Kerstin Trunzer

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

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.).

Safety and efficacy of vemurafenib in BRAFV600E and BRAFV600K mutation-positive melanoma (BRIM-3): extended follow-up of a phase 3, randomised, open-label study

BACKGROUND In the BRIM-3 trial, vemurafenib was associated with risk reduction versus dacarbazine of both death and progression in patients with advanced BRAF(V600) mutation-positive melanoma. We present an extended follow-up analysis of the total population and in the BRAF(V600E) and BRAF(V600K) mutation subgroups. METHODS Patients older than 18 years, with treatment-naive metastatic melanoma and whose tumour tissue was positive for BRAF(V600) mutations were eligible. Patients also had to have a life expectancy of at least 3 months, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and adequate haematological, hepatic, and renal function. Patients were randomly assigned by interactive voice recognition system to receive either vemurafenib (960 mg orally twice daily) or dacarbazine (1000 mg/m(2) of body surface area intravenously every 3 weeks). Coprimary endpoints were overall survival and progression-free survival, analysed in the intention-to-treat population (n=675), with data censored at crossover. A sensitivity analysis was done. This trial is registered with ClinicalTrials.gov, NCT01006980. FINDINGS 675 eligible patients were enrolled from 104 centres in 12 countries between Jan 4, 2010, and Dec 16, 2010. 337 patients were randomly assigned to receive vemurafenib and 338 to receive dacarbazine. Median follow-up was 12·5 months (IQR 7·7-16·0) on vemurafenib and 9·5 months (3·1-14·7) on dacarbazine. 83 (25%) of the 338 patients initially randomly assigned to dacarbazine crossed over from dacarbazine to vemurafenib. Median overall survival was significantly longer in the vemurafenib group than in the dacarbazine group (13·6 months [95% CI 12·0-15·2] vs 9·7 months [7·9-12·8]; hazard ratio [HR] 0·70 [95% CI 0·57-0·87]; p=0·0008), as was median progression-free survival (6·9 months [95% CI 6·1-7·0] vs 1·6 months [1·6-2·1]; HR 0·38 [95% CI 0·32-0·46]; p<0·0001). For the 598 (91%) patients with BRAF(V600E) disease, median overall survival in the vemurafenib group was 13·3 months (95% CI 11·9-14·9) compared with 10·0 months (8·0-14·0) in the dacarbazine group (HR 0·75 [95% CI 0·60-0·93]; p=0·0085); median progression-free survival was 6·9 months (95% CI 6·2-7·0) and 1·6 months (1·6-2·1), respectively (HR 0·39 [95% CI 0·33-0·47]; p<0·0001). For the 57 (9%) patients with BRAF(V600K) disease, median overall survival in the vemurafenib group was 14·5 months (95% CI 11·2-not estimable) compared with 7·6 months (6·1-16·6) in the dacarbazine group (HR 0·43 [95% CI 0·21-0·90]; p=0·024); median progression-free survival was 5·9 months (95% CI 4·4-9·0) and 1·7 months (1·4-2·9), respectively (HR 0·30 [95% CI 0·16-0·56]; p<0·0001). The most frequent grade 3-4 events were cutaneous squamous-cell carcinoma (65 [19%] of 337 patients) and keratoacanthomas (34 [10%]), rash (30 [9%]), and abnormal liver function tests (38 [11%]) in the vemurafenib group and neutropenia (26 [9%] of 287 patients) in the dacarbazine group. Eight (2%) patients in the vemurafenib group and seven (2%) in the dacarbazine group had grade 5 events. INTERPRETATION Inhibition of BRAF with vemurafenib improves survival in patients with the most common BRAF(V600E) mutation and in patients with the less common BRAF(V600K) mutation. FUNDING F Hoffmann-La Roche-Genentech.

