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NRAS-BRAF Mutation Analysis by PCR

Clinical Significance

NRAS
The NRAS proto-oncogene encodes a GTPase that functions in signal transduction and is a member of the RAS superfamily which also includes KRAS and HRAS. RAS proteins mediate the transmission of growth signals from the cell surface to the nucleus via the PI3K/AKT/MTOR and RAS/RAF/MEK/ERK pathways, which regulate cell division, differentiation, and survival[PMID: 21993244;PMID: 18568040;PMID: 27341593]. Recurrent mutations in RAS oncogenes cause constitutive activation and are found in 20-30% of cancers. NRAS mutations are particularly common in melanomas (up to 25%) and are observed at frequencies of 5-10% in acute myeloid leukemia, colorectal, and thyroid cancers[PMID: 24071849;PMID: 21829508]. The majority of NRAS mutations consist of point mutations at G12, G13, and Q61[PMID: 24071849;PMID: 23515407]. Mutations at A59, K117, and A146 have also been observed but are less frequent[PMID: 22588877;PMID: 26438111].
Currently, no therapies are approved for NRAS aberrations. The EGFR antagonists, cetuximab[FDA-cetuximab: ERBITUX] and panitumumab[FDA-panitumumab: VECTIBIX], are contraindicated for treatment of colorectal cancer patients with NRAS mutations in exon 2 (codons 12 and 13), exon 3 (codons 59 and 61), and exon 4 (codons 117 and 146)[PMID: 26438111]. NRAS mutations are associated with poor prognosis in patients with low-risk myelodysplastic syndrome[NCCN-Myelodysplastic Syndromes] as well as melanoma[PMID: 25796376]. In a phase III clinical trial in patients with advanced NRAS-mutant melanoma, binimetinib improved progression free survival (PFS) relative to dacarbazine with median PFS of 2.8 and 1.5 months, respectively[PMID: 28284557].

