Biomarkers of resistance to anti-EGFR therapy in patients with left-sided colorectal cancer: a retrospective NGS analysis

Background: Routine analysis of KRAS, NRAS, BRAF V600 mutations, as well as MSI and HER2 guides treatment selection in colorectal cancer (CRC). Recent findings from the PRESSING and PARADIGM trials have demonstrated that the negative hyperselection of patients based on the results of comprehensive genomic profiling results in better treatment outcomes following anti-EGFR therapy. Study objective: The study aimed to retrospectively analyze the occurrence of alterations associated with potential resistance in samples from CRC patients treated with anti-EGFR therapy.

Materials and methods: Patients with confirmed left-sided CRC treated with anti-EGFR therapy due to the lack of RAS / BRAFV600 mutations as per routine PCR were included in the study. FFPE samples were analyzed via NGS (Solo-test Atlas Pro, 38 genes, MSI). Samples determined as RAS / BRAFV600positive by NGS were validated with PCR.

Results: A total of 111 samples were analyzed via NGS. A total of 172 alterations in 17 genes were found; alterations in any of the genes covered by the panel were found in 96 (86.5 %) samples. The variant allele frequency ranged 1.3–93.0 %. NGS identified 29 (26.1 %) samples with KRAS (n = 24), NRAS (n = 3) or BRAF p. V600E (n = 4) mutations. Confirmatory PCR testing of 16 RAS / BRAF p. V600E-positive samples resulted in 100 % agreement with NGS. Among RAS / BRAFV600negative samples, 10 (9 %) samples harbored other alterations (ERBB2 amplification, n = 3; PIK3CA, n = 2; MSI, n = 1; BRAF class II mutation, n = 1; ERBB3, n = 1). A total of 11 samples harbored more than 1 alterations associated with potential resistance. BRAF class II / III mutations were found in 4 samples, PIK3CA mutations — in 17 (15.3 %) samples (of those, 5 samples harbored mutations in exon 21).

Conclusions: The results of this retrospective analysis demonstrate that a high frequency of false-positive routine PCR results may lead to incorrect indication of anti-EGFR therapy in ~ 26 % cases. Analysis of alterations beyond RAS / BRAFV600 might identify an additional 9 % of patients whose tumors are potentially resistant to anti-EGFR therapy.

1. Malignant tumors in Russia in 2021 (morbidity and mortality). Eds.: А.D. Kaprin, V.V. Starinskiy, A.O. Shachzadova. Moscow: MNIOI im. P.A. Gertsena – filial FGBU “NMITS radiologii” Minzdrava Rossii, 2022.252 p (In Russ.)

2. Bray F., Laversanne M., Sung H., et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229–263. https://doi.org/10.3322/caac.21834 PMID: 38572751

3. Kazachenko E.A., Shubin V.P., Otstanov S.S., et al. The RAS/BRAF genes status in patients with colorectal cancer (review). Koloproktologia. 2024;23(3):112–125 (In Russ.). https://doi.org/10.33878/2073-7556-2024-23-3-112-125

4. Davies H., Bignell G.R., Cox C., et al. Mutations of the BRAF gene in human cancer. Nature 2002;417(6892):949–954. https://doi.org/10.1038/nature00766 PMID: 12068308

5. Barras D., Missiaglia E., Wirapati P., et al. BRAF V600E Mutant Colorectal Cancer Subtypes Based on Gene Expression. Clin Cancer Res 2017;23(1):104–115. https://doi.org/10.1158/1078-0432.CCR-16-0140

6. De Roock W., Claes B., Bernasconi D., et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol 2010;11(8):753–762. https://doi.org/10.1016/S1470-2045(10)70130-3

7. Cantwell-Dorris E.R., O’Leary J.J., Sheils O.M. BRAFV600E: implications for carcinogenesis and molecular therapy. Mol Cancer Ther 2011;10(3):385–394. https://doi.org/10.1158/1535-7163.MCT-10-0799

8. Arnold D., Lueza B., Douillard J.Y., et al. Prognostic and predictive value of primary tumour side in patients with RAS wild-type metastatic colorectal cancer treated with chemotherapy and EGFR directed antibodies in six randomized trials. Ann Oncol 2017;28(8):1713–1729. https://doi.org/10.1093/annonc/mdx175

9. Cervantes A., Adam R., Roselló S., et al. Metastatic colorectal cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up. Ann Oncol 2023;34(1):10–32. https://doi.org/10.1016/j.annonc.2022.10.003

