Liquid biopsy of colorectal cancer: a new approach to evaluation of aberrant methylation of the SEPT9 gene

Introduction. Hypermethylated CpG islands in the promoters of suppressor genes (in particular, SEPT9) are clinically significant markers of malignant growth that are widely used in liquid biopsy. Real-time polymerase chain reaction with methylation-specific primers is commonly used to quantify hypermethylated DNA. The method requires data normalization, depends on copy number variability of the calibrator genes, and is rather laborious.

The study subject is to develop an alternative qDMA method (quantitative DNA Melting Analysis).

Materials and methods. DNA samples isolated from blood plasma of healthy donors and colorectal cancer patients were analyzed by the method including: 1) asymmetric polymerase chain reaction with methylation-independent individually selected primers for the SEPT9 gene; 2) using the TaqMan probe hybridizing to two CpG dinucleotides in the amplicon; 3) post-amplification melting of probe/amplicon hybrids; 4) quantitative analysis of DNA melting.

Results. The method was tested on the SEPT9 gene in liquid biopsy of colorectal cancer. Differences in SEPT9 methylation in healthy donors (n = 41) and cancer patients (n = 39) were statistically significant (p <0.0001). Analytical sensitivity and diagnostic efficiency of qDMA were determined: AUC (area under curve) ROC– 0.812 (according to the result of 10-fold cross-validation AUC ROC – 0.801), sensitivity – 90%, specificity – 66%.

Conclusion. The proposed method for the quantitative assessment of aberrantly methylated DNA is simple, implemented in the closed-tube format, does not require normalization and usage of standard curves. The possibility of optimization through the use of a multiplex variant with simultaneous analysis of several markers is assumed.

1. Serrano M.J., Garrido-Navas M.C., Diaz Mochon J.J. et al. Precision prevention and cancer interception: the new challenges of liquid biopsy. Cancer Discovery 2020;10(11):1635–44. DOI: 10.1158/2159-8290.CD-20-0466.

2. Cescon D.W., Bratman S.V., Chan S.M. et al. Circulating tumor DNA and liquid biopsy in oncology. Nature Cancer 2020;1:276–90. DOI: 10.1038/s43018-020-0043-5.

3. Xiong Z., Laird P.W. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 1997;25(12):2532–4. DOI: 10.1093/nar/25.12.2532.

4. Worm J., Aggerholm A., Guldberg P. In-tube DNA methylation profiling by fluorescence melting curve analysis. Clinical Chemistry 2001;47(7):1183–9.

5. Tse M.Y., Ashbury J.E., Zwingerman N. et al. A refined, rapid and reproducible high resolution melt (HRM)-based method suitable for quantification of global LINE-1 repetitive element methylation. BMC Research Notes 2011;4:565. DOI: 10.1186/1756-0500-4-565.

6. Quillien V., Lavenu A., Karayan-Tapon L. et al. Comparative assessment of 5 methods (methylation-specific polymerase chain reaction, MethyLight, pyrosequencing, methylation-sensitive high-resolution melting, and immunohistochemistry) to analyze O6-methylguanine-DNA-methyltranferase in a series of 100 glioblastoma patients. Cancer 2012;118(17):4201–11. DOI: 10.1002/cncr.27392.

7. Korshunova Y., Maloney R.K., Lakey N. et al. Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA. Genome Res 2008;18(1):19–29. DOI: 10.1101/gr.6883307.

8. Sepulveda A.R., Jones D., Ogino S. et al. CpG methylation analysis-current status of clinical assays and potential applications in molecular diagnostics: a report of the Association for Molecular Pathology. J Mol Diagn 2009;11(4):266–78. DOI: 10.2353/jmoldx.2009.080125.

9. Egger G., Wielscher M., Pulverer W. et al. DNA methylation testing and marker validation using PCR: diagnostic applications. Expert Rev Mol Diagn 2012;12(1):75–92. DOI: 10.1586/erm.11.90.

10. Dietrich D., Jung M., Puetzer S. et al. Diagnostic and prognostic value of SHOX2 and SEPT9 DNA methylation and cytology in benign, paramalignant and malignant pleural effusions. PLoS ONE 2013;8(12):e84225. DOI: 10.1371/journal.pone.0084225.

11. De Vos L., Gevensleben H., Schrock A. et al. Comparison of quantification algorithms for circulating cell-free DNA methylation biomarkers in blood plasma from cancer patients. Clin Epigenetics 2017;9(1):125. DOI: 10.1186/s13148-017-0425-4.

12. Pharo H.D., Honne H., Vedeld H.M. et al. Experimental factors affecting the robustness of DNA methylation analysis. Sci Rep 2016;6:33936. DOI: 10.1038/srep33936.

