Implications of BRAF Mutations in Cancer

Christos Fountzilas, MD; Virginia G. Kaklamani, MD, DSc
Department of Medicine, Division of Hematology/Oncology, Cancer Treatment Research Center,
The University of Texas Health Science Center at San Antonio, TX

BRAF Mutations

Sustaining proliferative signaling is one of the hallmarks of cancer as described by Hanahan and Weinberg.^1,2 Signals from multiple receptor tyrosine kinases converge to the mitogen-activated protein kinase (MAPK) signal transduction pathway and regulate multiple aspects of cell biology (Figure).³ Guanine triphosphate–binding proteins of the RAS family and regulators of α-fetoprotein (RAF) serine-threonine kinases are altered and constitutively activated in a significant number of human malignancies,^4,5 leading to activation of downstream molecules and sustained cell proliferation and survival.³ There are 3 different RAF kinases in humans: ARAF, BRAF, and CRAF (RAF1).⁶ Substitutions of BRAF glutamic acid to valine (V600E) or lysine (V600K) are the most common RAF mutations in human neoplasia.⁴

Over the past decade, significant advances have been made in pharmacologic inhibition of BRAF as a treatment strategy for malignant melanoma, the tumor with the highest frequency of BRAF mutations.⁷ In this article, we review recent clinical progress in the use of BRAF as a diagnostic as well as a therapeutic target.

Melanoma

BRAF V600E/K is present in 50% to 60% of malignant melanoma.⁴ BRAF inhibition with small molecules such as vemurafenib at a dose of 960 mg orally twice daily, or dabrafenib at a dose of 150 mg orally twice daily, has been proved to be a very effective treatment strategy with an overall response rate (ORR) of 51% in phase 2 trials.⁸ In phase 3 trials, BRAF inhibition was superior to cytotoxic therapy with dacarbazine 1000 mg/m² intravenously (IV) every 3 weeks, with a progression-free survival (PFS) benefit of approximately 3.7 months (hazard ratio [HR] for progression or death 0.26 for vemurafenib [P <.001] and 0.30 for dabrafenib [P <.0001]), as well as a 6-month overall survival (OS) benefit in the BRIM-3 trial.^9,10 The major toxicities of BRAF inhibitors are hyperkeratosis, nausea/vomiting, arthralgias, palmoplantar dysesthesia (hand-foot syndrome), pyrexia, and fatigue. An important toxicity is the development of squamous abnormalities, including squamous cell carcinomas, of the skin in 6% to 12% of the patients, necessitating close dermatologic follow-up for excision of those lesions should they occur. The mechanism is thought to be paradoxical hyperactivation of the MAPK pathway in BRAF wild-type cells.¹¹

Inhibition of the downstream serine-threonine kinase MEK has also been proved efficacious in the treatment of BRAF-mutated melanoma. In a phase 3 study, Flaherty and colleagues randomized 322 patients with BRAFV600E/K mutation–positive melanoma naive to BRAF or MEK inhibition to the MEK inhibitor trametinib 2 mg orally once daily or dacarbazine 1000 mg/m² IV every 3 weeks.¹² The study met its primary end point with a PFS of 4.8 versus 1.5 months with trametinib and dacarbazine, respectively (HR, 0.45; P <.001). Response rates were 22% versus 8% for trametinib and dacarbazine, respectively. The major side effects of trametinib are nausea/vomiting, diarrhea, rash, fatigue, peripheral edema, and hypertension. Ocular events occur in 9% of the patients, with reversible chorioretinitis being the most severe. Retinopathy and retinal vein occlusion are rare but serious adverse events, and serial ophthalmologic evaluation is warranted.

Combination strategies with BRAF and MEK inhibition have been developed and proved superior to BRAF inhibition alone, setting a new standard for BRAF-mutated melanoma. In a phase 1/2 study, the combination of dabrafenib/trametinib was superior to dabrafenib alone, with a response rate of 76% versus 54% and a PFS of 9.4 versus 5.8 months, respectively.¹³ Subsequent phase 3 trials randomizing more than 1500 patients with advanced melanoma to a BRAF/MEK combination versus monotherapy with a BRAF inhibitor confirmed the superiority of the combination approach (Table 1).^14-16 Toxicities are not significantly increased with combination therapy, and there was a decrease in cases of cutaneous squamous lesions with BRAF/MEK combination strategies. Unfortunately, resistance is a common theme, and the disease does progress after approximately 10 months of therapy.