BRAF
The BRAF gene encodes the B-Raf proto-oncogene serine/threonine kinase, a member of the RAF family of serine/threonine protein kinases which also includes ARAF and RAF1 (CRAF). BRAF is among the most commonly mutated kinases in cancer. Activation of the MAPK pathway occurs through BRAF mutations and leads to an increase in cell division, dedifferentiation, and survival[PMID: 29148538;PMID: 30315274]. BRAF mutations are categorized into three distinct functional classes namely, class 1, 2, and 3, and are defined by the dependency on the RAS pathway. Class 1 and 2 BRAF mutants are RAS-independent in that they signal as active monomers (Class 1) or dimers (Class 2) and become uncoupled from RAS GTPase signaling, resulting in constitutive activation of BRAF[PMID: 31470866]. Class 3 mutants are RAS dependent as the kinase domain function is impaired or dead[PMID: 28783719; PMID: 31470866; PMID: 31533235].
Recurrent somatic mutations in BRAF are observed in 40-60% of melanoma and thyroid cancer, approximately 10% of colorectal cancer, and about 2% of non-small cell lung cancer (NSCLC)[PMID: 25417114;PMID: 22588877;PMID: 22810696;PMID: 25079552;PMID: 24071849]. Mutations at V600 belong to class 1 and include V600E, the most recurrent somatic BRAF mutation across diverse cancer types[PMID: 28783719;PMID: 15035987]. Class 2 mutations include K601E/N/T, L597Q/V, G469A/V/R/, G464V/E/, and BRAF fusions[PMID: 28783719]. Class 3 mutations include D287H, V459L, G466V/E/A, S467L, G469E, and N581S/I[PMID: 28783719]. BRAF V600E is universally present in hairy cell leukemia, mature B-cell cancer, and prevalent in histiocytic neoplasms[PMID: 21663470;PMID: 26566875;PMID: 24569458]. Other recurrent BRAF somatic mutations cluster in the glycine-rich phosphate-binding loop at codons 464-469 in exon 11 as well as additional codons flanking V600 in the activation loop[PMID: 15035987]. In primary cancers, BRAF amplification is observed in 8% of ovarian cancer and about 1% of breast cancer[PMID: 22588877;PMID: 24071849]. BRAF fusions are mutually exclusive to BRAF V600 mutations and have been described in melanoma, thyroid cancer, pilocytic astrocytoma, NSCLC, and several other cancer types[PMID: 15630448;PMID: 20526349;PMID: 18974108;PMID: 21424530;PMID: 26314551]. Part of the oncogenic mechanism of BRAF gene fusions is the removal of the N-terminal auto-inhibitory domain leading to constitutive kinase activation[PMID: 15630448;PMID: 18974108;PMID: 31533235].
Vemurafenib[FDA-vemurafenib: ZELBORAF] (2011) was the first targeted therapy approved for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E mutation. BRAF class 1 mutations, including V600E, are sensitive to vemurafenib, whereas class 2 and 3 mutations are insensitive[PMID: 28783719]. BRAF kinase inhibitors including dabrafenib[FDA-dabrafenib: TAFINLAR] (2013) and encorafenib[FDA-encorafenib: BRAFTOVI] (2018) are also approved for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E/K mutations. Encorafenib[FDA-encorafenib: BRAFTOVI] is approved in combination with cetuximab[FDA-cetuximab: ERBITUX] (2020) for the treatment of BRAF V600E mutated colorectal cancer. Due to the tight coupling of RAF and MEK signaling, several MEK inhibitors have been approved for patients harboring BRAF alterations[PMID: 28783719]. Trametinib[FDA-trametinib: MEKINIST] (2013) and binimetinib[FDA-binimetinib: MEKTOVI] (2018) were approved for the treatment of metastatic melanoma with BRAF V600E/K mutations. Combination therapies of BRAF plus MEK inhibitors have been approved in melanoma and NSCLC. The combinations of dabrafenib/trametinib (2015) and vemurafenib/cobimetinib[FDA-cobimetinib: COTELLIC] (2015) were approved for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E/K mutation. Subsequently, the combination of dabrafenib and trametinib was approved for metastatic NSCLC (2017) with a BRAF V600E mutation. The PD-L1 antibody, atezolizumab[FDA-atezolizumab: TECENTRIQ], has also been approved in combination with cobimetinib and vemurafenib for BRAF V600 mutation-positive unresectable or metastatic melanoma. In 2018, binimetinib[FDA-binimetinib: Breakthrough] was also granted breakthrough designation in combination with cetuximab and encorafenib for BRAF V600E mutant metastatic colorectal cancer. The pan-RAF kinase inhibitor, tovorafenib (DAY-101), was granted breakthrough therapy designation (2020) by the FDA for pediatric patients with advanced low-grade glioma harboring activating RAF alterations[FDA-tovorafenib: Breakthrough]. The ERK inhibitor ulixertinib[FDA-ulixertinib: Fast Track] was also granted a fast track designation in 2020 for the treatment of patients with non-colorectal solid tumors harboring BRAF mutations G469A/V, L485W, or L597Q. BRAF fusion is a suggested mechanism of resistance to BRAF targeted therapy in melanoma[PMID: 28539463]. Additional mechanisms of resistance to BRAF targeted therapy include BRAF amplification and alternative splice transcripts as well as activation of PI3K signaling and activating mutations in KRAS, NRAS, and MAP2K1/2 (MEK1/2)[PMID: 26608120;PMID: 21107323;PMID: 24463458;PMID: 24265152;PMID: 24265153;PMID: 24055054;PMID: 24265155;PMID: 24463458]. Clinical responses to sorafenib and trametinib in limited case studies of patients with BRAF fusions have been reported[PMID: 26314551].

