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Topical 5-Fluorouracil Elicits Regressions of BRAF Inhibitor–Induced Cutaneous Squamous Cell Carcinoma

2012; Elsevier BV; Volume: 133; Issue: 1 Linguagem: Inglês

10.1038/jid.2012.268

1523-1747

Amaya Virós, Robert Hayward, Matthew J. Martin, Sharona Yashar, Clarissa C. Yu, Berta Sánchez-Laorden, Alfonso Zambon, Dan Niculescu‐Duvaz, Caroline J. Springer, Roger S. Lo, Richard Marais,

Cellular Mechanics and Interactions

cutaneous squamous cell carcinomas 7,12-dimethylbenz-(a)anthracene keratoacanthomas mitogen-activated protein kinase 12-O-tetradecanoyl-phorbol-13-acetate 5-fluorouracil TO THE EDITOR The protein kinase BRAF regulates cell growth through the mitogen-activated protein kinase (MAPK) pathway. In about half of the melanomas, BRAF is mutated and acts as a driver oncogene that stimulates cell proliferation, survival, and tumor progression (Davies et al., 2002Davies H. Bignell G.R. Cox C. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8271) Google Scholar; Gray-Schopfer et al., 2007Gray-Schopfer V. Wellbrock C. Marais R. Melanoma biology and new targeted therapy.Nature. 2007; 445: 851-857Crossref PubMed Scopus (1042) Google Scholar. The anti-BRAF drugs vemurafenib (PLX4032/RG7204) and dabrafenib (GSK2118436) achieve objective clinical responses in about 60% of melanoma patients whose tumors express mutant BRAF (Flaherty et al., 2010Flaherty K.T. Puzanov I. Kim K.B. et al.Inhibition of mutated, activated BRAF in metastatic melanoma.N Engl J Med. 2010; 363: 809-819Crossref PubMed Scopus (2953) Google Scholar; Chapman et al., 2011Chapman P.B. Hauschild A. Robert C. et al.Improved survival with vemurafenib in melanoma with BRAF V600E mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6115) Google Scholar; Sosman et al., 2012Sosman J.A. Kim K.B. Schuchter L. et al.Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib.N Engl J Med. 2012; 366: 707-714Crossref PubMed Scopus (1769) Google Scholar, validating these drugs as a therapeutic option for BRAF-mutant melanoma patients. An unexpected side effect of vemurafenib is that it induces keratoacanthomas (KA) and well-differentiated cutaneous squamous cell carcinomas (cuSCCs) in ∼26% of patients (Flaherty et al., 2010Flaherty K.T. Puzanov I. Kim K.B. et al.Inhibition of mutated, activated BRAF in metastatic melanoma.N Engl J Med. 2010; 363: 809-819Crossref PubMed Scopus (2953) Google Scholar; Sosman et al., 2012Sosman J.A. Kim K.B. Schuchter L. et al.Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib.N Engl J Med. 2012; 366: 707-714Crossref PubMed Scopus (1769) Google Scholar. This is because, although BRAF inhibitors block MAPK signaling in cells harboring BRAF mutations, they activate the pathway in cells expressing mutant RAS (Rat sarcoma) or when RAS is activated by receptor tyrosine kinases (Hatzivassiliou et al., 2010Hatzivassiliou G. Song K. Yen I. et al.RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.Nature. 2010; 464: 431-435Crossref PubMed Scopus (1280) Google Scholar; Heidorn et al., 2010Heidorn S.J. Milagre C. Whittaker S. et al.Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.Cell. 2010; 140: 209-221Abstract Full Text Full Text PDF PubMed Scopus (1191) Google Scholar; Poulikakos et al., 2010Poulikakos P.I. Zhang C. Bollag G. et al.RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.Nature. 2010; 464: 427-430Crossref PubMed Scopus (1396) Google Scholar. Critically, 21–57% of the nonmelanoma skin lesions that develop in vemurafenib-treated patients carry somatic mutations in HRAS or KRAS (Oberholzer et al., 2011Oberholzer P.A. Kee D. Dziunycz P. et al.RAS mutations are associated with the development of cutaneous squamous cell tumors in patients treated with RAF inhibitors.J Clin Oncol. 2011; 30: 316-321Crossref PubMed Scopus (345) Google Scholar; Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. We recently used a mouse two-stage skin carcinogenesis model to investigate how BRAF inhibitors drive the development of these nonmelanoma skin lesions (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. FVB/N mice were treated with a single topical application of the carcinogen 7,12-dimethylbenz-(a)anthracene (DMBA), which induces HRAS Q61L mutations in keratinocytes. The mice were then treated weekly with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and daily with oral doses of PLX4270 (see Supplementary Experimental Procedures online), a BRAF inhibitor that is preferred for mouse studies because of its superior oral bioavailability (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. We demonstrated that PLX4720 accelerated the growth of squamoproliferative lesions in DMBA/TPA-treated mice but did not induce lesions in mice treated with DMBA alone (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. These data demonstrated that vermurafenib is not a tumor promoter per se; rather, it acts by accelerating the progression of preexisting, premalignant lesions in susceptible individuals. Download .pdf (.05 MB) Help with pdf files Supplementary Information It is not known whether the cuSCCs that develop in vemurafenib-treated patients have metastatic potential different from cuSCCs that develop in the absence of BRAF inhibition, because in both the clinical settings these lesions are treated by surgical excision. However, advanced-stage melanoma patients on BRAF inhibitors can develop multiple eruptive lesions early during the course of BRAF inhibitor therapy and these can be challenging to manage surgically. We therefore sought to develop a noninvasive approach to treat these particular patients. Previous studies have shown that BRAF inhibitors accelerate the proliferation of RAS-transformed cells (Hatzivassiliou et al., 2010Hatzivassiliou G. Song K. Yen I. et al.RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.Nature. 