Pharmacodynamic Effects and Mechanisms of Resistance to Vemurafenib in Patients With Metastatic Melanoma

PURPOSE To assess pharmacodynamic effects and intrinsic and acquired resistance mechanisms of the BRAF inhibitor vemurafenib in BRAF(V600)-mutant melanoma, leading to an understanding of the mechanism of action of vemurafenib and ultimately to optimization of metastatic melanoma therapy. METHODS In the phase II clinical study NP22657 (BRIM-2), patients received oral doses of vemurafenib (960 mg twice per day). Serial biopsies were collected to study changes in mitogen-activated protein kinase (MAPK) signaling, cell-cycle progression, and factors causing intrinsic or acquired resistance by immunohistochemistry, DNA sequencing, or somatic mutation profiling. Results Vemurafenib inhibited MAPK signaling and cell-cycle progression. An association between the decrease in extracellular signal-related kinase (ERK) phosphorylation and objective response was observed in paired biopsies (n = 22; P = .013). Low expression of phosphatase and tensin homolog showed a modest association with lower response. Baseline mutations in MEK1(P124) coexisting with BRAF(V600) were noted in seven of 92 samples; their presence did not preclude objective tumor responses. Acquired resistance to vemurafenib associated with reactivation of MAPK signaling as observed by elevated ERK1/2 phosphorylation levels in progressive lesions and the appearance of secondary NRAS(Q61) mutations or MEK1(Q56P) or MEK1(E203K) mutations. These two activating MEK1 mutations had not previously been observed in vivo in biopsies of progressive melanoma tumors. CONCLUSION Vemurafenib inhibits tumor proliferation and oncogenic BRAF signaling through the MAPK pathway. Acquired resistance results primarily from MAPK reactivation driven by the appearance of secondary mutations in NRAS and MEK1 in subsets of patients. The data suggest that inhibition downstream of BRAF should help to overcome acquired resistance.

Prospective Molecular Marker Analyses of EGFR and KRAS From a Randomized, Placebo-Controlled Study of Erlotinib Maintenance Therapy in Advanced Non–Small-Cell Lung Cancer

PURPOSE The phase III, randomized, placebo-controlled Sequential Tarceva in Unresectable NSCLC (SATURN; BO18192) study found that erlotinib maintenance therapy extended progression-free survival (PFS) and overall survival in patients with advanced non-small-cell lung cancer (NSCLC) who had nonprogressive disease following first-line platinum-doublet chemotherapy. This study included prospective analysis of the prognostic and predictive value of several biomarkers. PATIENTS AND METHODS Mandatory diagnostic tumor specimens were collected before initiating first-line chemotherapy and were tested for epidermal growth factor receptor (EGFR) protein expression by using immunohistochemistry (IHC), EGFR gene copy number by using fluorescent in situ hybridization (FISH), and EGFR and KRAS mutations by using DNA sequencing. An EGFR CA simple sequence repeat in intron 1 (CA-SSR1) polymorphism was evaluated in blood. RESULTS All 889 randomly assigned patients provided tumor samples. EGFR IHC, EGFR FISH, KRAS mutation, and EGFR CA-SSR1 repeat length status were not predictive for erlotinib efficacy. A profound predictive effect on PFS of erlotinib relative to placebo was observed in the EGFR mutation-positive subgroup (hazard ratio [HR], 0.10; P < .001). Significant PFS benefits were also observed with erlotinib in the wild-type EGFR subgroup (HR, 0.78; P = .0185). KRAS mutation status was a significant negative prognostic factor for PFS. CONCLUSION This large prospective biomarker study found that patients with activating EGFR mutations derive the greatest PFS benefit from erlotinib maintenance therapy. No other biomarkers were predictive for outcomes with erlotinib, although the study was not powered for clinical outcomes in biomarker subgroups other than EGFR IHC-positive [corrected]. KRAS mutations were prognostic for reduced PFS. The study demonstrated the feasibility of prospective tissue collection for biomarker analyses in NSCLC.

Analysis of Dermatologic Events in Vemurafenib-Treated Patients With Melanoma

BACKGROUND Vemurafenib has been approved for the treatment of patients with advanced BRAF(V600E)-mutant melanoma. This report by the Vemurafenib Dermatology Working Group presents the characteristics of dermatologic adverse events (AEs) that occur in vemurafenib-treated patients, including cutaneous squamous cell carcinoma (cuSCC). METHODS Dermatologic AEs were assessed from three ongoing trials of BRAF(V600E) mutation-positive advanced melanoma. Histologic central review and genetic characterization were completed for a subset of cuSCC lesions. RESULTS A total of 520 patients received vemurafenib. The most commonly reported AEs were dermatologic AEs, occurring in 92%-95% of patients. Rash was the most common AE (64%-75% of patients), and the most common types were rash not otherwise specified, erythema, maculopapular rash, and folliculitis. Rash development did not appear to correlate with tumor response. Photosensitivity occurred in 35%-63% of patients, and palmar-plantar erythrodysesthesia (PPE) occurred in 8%-10% of patients. The severity of rash, photosensitivity, and PPE were mainly grade 1 or 2. In all, 19%-26% of patients developed cuSCC, mostly keratoacanthomas (KAs). The majority of patients with cuSCC continued therapy without dose reduction after resection. Genetic analysis of 29 cuSCC/KA samples demonstrated HRAS mutations in 41%. CONCLUSIONS Dermatologic AEs associated with vemurafenib treatment in patients with melanoma were generally manageable with supportive care measures. Dose interruptions and/or reductions were required in <10% of patients.