10. Sun H., Li Y., Su Y., et al. Efficacy and safety of anti-EGFR monoclonal antibodies combined with different chemotherapy regimens in patients with RAS wild-type metastatic colorectal cancer: A meta-analysis. J Evid Based Med 2019;12(4):300–312. https://doi.org/10.1111/jebm.12360

11. Douillard J.Y., Siena S., Cassidy J., et al. Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the PRIME study. J Clin Oncol 2010;28(31):4697–4705. https://doi.org/10.1200/JCO.2009.27.4860

12. Peeters M., Price T.J., Cervantes A., et al. Randomized phase III study of panitumumab with fluorouracil, leucovorin, and irinotecan (FOLFIRI) compared with FOLFIRI alone as second-line treatment in patients with metastatic colorectal cancer. J Clin Oncol 2010;28(31):4706–4713. https://doi.org/10.1200/JCO.2009.27.6055

13. Van Cutsem E., Köhne C.H., Hitre E., et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med 2009;360(14):1408–1417. https://doi.org/10.1056/NEJMoa0805019

14. Amado R.G., Wolf M., Peeters M., et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008;26(10):1626–1634. https://doi.org/10.1200/JCO.2007.14.7116

15. Misale S., Yaeger R., Hobor S., et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 2012;486(7404):532–536. https://doi.org/10.1038/nature11156

16. Venook A.P., Niedzwiecki D., Lenz H.J., et al. Effect of first-line chemotherapy combined with cetuximab or bevacizumab on overall survival in patients with KRAS wild-type advanced or metastatic colorectal cancer: a randomized clinical trial. JAMA 2017;317(23):2392–2401. https://doi.org/10.1001/jama.2017.7105

17. Cremolini C., Antoniotti C., Lonardi S., et al. Activity and safety of cetuximab plus modified FOLFOXIRI followed by maintenance with cetuximab or bevacizumab for RAS and BRAF wild-type metastatic colorectal cancer: a randomized phase 2 clinical trial. JAMA Oncol 2018;4(4):529–536. https://doi.org/10.1001/jamaoncol.2017.5314

18. Bokemeyer C., Köhne C.H., Ciardiello F., et al. FOLFOX4 plus cetuximab treatment and RAS mutations in colorectal cancer. Eur J Cancer 2015;51(10):1243–1252. https://doi.org/10.1016/j.ejca.2015.04.007

19. Kim T.W., Elme A., Kusic Z., et al. A phase 3 trial evaluating panitumumab plus best supportive care vs best supportive care in chemorefractory wild-type KRAS or RAS metastatic colorectal cancer. Br J Cancer 2016;115(10):1206–1214. https://doi.org/10.1038/bjc.2016.309 PMID: 27736842

20. Douillard J.Y., Oliner K.S., Siena S., et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med 2013;369(11):1023–1034. https://doi.org/10.1056/NEJMoa1305275

21. Benson A.B., Venook A.P., Adam M., et al. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) Colon Cancer Version 3.2024 — May 24, 2024. Available on: https://www.nccn.org/professionals/physician_gls/pdf/colon.pdf

22. Morano F., Corallo S., Lonardi S., et al. Negative hyperselection of patients with RAS and BRAF wild-type metastatic colorectal cancer who received panitumumab-based maintenance therapy. J Clin Oncol 2019;37(33):3099–3110. https://doi.org/10.1200/JCO.19.01254

23. Watanabe J., Muro K., Shitara K., et al. Panitumumab vs bevacizumab added to standard first-line chemotherapy and overall survival among patients with RAS wild-type, left-sided metastatic colorectal cancer: a randomized clinical trial [published correction appears in JAMA. 2023 Jun 27;329(24):2196. JAMA 2023;329(15):1271–1282. https://doi.org/10.1001/jama.2023.4428

24. Vidal J., Bellosillo B., Santos Vivas C., et al. Ultra-selection of metastatic colorectal cancer patients using next-generation sequencing to improve clinical efficacy of anti-EGFR therapy. Ann Oncol 2019;30(3):439–446. https://doi.org/10.1093/annonc/mdz005

25. Ma W., Brodie S., Agersborg S., et al. Significant Improvement in Detecting BRAF, KRAS, and EGFR Mutations Using Next-Generation Sequencing as Compared with FDA-Cleared Kits. Mol Diagn Ther 2017;21(5):571–579. https://doi.org/10.1007/s40291-017-0290-z

26. Wolf J., Seto T., Han J.Y., et al. Capmatinib in MET Exon 14-Mutated or MET-Amplified Non-Small-Cell Lung Cancer. N Engl J Med 2020;383(10):944–957. https://doi.org/10.1056/NEJMoa2002787