Thyroid Cancer

BRAF is commonly mutated in well-differentiated thyroid cancer.⁷ In DECISION, a randomized, double-blind, placebo-controlled phase 3 trial, 417 patients with iodine-refractory disease were randomized 1:1 to sorafenib, a multikinase (including RAF) small molecule inhibitor, or placebo.¹⁷ The study reached its primary end point with a significant improvement in PFS with sorafenib compared with placebo (10.8 vs 5.8 months, respectively; HR, 0.59; P <.0001). Clinical benefit was noted for all patients regardless of mutation status, and there was no difference in OS between groups. The benefit of sorafenib in thyroid cancer may not have been secondary to BRAF inhibition per se, but more a result of inhibition of multiple targets, especially the vascular endothelial growth factor (VEGF) receptor pathway.

Non–Small Cell Lung Cancer (NSCLC)

The relative success of BRAF inhibition in melanoma opened the road for evaluation in other malignancies with various levels of success (Table 2). BRAF is uncommonly mutated in lung cancer (2% of cases, in the range of ALK and ROS1 mutations). In a phase 2 basket trial of vemurafenib 960 mg orally twice daily in BRAF-mutated nonmelanoma cancers, 8 of 20 patients in the NSCLC cohort attained a partial response (PR) and another 8 had stable disease, for a disease control rate of 80%.¹⁸ In a retrospective review of off-label use of da­brafenib, vemurafenib, or sorafenib in a cohort of patients with NSCLC harboring a BRAF V600E/K mutation, overall response and disease control rates were 53% and 85%, respectively; median PFS and OS were 5.0 and 10.8 months, respectively.¹⁹ In a recently reported nonrandomized phase 2 study of patients with BRAF V600E mutation–positive NSCLC, naive to BRAF or MEK inhibition, the response rate with dabrafenib monotherapy at a dose of 150 mg orally twice daily was 33%, and the disease control rate was 53%. Duration of response was 9.9 months and PFS 5.5 months.²⁰ There were no new safety concerns. Twelve percent of the patients developed squamous cell carcinoma of the skin. The interim results of the BRAF/MEK combination therapy cohort have been presented in abstract form.²¹ The response rate with dabrafenib 150 mg orally twice daily and trametinib 2 mg orally once daily was 63%, and 88% of patients had their disease controlled for more than 3 months. A total of 6% of the patients developed squamous cell carcinoma of the skin or keratoacanthoma. Final data have not yet been reported.

Colorectal Cancer (CRC)

BRAF mutations are present in <10% of patients with CRC.²² Whereas in the early disease setting, the presence of a BRAF mutation correlates with the presence of microsatellite instability and nongermline mutations in mismatch repair (MMR) proteins and a good prognosis, in the advanced setting the prognosis in this patient population is poor with a median OS of up to 14 months.^23,24 BRAF inhibition was thought to be a reasonable therapeutic target in this patient population, but results from early studies were disappointing with a very low observed response rate and a PFS of only 2 months.^25,26

Preclinical studies revealed a rapid feedback activation of the epidermal growth factor receptor (EGFR) as a potential mechanism of resistance.^27,28 In the phase 2 study by Hyman et al, of the 27 patients with BRAF-mutated CRC treated with vemurafenib 960 mg orally twice daily and the anti-EGFR antibody cetuximab (400 mg/m² IV loading dose followed by 250 mg/m² IV weekly thereafter), 4% had a PR and 69% had stable disease.¹⁸ Seventy-four percent of the patients developed a rash (grade 3 in 4% of the patients), 50% had grade 1/2 fatigue, and 33% grade 1/2 photosensitivity reaction; 15% of the patients developed a squamous carcinoma, mainly in the skin. Yeager and colleagues reported in abstract form the results of a phase 2 trial of the anti-EGFR antibody panitumumab in combination with vemurafenib in a similar patient population.²⁹ The ORR was 16%, and another 16% of the patients had disease stabilization for more than 2 months. Minor responses (not fulfilling the criteria for PR by RECIST) were noted in 66% of the patients. No cases of squamous cell cancers were noted, and the treatment was generally well tolerated.