13. Pharo H.D., Andresen K., Berg K.C.G. et al. A robust internal control for high-precision DNA methylation analyses by droplet digital PCR. Clin Epigenetics 2018;10:24. DOI: 10.1186/s13148-018-0456-5.

14. Warnecke P.M., Stirzaker C., Melki J.R. et al. Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res 1997;25(21):4422–6. DOI: 10.1093/nar/25.21.4422.

15. Wojdacz T.K., Hansen L.L., Dobrovic A. A new approach to primer design for the control of PCR bias in methylation studies. BMC Res Notes 2008;1:54. DOI: 10.1186/1756-0500-1-54.

16. Wojdacz T.K., Borgbo T., Hansen L.L. Primer design versus PCR bias in methylation independent PCR amplifications. Epigenetics 2009;4(4):231–4. DOI: 10.4161/epi.9020.

17. Botezatu I.V., Kondratova V.N., Shelepov V.P. et al. Asymmetric mutantenriched polymerase chain reaction and quantitative DNA melting analysis of KRAS mutation in colorectal cancer. Anal Biochem 2020;590:1–9. DOI: 10.1016/j.ab.2019.113517.

18. Kondratova V.N., Botezatu I.V., Shelepov V.P. et al. SLAM-MS: mutation scanning of stem-loop amplicons with TaqMan probes by quantitative DNA melting analysis. Sci Rep 2020;10:5476. DOI: 10.1038/s41598-020-62173-x.

19. Lazarevich N.L., Abramov P.M., Fedorova M.D. et al. Identification of a new methylation site in the Sept9 promoter region for the diagnosis of hepatocellular carcinoma. Uspekhi molekulyarnoy onkologii = Advances in Molecular Oncology 2019;6:26–37. (In Russ.). DOI: 10.17650/2313-805X-2019-6-4-26-37.

20. Sui J., Wu X., Wang C. et al. Discovery and validation of methylation signatures in blood-based circulating tumor cell-free DNA in early detection of colorectal carcinoma: a case-control study. Clin Epigenetics 2021;13:26. DOI: 10.1186/s13148-020-00985-4.

21. Wasserkort R., Kalmar A., Valcz G. et al. Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island. BMC Cancer 2013;13:398. DOI: 10.1186/1471-2407-13-398.

22. Schutz E., von Ahsen N. Spreadsheet software for thermodynamic melting point prediction of oligonucleotide hybridization with and without mismatches. Biotechniques 1999;27(6):1218–22, 1224. DOI: 10.2144/99276bc04.

23. Mazzara S., Rossi R.L., Grifantini R. et al. CombiROC: an interactive web tool for selecting accurate marker combinations of omics data. Sci Rep 2017;7:45477. DOI: 10.1038/srep45477.

24. Botezatu I.V., Panchuk I.O., Stroganova A.M. et al. TaqMan probes as blocking agents for enriched PCR amplification and DNA melting analysis of mutant genes. Biotechniques 2017;62(2):62–8. DOI: 10.2144/000114515.

25. Montgomery J.L., Rejali N., Wittwer C.T. The influence of nucleotide sequence and temperature on the activity of thermostable DNA polymerases. J MolDiagn 2014;16(3):305–13. DOI: 10.1016/j.jmoldx.2014.01.006.

26. Livak K.J., Flood S.J., Marmaro J. et al. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 1995;4(6):357–62. DOI: 10.1101/gr.4.6.357.

27. Huang Q., Liu Z., Liao Y. et al. Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes. PLoS ONE 2011;6(4):e19206. DOI: 10.1371/journal.pone.0019206.

28. Botezatu I.V., Nechaeva I.O., Stroganova C.A. et al. Optimization of melting analysis with Taqman probes for detection of KRAS, NRAS and BRAF mutations. Anal Biochem 2015;491:75–83. DOI: 10.1016/j.ab.2015.09.005.

29. Armbruster D.A., Pry T. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev 2008;29(Suppl 1):S49–52.

30. Wojdacz T.K., Dobrovic A. Methylation-sensitive high resolution melting (MS- HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res 2007;35(6):e41. DOI: 10.1093/nar/gkm013.

31. Malentacchi F., Forni G., Vinci S. et al. Quantitative evaluation of DNA methylation by optimization of a differential-high resolution melt analysis protocol. Nucleic Acids Res 2009;37(12):e86. DOI: 10.1093/nar/gkp383.

32. Spitzwieser M., Entfellner E., Werner B. et al. Hypermethylation of CDKN2A exon 2 in tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients. BMC Cancer 2017;17:260. DOI: 10.1186/s12885-017-3244-2.