REFERENCES

NRAS

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  2. [PMID: 18568040]Karnoub et al. Ras oncogenes: split personalities. Nat. Rev. Mol. Cell Biol. 2008 Jul;9(7):517-31. PMID: 18568040
  3. [PMID: 27341593]Scott et al. Therapeutic Approaches to RAS Mutation. Cancer J. 2016 May-Jun;22(3):165-74. doi: 10.1097/PPO.0000000000000187. PMID: 27341593
  4. [PMID: 24071849]Weinstein et al. The Cancer Genome Atlas Pan-Cancer analysis project. Nat. Genet. 2013 Oct;45(10):1113-20. PMID: 24071849
  5. [PMID: 21829508]Janku et al. PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers. PLoS ONE. 2011;6(7):e22769. PMID: 21829508
  6. [PMID: 23515407]Ohashi et al. Characteristics of lung cancers harboring NRAS mutations. Clin. Cancer Res. 2013 May 1;19(9):2584-91. PMID: 23515407
  7. [PMID: 22588877]Cerami et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012 May;2(5):401-4. PMID: 22588877
  8. [PMID: 26438111]Allegra et al. Extended RAS Gene Mutation Testing in Metastatic Colorectal Carcinoma to Predict Response to Anti-Epidermal Growth Factor Receptor Monoclonal Antibody Therapy: American Society of Clinical Oncology Provisional Clinical Opinion Update 2015. J. Clin. Oncol. 2016 Jan 10;34(2):179-85. PMID: 26438111
  9. [FDA-cetuximab: ERBITUX]https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/125084s279lbl.pdf
  10. [FDA-panitumumab: VECTIBIX]https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/125147s210lbl.pdf
  11. [NCCN-Myelodysplastic Syndromes]NCCN Guidelines® – NCCN-Myelodysplastic Syndromes [Version 3.2022]
  12. [PMID: 25796376]Johnson et al. Treatment of NRAS-Mutant Melanoma. Curr Treat Options Oncol. 2015 Apr;16(4):15. doi: 10.1007/s11864-015-0330-z. PMID: 25796376
  13. [PMID: 28284557]Dummer et al. Binimetinib versus dacarbazine in patients with advanced NRAS-mutant melanoma (NEMO): a multicentre, open-label, randomised, phase 3 trial. Lancet Oncol. 2017 Apr;18(4):435-445. PMID: 28284557

 