BRAF gene amplification is also a possible resistance mechanism that can potentially be bypassed by combined BRAF/MEK inhibition.³⁰ In a phase 2 trial of patients with BRAF-mutated CRC, the combined BRAF/MEK inhibition with dabrafenib 150 mg orally twice daily and trametinib 2 mg orally once daily resulted in a response rate of 12%, and a total of 56% of patients had stable disease as best response. The median PFS was 3.5 months.³¹ Toxicities were as expected for BRAF/MEK combination therapy, with the most common adverse events being nausea/vomiting, pyrexia/chills, diarrhea, and fatigue. The only grade 3/4 adverse events in >10% of patients were anemia (16%) and pyrexia (12%). In a phase 1 study in a similar patient population reported in abstract form, panitumumab was safely combined with dabrafenib or the dabrafenib/trametinib combination.³² The most common toxicity was acneiform rash (grade 1 or 2). Preliminary efficacy results show response in 4 of 6 patients in the 3-drug combination cohort and stable disease in 7 of 8 patients with the 2-drug combination.

Combined BRAF and phosphatidylinositol 3-kinase (PI3K) inhibition may be another strategy to overcome resistance of BRAF-mutated CRC cells to BRAF inhibition.³³ A phase 1 study evaluated the combination of the BRAF inhibitor encorafenib in combination with cetuximab and the PI3K inhibitor BYL179 in BRAF-mutated CRC.³⁴ Treatment was well tolerated, and the preliminary efficacy results were promising (1 of 3 patients treated with all 3 medications attained a PR). The study is ongoing.

The most promising results for the treatment of patients with BRAF-mutated metastatic CRC come from a small phase 2 study from Italy, where 15 patients with BRAF-mutated metastatic CRC were treated with 5-fluorouracil/leukovorin, oxaliplatin, irinotecan, and the VEGF-A–targeting antibody bevacizumab (FOLFOXIRI-Bev).³⁵ The median survival was 24 months and the PFS 9 months, exceeding historical controls for this patient population.

Other Disease Types

BRAF V600E mutation is a very specific (100%) genetic abnormality for hairy cell leukemia (HCL),³⁶ and it is present in 60% of Langerhans cell histiocytosis (LCH) cases.³⁷In 2 phase 2 studies in patients with relapsed/refractory HCL, the ORR was 98% (48% with complete response) with vemurafenib 960 mg orally twice daily; all patients with complete response had minimal residual disease in the bone marrow by immunohistochemistry.³⁸ The median relapse-free survival was 9 months and duration of response 24 months. Responses have also been observed with a lower dose.³⁹ In the study by Hyman et al, the ORR to vemurafenib in the LCH cohort was 43% (7% with complete response), and 57% of patients had stable disease.¹⁸

BRAF mutations are present in 22% of cholangiocarcinomas.⁴⁰ In the cholangiocarcinoma cohort of the basket study by Hyman et al, of the 8 patients enrolled, 1 had a PR with vemurafenib (12%); 50% of the patients had stable disease.¹⁸

Conclusion

RAF serine-threonine kinase has a pivotal role in signal transduction through the MAPK pathway. BRAF V600E/K inhibition has been a successful strategy for the treatment of melanoma positive for these mutations either alone or in combination with inhibition of downstream MEK. BRAF inhibition appears to be an effective treatment for patients with relapsed/refractory HCL and a promising treatment strategy for the small subgroup of BRAF-mutated NSCLC, but its efficacy in BRAF-mutated metastatic CRC is limited. Feedback activation of EGFR, BRAF amplification, and PI3K mutations have been implicated as potential factors for resistance explaining the low efficacy of single-agent BRAF inhibitors as discussed above. In the study by Kopetz et al, there was no difference in PFS based on the presence or not of PI3K pathway activation or expression of EGFR by immunohistochemistry.²⁶ In the study by Corcoran et al, the majority of patients with hotspot exon 9 or 20 PI3K mutations, phosphate and tensin homolog (PTEN) mutations, or transforming growth factor beta pathway alterations, had some decrease in the target lesion size by RECIST with BRAF/MEK combination therapy, there was no correlation between PTEN or microsatellite status or total EGFR and PFS.³¹ The number of patients was small for definitive conclusions. This is a cautionary tale for treatment strategies that rely only on the molecular composition of each tumor without taking histology into consideration.

Disclosure

The authors have no conflicts of interest.