Resistance to Selective BRAF Inhibition Can Be Mediated by Modest Upstream Pathway Activation

A high percentage of patients with BRAF(V600E) mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAF(V600E) melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAF(V600E) inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance.

Abstract 655: Development of IHC staining protocols for assessment of PD-L1 expression in cytological samples

Immunohistochemistry (IHC) staining of non-small cell lung cancer (NSCLC) samples for programmed cell death ligand 1 (PD-L1) can help identify patients that may benefit from anti-PDL1 therapy. However, resection or biopsy samples cannot be obtained for some patients with NSCLC. Fine needle aspiration (FNA) is a less invasive method for obtaining samples in such patients, and it is yet to be determined if PD-L1 can be assessed using cytological samples. In this study we had three objectives 1. Develop staining protocols on the VENTANA BenchMark ULTRA automated staining platform for cytology samples fixed with the most common methods using cell lines as a model. 2, Determine the optimal cytology fixation method for VENTANA PD-L1 (SP142) IHC Assay (PD-L1 (SP142)) staining, and 3. Assess PD-L1 expression in tumor cells (TC) and tumor-infiltrating immune cells (IC) in cytological samples prepared from NSCLC patients. KARPAS 299 cell line was used as a model system for the fixation studies. Liquid-based preparations (LBPs) fixed in PreservCyt, conventional smears (unstained and pap-stained) fixed in 95% ethanol, and paraffin embedded cell blocks (CBs) fixed in 95% ethanol, 10% neutral buffered formalin (NBF), PreservCyt, and SurePath preservative were optimized on the BenchMark ULTRA using PD-L1 (SP142) antibody and OptiView DAB Detection and Amplification Kits. NSCLC FNA CBs fixed in 10% NBF (N=69) and a subset of FNAs with matched resections (N=20) were stained with the optimized protocol. FNAs and resections were assessed for percentage of TC and IC with PD-L1 staining. Staining parameters were optimized on the BenchMark ULTRA for all sample types tested, and they all produced a range of moderate to strong PD-L1 expression. CBs fixed in 95% ethanol and 10% NBF produced the highest percentage of staining, with expression in 90% of cells. CBs fixed in PreservCyt and SurePath preservative had a lower percentage of staining, 40% and 75% respectively. LBPs had 25% cells staining and smears ranged from 20%-60%. NSCLC FNA CBs fixed in 10% NBF produced interpretable results when stained with PD-L1 (SP142). IC staining was seen in 8.7% (6/69) of samples, TC staining was seen in 8.7% (6/69) of samples, both IC and TC staining was seen in 14.5% (10/69) of samples. The subset of FNAs with matched resections showed concordance with PD-L1 IC staining in 60% (12/20) of samples and TC staining in 75% (15/20) of samples. The discordant cases showed that FNAs were negative for IC when the resection was negative and positive for TC in 5% of cases when the resection was negative. The VENTANA PD-L1 (SP142) Assay staining parameters were found to be optimal for staining NSCLC FNA CB fixed in 10% NBF. PD-L1 staining was detected in both TC and IC in FNA samples and concordance of FNA and matched resections was high for both IC and TC. A larger study is necessary to validate the use of FNAs for assessment of PD-L1 expression in a clinical setting. Citation Format: Christine Boyiddle, Mark Ruboyianes, Ed Del Valle, Lukas Bubendorf, Kerstin Trunzer, Judith Pugh, Jennifer Boone. Development of IHC staining protocols for assessment of PD-L1 expression in cytological samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 655. doi:10.1158/1538-7445.AM2017-655