27. Chen J., Xu C., Wang Q., et al. Exploration on the first-line treatment of ERBB2-altered advanced non-small cell lung cancer: A multicenter retrospective study. Lung Cancer 2023;183:107315. https://doi.org/10.1016/j.lungcan.2023.107315

28. Song Z., Lv D., Chen S.Q., et al. Pyrotinib in patients with HER2-amplified advanced non-small cell lung cancer: a prospective, multicenter, single-arm trial. Clin Cancer Res 2022;28(3):461–467. https://doi.org/10.1158/1078-0432.CCR-21-2936

29. Camidge D.R., Otterson G.A., Clark J.W., et al. Crizotinib in patients with MET-amplified NSCLC. J Thorac Oncol 2021;16(6):1017–1029. https://doi.org/10.1016/j.jtho.2021.02.010

30. Rodón J., Damian S., Furqan M., et al. Pemigatinib in previously treated solid tumors with activating FGFR1-FGFR3 alterations: phase 2 FIGHT-207 basket trial. Nat Med 2024;30(6):1645–1654. https://doi.org/10.1038/s41591-024-02934-7. Erratum in: Nat Med. 2024 Aug;30(8):2377. https://doi.org/10.1038/s41591-024-03072-w

31. Richards S., Aziz N., Bale S., et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17(5):405–424. https://doi.org/10.1038/gim.2015.30

32. Horak P., Griffith M., Danos A.M., et al. Standards for the classification of pathogenicity of somatic variants in cancer (oncogenicity): Joint recommendations of Clinical Genome Resource (ClinGen), Cancer Genomics Consortium (CGC), and Variant Interpretation for Cancer Consortium (VICC) [published correction appears in Genet Med. 2022 Sep;24(9):1991. Genet Med 2022;24(5):986–998. https://doi.org/10.1016/j.gim.2022.01.001

33. Van Cutsem E., Lenz H.J., Köhne C.H., et al. Fluorouracil, leucovorin, and irinotecan plus cetuximab treatment and RAS mutations in colorectal cancer. J Clin Oncol 2015;33(7):692–700. https://doi.org/10.1200/JCO.2014.59.4812

34. Shitara K., Muro K., Watanabe J., et al. Baseline ctDNA gene alterations as a biomarker of survival after panitumumab and chemotherapy in metastatic colorectal cancer. Nat Med 2024;30(3):730–739. https://doi.org/10.1038/s41591-023-02791-w

35. Innocenti F., Ou F.S., Qu X., et al. Mutational analysis of patients with colorectal cancer in CALGB/SWOG 80405 identifies new roles of microsatellite instability and tumor mutational burden for patient outcome. J Clin Oncol 2019;37(14):1217–1227. https://doi.org/10.1200/JCO.18.01798

36. Доступно по: https://roszdravnadzor.gov.ru/

37. Nguyen B., Fong C., Luthra A., et al. Genomic characterization of metastatic patterns from prospective clinical sequencing of 25,000 patients. Cell 2022;185(3):563–575.e11. https://doi.org/10.1016/j.cell.2022.01.003

38. Pietrantonio F., Bergamo F., Rossini D., et al. Negative hyperselection of elderly patients with RAS and BRAF wild-type metastatic colorectal cancer receiving initial panitumumab plus FOLFOX or 5-FU/LV. Eur J Cancer 2023;195:113396. https://doi.org/10.1016/j.ejca.2023.113396

39. Su W.C., Tsai Y.C., Tsai H.L., et al. Comparison of next-generation sequencing and polymerase chain reaction for personalized treatment-related genomic status in patients with metastatic colorectal cancer. Curr Issues Mol Biol 2022;44(4):1552–1563. https://doi.org/10.3390/cimb44040106

40. Kothari N., Schell M.J., Teer J.K., et al. Comparison of KRAS mutation analysis of colorectal cancer samples by standard testing and next-generation sequencing. J Clin Pathol 2014;67(9):764–767. https://doi.org/10.1136/jclinpath-2014-202405

41. Darwanto A., Hein A.M., Strauss S., et al. Use of the QIAGEN GeneReader NGS system for detection of KRAS mutations, validated by the QIAGEN Therascreen PCR kit and alternative NGS platform. BMC Cancer 2017;17(1):358. https://doi.org/10.1186/s12885-017-3328-z

42. Laurent-Puig P., Pekin D., Normand C., et al. Clinical relevance of KRAS-mutated subclones detected with picodroplet digital PCR in advanced colorectal cancer treated with anti-EGFR therapy. Clin Cancer Res 2015;21(5):1087–1097. https://doi.org/10.1158/1078-0432.CCR-14-0983.