Acknowledgments

This work was supported by the Dolores Knes Fund. Dr Christos Fountzilas is a recipient of a Cancer Prevention and Research Institute of Texas (CPRIT) Cancer Research Training Award (RP140105).

Key Points

1. BRAF mutations are common in a variety of human malignancies
2. Inhibition of BRAF is an attractive treatment strategy for melanoma and lung cancer harboring activating mutations
3. Treatment with BRAF inhibitors has not been successful in all disease types, the best example being colorectal cancer

---

Dr Fountzilas is a Clinical Fellow in The Division of Hematology/Oncology at The University of Texas Health Science Center in San Antonio. Dr Fountzilas completed his medical training with honors at the Aristotle University of Thessaloniki School of Medicine in Greece, and his residency in Internal Medicine at Lenox Hill Hospital in New York, NY. His research interests include oncolytic virotherapy and use of immunotherapy for gastrointestinal and thoracic malignancies.

Dr Kaklamani is Professor of Medicine in the Division of Hematology/Oncology at The University of Texas Health Science Center in San Antonio and is the Leader of the Breast Cancer Program at the Cancer Therapy and Research Center. Dr Kaklamani completed her medical training with honors at the University of Athens and her residency in Internal Medicine at Newton-Wellesley Hospital in Boston, MA. She completed her fellowship in hematology/oncology at Northwestern University. She also received a Master of Science in Clinical Investigation from Northwestern University. She was Head of the Translational Breast Cancer Program at Northwestern University and Codirector of the Cancer Genetics Program at the same institution. Her research interests include studying high-risk families and identifying genetic mutations that are associated with an increased risk for breast, colon, and prostate cancers

