Purpose: The limited knowledge on the molecular profile of patients with BRAF-mutant non-small cell lung cancer (NSCLC) who progress under BRAF-targeted therapies (BRAF-TT) has hampered the development of subsequent therapeutic strategies for these patients. Here, we evaluated the clinical utility of circulating tumor DNA (ctDNA)-targeted sequencing to identify canonical BRAF mutations and genomic alterations potentially related to resistance to BRAF-TT, in a large cohort of patients with BRAFmutant NSCLC.Experimental Design: This was a prospective study of 78 patients with advanced BRAF-mutant NSCLC, enrolled in 27 centers across France. Blood samples (n ¼ 208) were collected from BRAF-TT-naïve patients (n ¼ 47), patients nonprogressive under treatment (n ¼ 115), or patients at disease progression (PD) to BRAF-TT (24/46 on BRAF monotherapy and 22/46 on BRAF/ MEK combination therapy). ctDNA sequencing was performed using InVisionFirst-Lung. In silico structural modeling was used to predict the potential functional effect of the alterations found in ctDNA.Results: BRAF V600E ctDNA was detected in 74% of BRAF-TTnaïve patients, where alterations in genes related with the MAPK and PI3K pathways, signal transducers, and protein kinases were identified in 29% of the samples. ctDNA positivity at the first radiographic evaluation under treatment, as well as BRAF-mutant ctDNA positivity at PD were associated with poor survival. Potential drivers of resistance to either BRAF-TT monotherapy or BRAF/MEK combination were identified in 46% of patients and these included activating mutations in effectors of the MAPK and PI3K pathways, as well as alterations in U2AF1, IDH1, and CTNNB1.Conclusions: ctDNA sequencing is clinically relevant for the detection of BRAF-activating mutations and the identification of alterations potentially related to resistance to BRAF-TT in BRAFmutant NSCLC.

All 30 patients received prior EGFR TKIs, of which 28 (93%) received prior osimertinib, and 15 (50%) prior chemotherapy. Activating EGFR mutations were reported in all patients (Ex19del: 57%; L858R: 40%; L861Q: 3%). All 25 evaluable tumors demonstrated HER3 expression (median HER3 membrane H-score, 183 [range, 56e290]). History of central nervous system (CNS) metastases was reported in 15 patients (50%). Treatment-emergent AEs were reported in 29 patients (97%; 13 patients [43%] reported grade 3/4). Two DLTs (grade 3 febrile neutropenia and grade 4 platelet count decrease) were reported in 1 patient (5.6 mg/kg) and 3 DLTs (all grade 4 platelet count decrease) in 3 patients (6.4 mg/kg). Of patients with a history of CNS metastases, 9 have progressed (2 with CNS progression; 3 with both CNS and non-CNS progression). One patient without a history of CNS metastasis progressed with new CNS disease. Of 26 efficacy-evaluable patients, 6 had confirmed partial responses (2 each at 4.8, 5.6, and 6.4 mg/kg), including 2 patients with an EGFR C797S mutation. Median best percentage change in sum of diameters (SoD) was À25.7% (range, À82.6% to 13.3%), including decreases in SoD in patients with CDK4 amplification (e25.7% and e17.8%), HER2 amplification (e28.6%), and both CCNE1 amplification and PIK3CA mutation (e28.8%). Conclusion: U3-1402 demonstrated tolerable safety and antitumor activity in this ongoing study. Antitumor activity of U3-1402 was seen in cancers with EGFR-mediated and other resistance mechanisms. These findings support the hypothesis that targeting HER3 with U3-1402 may provide clinical benefit to patients with EGFR-mutant NSCLC with diverse mechanisms of resistance. RDE evaluation is ongoing.

Background: BRAF mutations occur in 2 to 3% of patients (pts) with non-small cell lung cancer (NSCLC). In these pts vemurafenib, a selective oral BRAF inhibitor is associated with a response rate (RR) of 42%, rising to 64% for combination treatment with dabrafenib and trametinib. Despite initial responses, most pts ultimately develop resistance to therapy. Mechanisms of resistance to BRAF inhibitors in NSCLC have only been reported in 2 pts (acquired KRAS G12D and primary resistance due to BRAF G469L) Objective: To assess the molecular mechanisms of resistance and to monitor disease response to treatment using liquid biopsies in NSCLC pts treated with BRAF inhibitors. Strategy: We performed a longitudinal genomic analysis of circulating-tumor DNA (ctDNA) in BRAF-mutated NSCLC pts treated in the AcSé vemurafenib program (NCT02304809) (n=44), or with the combination of dabrafenib and trametinib (n=6). We have collected 24 samples at baseline, 45 during follow-up and 9 at progressive disease (PD). ctDNA genotyping of 36 genes was performed using the Inivata InVisionFirst™ assay. Functional analyses of potentially resistant mutations and in vitro strategies to revert the resistant phenotype are ongoing. Results: Our preliminary analyses showed that BRAF mutations were detected at diagnosis in 16/24 pts, including 12 BRAF V600E mutations and 4 non-V600E mutations (i.e. G466V, G596R, G469A and K601E). 4/12 (34%) of BRAF V600E-mutated pts presented coexistent mutations, in FGFR2, CTNNB1, IDH1 or PI3KCA, whereas concomitant mutations in KRAS, NRAS or MYC were found in 3/4 (75%) of non-V600E cases. Analyses of response to treatment vs mutational profile will be presented. For the remaining 8/24 pts, TP53 mutations were found in 5 pts in absence of BRAF mutations, and no mutations were detected in 3 pts. Mechanisms of resistance were evaluated in 9 pts. One patient who progressed after 11 months on vemurafenib had MAP2K1 C121S and NFE2L2 p.31-32:GV/X mutations. In this patient, longitudinal ctDNA profiling revealed agreement between the %AF of BRAF and TP53 mutations and response to treatment, and detectable levels of the BRAF V600E and the MAP2K1 C121S mutations up to 6 months before the clinical confirmation of PD. Acquired PI3KCA H1047R and E545K mutations were seen in two pts, respectively, who progressed after 15 and 7 months of vemurafenib. Finally, a fourth patient who relapsed after 3 months on vemurafenib, presented a KRAS G12C mutation. All 4 cases also presented detectable levels of the BRAF V600E mutation at PD. In 3/8 pts, we detected the BRAF V600E mutation at PD but no other mutations; drivers of resistance may be present in genes outside this panel. ctDNA sequencing data on additional 7 pts at PD will be presented. Conclusion: Our results suggest that ctDNA genotyping might be an informative tool for monitoring disease response and resistance in NSCLC pts treated with BRAF-targeted therapies. Citation Format: Sandra Ortiz-Cuaran, Julien Mazières, Aurélie Swalduz, Washington René Chumbi Flores, Yohan Loriot, Virginie Westeel, Anne Pradines, Claire Tissot, Christelle Clement Duchene, Christine Raynaud, Xavier Quantin, Radj Gervais, Etienne Brain, Isabelle Monnet, Etienne Giroux Leprieur, Séverine Neymarc, Virginie Avrillon, Solène Marteau, Séverine Martinez, Gilles Clapisson, Nathalie Girerd-Chambaz, Celine Mahier, Nathalie Hoog-Labouret, Frank de Kievit, Karen Howarth, Emma Green, Clive Morris, Maurice Pérol, Jean-Yves Blay, Pierre Saintingy. Integrative analysis of resistance to BRAF-targeted therapies in lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1841.

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