RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors

Abstract

Background: Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors.

Methods: We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed.

Results: Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor.

Conclusions: Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).

Figure 1. Cutaneous Squamous-Cell Carcinomas or Keratoacanthomas in Patients Treated with Vemurafenib

Representative photographs and photomicrographs of nonmelanoma skin lesions in vemurafenib-treated patients are shown. The upper image in Panel A shows a lesion with the clinical features of keratoacanthoma, noted on day 98 after the patient had begun taking vemurafenib at a dose of 960 mg twice daily, in the centrally analyzed initial series. The lower image in Panel A is a low-power view of a section of a lesion obtained from the skin of the torso of the same patient with no RAS mutation, reported as squamous-cell carcinoma of the keratoacanthoma subtype (hematoxylin and eosin). Panel B shows the clinical appearance (upper image) and histopathological appearance (lower image, hematoxylin and eosin) of a keratoacanthoma from the chin of a patient with HRAS Q61R in the validation series.

Figure 2. Examples of the Mutation and MAPK-Signaling Analysis in Cutaneous Squamous-Cell Carcinomas or Keratoacanthomas

Immunohistochemical staining for pERK (brown) of samples of normal adjacent skin (left) and of cutaneous squamous-cell carcinomas (right) is shown from two patients in the validation set who were treated with vemurafenib. In these patients, both of whom had KRAS G12 mutations, the lesions were diagnosed as well-differentiated squamous-cell carcinomas that appeared 87 days (Panel A) and 51 days (Panel B) after vemurafenib therapy was begun.

Figure 3. Mechanistic Studies Showing Paradoxical MAPK Activation in Cell Lines

Panel A shows stimulation of B9 cell growth in soft agar after exposure to vemurafenib or PLX4720. Panel B shows differential expression of selected MAPK-regulated output genes in B9 cells after exposure to vemurafenib or PLX4720 overnight, as compared with gene expression of five human melanoma cell lines used as a reference. Red indicates higher expression and green indicates lower expression than untreated cells. Panel C shows the results of Western blot analysis of NIH3T3 cells transfected with a control vector or with mutated HRAS Q61L and treated with different concentrations of vemurafenib.

Figure 4. Acceleration of Growth of Nonmelanoma Skin Tumors in Mice with BRAF Inhibition

Panel A shows the number of palpable tumors arising over time in mice treated with the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA), the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the BRAF inhibitor PLX4720 (PLX) (25 mg per kilogram of body weight per day), and the MEK inhibitor PD184352 (PD) (25 mg per kilogram per day). Each cohort consisted of six mice, and the mean number of tumors per mouse is shown. I bars represent standard deviations. Panel B shows the results at 90 days after mice were treated with four different regimens.