---

References

1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57-70.
2. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646-674.
3. Roskoski R Jr. ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol Res. 2012;66:105-143.
4. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949-954.
5. Bos JL. ras oncogenes in human cancer: a review. Cancer Res. 1989;49:4682-4689.
6. Marais R, Marshall CJ. Control of the ERK MAP kinase cascade by Ras and Raf. Cancer Surv. 1996;27:101-125.
7. Vakiani E, Solit DB. KRAS and BRAF: drug targets and predictive biomarkers. J Pathol. 2011;223:219-229.
8. Ascierto PA, Minor D, Ribas A, et al. Phase II trial (BREAK-2) of the BRAF inhibitor dabrafenib (GSK2118436) in patients with metastatic melanoma. J Clin Oncol. 2013;31:3205-3211.
9. Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364:2507-2516.
10. Hauschild A, Grob JJ, Demidov LV, et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet. 2012;380:358-365.
11. Hatzivassiliou G, Song K, Yen I, et al. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature. 2010;464:431-435.
12. Flaherty KT, Robert C, Hersey P, et al. Improved survival with MEK inhibition in BRAF-mutated melanoma. N Engl J Med. 2012;367:107-114.
13. Flaherty KT, Infante JR, Daud A, et al. Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med. 2012;367:1694-1703.
14. Long GV, Stroyakovskiy D, Gogas H, et al. Combined BRAF and MEK inhibition versus BRAF inhibition alone in melanoma. N Engl J Med. 2014;371:1877-1888.
15. Robert C, Karaszewska B, Schachter J, et al. Improved overall survival in melanoma with combined dabrafenib and trametinib. N Engl J Med. 2015;372:30-39.
16. Larkin J, Ascierto PA, Dréno B, et al. Combined vemurafenib and cobimetinib in BRAF-mutated melanoma. N Engl J Med. 2014;371:1867-1876.
17. Brose MS, Nutting CM, Jarzab B, et al. Sorafenib in radioactive iodine-refractory, locally advanced or metastatic differentiated thyroid cancer: a randomised, double-blind, phase 3 trial. Lancet. 2014;384:319-328.
18. Hyman DM, Puzanov I, Subbiah V, et al. Vemurafenib in multiple nonmelanoma cancers with BRAF V600 mutations. N Engl J Med. 2015;373:726-736.
19. Gautschi O, Milia J, Cabarrou B, et al. Targeted therapy for patients with BRAF-mutant lung cancer: results from the European EURAF cohort. J Thorac Oncol. 2015;10:1451-1457.
20. Planchard D, Kim TM, Mazieres J, et al. Dabrafenib in patients with BRAFV600E-positive advanced non-small-cell lung cancer: a single-arm, multicentre, open-label, phase 2 trial [published online April 11, 2016]. Lancet Oncol.
21. Planchard D, Groen HJM, Kim TM, et al. Interim results of a phase II study of the BRAF inhibitor (BRAFi) dabrafenib (D) in combination with the MEK inhibitor trametinib (T) in patients (pts) with BRAF V600E mutated (mut) metastatic non-small cell lung cancer (NSCLC). J Clin Oncol. 2015;33(suppl). Abstract 8006.
22. Tol J, Nagtegaal ID, Punt CJ. BRAF mutation in metastatic colorectal cancer. N Engl J Med. 2009;361:98-99.
23. Van Cutsem E, Köhne C-H, Láng I, et al. Cetuximab plus irinotecan, fluorouracil, and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status. J Clin Oncol. 2011;29:2011-2019.
24. Tie J, Gibbs P, Lipton L, et al. Optimizing targeted therapeutic development: analysis of a colorectal cancer patient population with the BRAF(V600E) mutation. Int J Cancer. 2011;128:2075-2084.
25. Kopetz S, Desai J, Chan E, et al. PLX4032 in metastatic colorectal cancer patients with mutant BRAF tumors. J Clin Oncol. 2010;28(suppl). Abstract 3534.
26. Kopetz S, Desai J, Chan E, et al. Phase II pilot study of vemurafenib in patients with metastatic BRAF-mutated colorectal cancer. J Clin Oncol. 2015;33:4032-4038.
27. Corcoran RB, Ebi H, Turke AB, et al. EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib. Cancer Discov. 2012;2:227-235.
28. Prahallad A, Sun C, Huang S, et al. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature. 2012;483:100-103.
29. Yaeger R, Cercek A, O’Reilly EM, et al. Pilot trial of combined BRAF and EGFR inhibition in BRAF-mutant metastatic colorectal cancer patients. Clin Cancer Res. 2015;21:1313-1320.
30. Corcoran RB, Dias-Santagata D, Bergethon K, et al. BRAF gene amplification can promote acquired resistance to MEK inhibitors in cancer cells harboring the BRAF V600E mutation. Sci Signal. 2010;3:ra84.
31. Corcoran RB, Atreya CE, Falchook GS, et al. Combined BRAF and MEK inhibition with dabrafenib and trametinib in BRAF V600-mutant colorectal cancer. J Clin Oncol. 2015;33:4023-4031.
32. Bendell JC, Atreya CE, André T, et al. Efficacy and tolerability in an open-label phase I/II study of MEK inhibitor trametinib (T), BRAF inhibitor dabrafenib (D), and anti-EGFR antibody panitumumab (P) in combination in patients (pts) with BRAF V600E mutated colorectal cancer (CRC). J Clin Oncol. 2014;32(suppl). Abstract 3515.
33. Mao M, Tian F, Mariadason JM, et al. Resistance to BRAF inhibition in BRAF-mutant colon cancer can be overcome with PI3K inhibition or demethylating agents. Clin Cancer Res. 2013;19:657-667.
34. Geel RV, Elez E, Bendell JC, et al. Phase I study of the selective BRAF V600 inhibitor encorafenib (LGX818) combined with cetuximab and with or without the α-specific PI3K inhibitor BYL719 in patients with advanced BRAF-mutant colorectal cancer. J Clin Oncol. 2014;32(suppl). Abstract 3514.
35. Loupakis F, Cremolini C, Salvatore L, et al. FOLFOXIRI plus bevacizumab as first-line treatment in BRAF mutant metastatic colorectal cancer. Eur J Cancer. 2014;50:57-63.
36. Tiacci E, Trifonov V, Schiavoni G, et al. BRAF mutations in hairy-cell leukemia. N Engl J Med. 2011;364:2305-2315.
37. Badalian-Very G, Vergilio JA, Degar BA, et al. Recurrent BRAF mutations in Langerhans cell histiocytosis. Blood. 2010;116:1919-1923.
38. Tiacci E, Park JH, De Carolis L, et al. Targeting mutant BRAF in relapsed or refractory hairy-cell leukemia. N Engl J Med. 2015;373:1733-1747.
39. Dietrich S, Pircher A, Endris V, et al. BRAF inhibition in hairy cell leukemia with low dose vemurafenib [published online March 3, 2016]. Blood.
40. Tannapfel A, Sommerer F, Benicke M, et al. Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma. Gut. 2003;52:706-712.

Related Articles