BRAF

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  5. [PMID: 31533235]Bracht et al. BRAF Mutations Classes I, II, and III in NSCLC Patients Included in the SLLIP Trial: The Need for a New Pre-Clinical Treatment Rationale. Cancers (Basel). 2019 Sep 17;11(9). PMID: 31533235
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  8. [PMID: 22810696]Donna et al. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012 Jul 18;487(7407):330-7. PMID: 22810696
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  11. [PMID: 15035987]Wan et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004 Mar 19;116(6):855-67. PMID: 15035987
  12. [PMID: 21663470]Tiacci et al. BRAF mutations in hairy-cell leukemia. N. Engl. J. Med. 2011 Jun 16;364(24):2305-15. PMID: 21663470
  13. [PMID: 26566875]Diamond et al. Diverse and Targetable Kinase Alterations Drive Histiocytic Neoplasms. Cancer Discov. 2016 Feb;6(2):154-65. doi: 10.1158/2159-8290.CD-15-0913. Epub 2015 Nov 13. PMID: 26566875
  14. [PMID: 24569458]Imielinski et al. Oncogenic and sorafenib-sensitive ARAF mutations in lung adenocarcinoma. J Clin Invest. 2014 Apr;124(4):1582-6. doi: 10.1172/JCI72763. Epub 2014 Feb 24. PMID: 24569458
  15. [PMID: 15630448]Ciampi et al. Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer. J. Clin. Invest. 2005 Jan;115(1):94-101. PMID: 15630448
  16. [PMID: 20526349]Palanisamy et al. Rearrangements of the RAF kinase pathway in prostate cancer, gastric cancer and melanoma. Nat. Med. 2010 Jul;16(7):793-8. PMID: 20526349
  17. [PMID: 18974108]Jones et al. Tandem duplication producing a novel oncogenic BRAF fusion gene defines the majority of pilocytic astrocytomas. Cancer Res. 2008 Nov 1;68(21):8673-7. PMID: 18974108
  18. [PMID: 21424530]Cin et al. Oncogenic FAM131B-BRAF fusion resulting from 7q34 deletion comprises an alternative mechanism of MAPK pathway activation in pilocytic astrocytoma. Acta Neuropathol. 2011 Jun;121(6):763-74. doi: 10.1007/s00401-011-0817-z. Epub 2011 Mar 20. PMID: 21424530
  19. [PMID: 26314551]Ross et al. The distribution of BRAF gene fusions in solid tumors and response to targeted therapy. Int. J. Cancer. 2016 Feb 15;138(4):881-90. PMID: 26314551
  20. [FDA-vemurafenib: ZELBORAF]https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/202429s019lbl.pdf
  21. [FDA-dabrafenib: TAFINLAR]https://www.accessdata.fda.gov/drugsatfda_docs/label/2022/202806s022lbl.pdf
  22. [FDA-encorafenib: BRAFTOVI]https://www.accessdata.fda.gov/drugsatfda_docs/label/2022/210496s013lbl.pdf
  23. [FDA-cetuximab: ERBITUX]https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/125084s279lbl.pdf
  24. [FDA-trametinib: MEKINIST]https://www.accessdata.fda.gov/drugsatfda_docs/label/2022/204114s024lbl.pdf
  25. [FDA-binimetinib: MEKTOVI]https://www.accessdata.fda.gov/drugsatfda_docs/label/2019/210498s001lbl.pdf
  26. [FDA-cobimetinib: COTELLIC]https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/206192s002lbl.pdf
  27. [FDA-atezolizumab: TECENTRIQ]https://www.accessdata.fda.gov/drugsatfda_docs/label/2022/761034s043lbl.pdf
  28. [FDA-binimetinib: Breakthrough]https://markets.businessinsider.com/news/stocks/array-biopharma-receives-fda-breakthrough-therapy-designation-for-braftovi-in-combination-with-mektovi-and-cetuximab-for-brafv600e-mutant-metastatic-colorectal-cancer-1027437791
  29. [FDA-tovorafenib: Breakthrough]https://ir.dayonebio.com/news-releases/news-release-details/day-one-receives-fda-rare-pediatric-disease-designation-day101
  30. [FDA-ulixertinib: Fast Track]https://biomed-valley.com/news/#press-releases
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  33. [PMID: 21107323]Nazarian et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature. 2010 Dec 16;468(7326):973-7. doi: 10.1038/nature09626. Epub 2010 Nov 24. PMID: 21107323
  34. [PMID: 24463458]Rizos et al. BRAF inhibitor resistance mechanisms in metastatic melanoma: spectrum and clinical impact. Clin. Cancer Res. 2014 Apr 1;20(7):1965-77. PMID: 24463458
  35. [PMID: 24265152]Shi et al. A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition. Cancer Discov. 2014 Jan;4(1):69-79. PMID: 24265152
  36. [PMID: 24265153]Van et al. The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma. Cancer Discov. 2014 Jan;4(1):94-109. doi: 10.1158/2159-8290.CD-13-0617. Epub 2013 Nov 21. PMID: 24265153
  37. [PMID: 24055054]Villanueva et al. Concurrent MEK2 mutation and BRAF amplification confer resistance to BRAF and MEK inhibitors in melanoma. Cell Rep. 2013 Sep 26;4(6):1090-9. PMID: 24055054
  38. [PMID: 24265155]Shi et al. Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy. Cancer Discov. 2014 Jan;4(1):80-93. PMID: 24265155