2010; 464: 431-435Crossref PubMed Scopus (1280) Google Scholar; Heidorn et al., 2010Heidorn S.J. Milagre C. Whittaker S. et al.Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.Cell. 2010; 140: 209-221Abstract Full Text Full Text PDF PubMed Scopus (1191) Google Scholar; Poulikakos et al., 2010Poulikakos P.I. Zhang C. Bollag G. et al.RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.Nature. 2010; 464: 427-430Crossref PubMed Scopus (1396) Google Scholar; Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. We confirmed that the nonmelanoma skin lesions in DMBA/TPA/PLX4720-treated mice have a high proliferative index by showing that they have a high mitotic index (5 × 10 high-power fields) and strong positive staining for Ki-67 (Figure 1a), and thus we investigated whether antiproliferative drugs could induce their regression. 5-Fluorouracil (5-FU) is an antimetabolite that blocks DNA synthesis and inhibits cell proliferation. Although topical application of 5-FU should be restricted to an area no greater than 500cm2 (approximately the area of an extended hand) (MedaPharmaceuticals, 2012MedaPharmaceuticals Summary of Product Characteristics. 2012http://www.medicines.org.uk/EMC/medicine/6219/SPC/Efudix+Cream/Google Scholar, 5-FU is inexpensive and achieves objective clinical responses in 46–87% of cases of solar keratosis, lesions that are the precursor of invasive cuSCC (Gray and Meland, 2000Gray R.J. Meland N.B. Topical 5-fluorouracil as primary therapy for keratoacanthoma.Ann Plast Surg. 2000; 44: 82-85Crossref PubMed Scopus (28) Google Scholar; Shimizu et al., 2011Shimizu I. Cruz A. Chang K.H. et al.Treatment of squamous cell carcinoma in situ: a review.Dermatol Surg. 2011; 37: 1394-1411Crossref PubMed Scopus (40) Google Scholar. We confirm that 5-FU inhibited the growth of PDV cells, an HRAS mutant keratinocyte line, with IC50 (half maximal inhibitory concentration 50) values of ∼4.43μM in the absence of PLX4720 and ∼1.67μM in its presence (Figure 1b). To test 5-FU in vivo, FVB/N mice (six animals per group) were treated with DMBA, TPA, and PLX4720 as described (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. As reported (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar, palpable lesions first appeared after 30 days, increasing in number to approximately 11 lesions per mouse in 55 days (Figure 1c). We then treated the individual lesions with topical 5% 5-FU cream twice a week. Left untreated, we have shown that the tumors continue to grow and reach tumor burden limits after approximately 90 days (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar; however, with 5-FU, we observed an immediate response, with tumor regression leading to complete remission within 25 days (Figure 1c and d). On the basis of these observations, we tested the efficacy of 5-FU in two melanoma patients who were being treated with the BRAF inhibitor dabrafenib (GSK2118436). Both patients rapidly developed multiple eruptive lesions consistent with actinic keratosis and/or cuSCC (Figure 2a and b). Individual lesions were treated with 5% 5-FU cream twice a day, respecting recommended safety guidelines for total skin treatment area. Photo-documented lesions presenting the characteristics of KA and cuSCCs at various stages responded to 5-FU, whereas untreated concurrent lesions progressed and after biopsy were confirmed to be bona fide cuSCCs (Figure 2b and c). During follow-up periods of 11 and 18 months, respectively, none of the treated lesions in patients 1 and 2 recurred. As noted in these anecdotal cases, nascent lesions tended to respond more quickly compared with the established hyperkeratotic lesions. Secondary malignancies induced by conventional chemotherapies generally take many years to develop (Curtis et al., 1992Curtis R.E. Boice Jr., J.D. Stovall M. et al.Risk of leukemia after chemotherapy and radiation treatment for breast cancer.N Engl J Med. 1992; 326: 1745-1751Crossref PubMed Scopus (383) Google Scholar, but the nonmelanoma skin lesions induced by BRAF inhibitors develop within weeks of initiating treatment (Su et al., 2012Su F. Viros A. Milagre C. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.N Engl J Med. 2012; 366: 207-215Crossref PubMed Scopus (866) Google Scholar. Our previous studies show that BRAF inhibitors are not carcinogens per se; rather, they act to accelerate the development of preexisting subclinical lesions in susceptible patients. The approval of vemurafenib raises a pressing clinical need to provide treatment guidelines to manage these secondary lesions in patients for whom surgery is an undesirable option. The observation that BRAF inhibitors drive proliferation of RAS-mutant keratinocytes led us to test the efficacy of antiproliferative agents. We show that 5-FU provides a safe, noninvasive, inexpensive, and effective alternative to surgical intervention in clinical cases in which multiple surgeries are difficult or in cases in which lesions are incipient. Details concerning the ethics of animal and human studies are provided in the Supplementary Materials online. We thank Mr Eric Ward, Dr Kay Savage (ICR), and Ms Annette Lane (ICR) for their technical assistance with immunohistochemical preparations. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid Correction to: Journal of Investigative Dermatology (2013) 133, 274–276; doi:10.1038/jid.2012.268; published online 16 August 2012Journal of Investigative DermatologyVol. 133Issue 6PreviewTopical 5-Fluorouracil Elicits Regressions of BRAF Inhibitor–Induced Cutaneous Squamous Cell Carcinoma Full-Text PDF Open Archive