD-12–0349

15. Iqbal N., Iqbal N. Human epidermal growth factor receptor 2 (HER2) in cancers: overexpression and therapeutic implications. Molecular Biology International. 2014;852748. https://doi:10.1155/2014/852748

16. Moasser M. M. The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis. Oncogene. 2007;26(45):6469–6487. https://doi:10.1038/sj.onc.1210477

17. Lee K., Kim H. J., Jang M. H., Lee S., Ahn S., Park Y. S. Centromere 17 copy number gain reflects chromosomal instability in breast cancer. Scientific Reports. 2019;9(1):17968. https://doi.org/10.1038/s41598–019–54471-w

18. Scherer W. F., Syverton J. T., Gey, G. O. Studies on the propagation in vitro of poliomyelitis viruses. IV. Viral multiplication in a stable strain of human malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix. Journal of Experimental Medicine. 1953;97(5):695–710. https://doi:10.1084/jem.97.5.695

19. Zhu Y., Lu D., Lira M. E., Xu Q., Du Y., Xiong J. et al. Droplet digital polymerase chain reaction detection of HER2 amplification in formalin fixed paraffin embedded breast and gastric carcinoma samples. Experimental and Molecular Pathology. 2015;100(2):287– 293. https://doi:10.1016/j.yexmp.2015.11.027

20. Romsos E. L., Kline M. C., Duewer D. L., Toman B., Farkas N. Certification of standard reference material 2372a; Human DNA quantitation standard. NIST Special Publication. 2018:260–189. https://doi:10.6028/NIST.SP.260–189

21. Stuppia L., Antonucci I., Palka G., Gatta V. Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. International Journal of Molecular Sciences. 2012;13(3):3245–3276. https://doi:10.3390/ijms13033245

22. Cousineau I., Belmaaza A. EMSY overexpression disrupts the BRCA2/ RAD51 pathway in the DNA-damage response: Implications for chromosomal instability/recombination syndromes as checkpoint diseases. Molecular Genetics and Genomics. 2011;285(4):325– 340. https://doi:10.1007/s00438-011-0612-5