Concurrent Acquired BRAF V600E Mutation and MET Amplification as Resistance Mechanism of First-Line Osimertinib Treatment in a Patient with EGFR-Mutated NSCLC

Recent results of the FLAURA trial showed a better progression-free survival for patients treated with osimertinib, a new third-generation EGFR tyrosine kinase inhibitor, than when gefitinib or erlotinib is used as first-line treatment of EGFR-mutated NSCLC, opening a new scenario for osimertinib as a first therapeutic option.1Soria J.C. Ohe Y. Vansteenkiste J. et al.Osimertinib in untreated EGFR-mutated advanced non-small-cell lung cancer.N Engl J Med. 2018; 378: 113-125Google Scholar Unfortunately, patients experienced disease progression after a median of 18.9 months, and actually, little is known about resistance mechanisms.2Ramalingam S.S. Yang J.C. Lee C.K. et al.Osimertinib as first-line treatment of EGFR mutation-positive advanced non-small-cell lung cancer.J Clin Oncol. 2018; 36: 841-849Google Scholar Therefore, understanding of the resistance mechanisms to first-line osimertinib is an emerging clinical need. Here, we report the case of a patient who was treated with osimertinib in the FLAURA trial and whose postprogression repeat tissue biopsy showed, for the first time, concurrent BRAF V600E mutation and MNNG HOS Transforming gene (MET) amplification as acquired mechanisms of resistance. In July 2015, after a diagnosis of stage IV lung adenocarcinoma with EGFR exon 19 deletion (right upper lobe lung mass, pleural effusion, mediastinal adenopathies, and bone metastases) (Fig. 1A), a 65-year-old female never-smoker started therapy in the FLAURA trial. Treatment was well tolerated, with a partial response (Fig. 1B) until April 2017, when a modest increment of bone lesions was noted. In July 2017, a new computed tomography scan showed progression of disease as a result of new bone lesions treated with radiotherapy (Fig. 1C). In October 2017, the patient presented with worsening dyspnea and a computed tomography scan showed new appearance of ground-glass consolidation involving the right lung (Fig. 1D). Treatment was interrupted and unblinded, confirming that the patient had been receiving osimertinib. New bronchoscopy confirmed intraalveolar spread of adenocarcinoma cells and revealed persistence of exon 19 deletion with new BRAF V600E mutation and MET amplification (see Figs. 1D and 2); other concomitant mechanisms (SCLC transformation; EGFR T790M, EGFR C797S, and KRAS mutations; and EGFR and erb-b2 receptor tyrosine kinase 2 gene [HER2] amplification) were excluded. Cell-free DNA samples were also tested; the first evidence of BRAF V600E mutation was discovered at the time of radiological progression in July 2017, with an increase found in the subsequent samples (Fig. 1E). Double immunohistochemistry was performed, and positivity both for BRAF V600E and MET primary antibody was demonstrated (Fig. 3) in different parts of the tumor (Fig. 3C and 3E). The patient's clinical condition worsened rapidly as a result of respiratory failure, and she died a few days later.Figure 2Molecular analysis. Analysis of BRAF V600E mutation (A–D). This figure shows negativity for BRAF V600E mutation in the two tissue biopsy samples obtained at diagnosis (pleura sample in A and lung sample in B), whereas specific amplification for a BRAF V600E probe was detected in both tissue (C) and cell-free DNA (D) postosimertinib samples. BRAF mutations were analyzed with the Easy BRAF Kit (Diatech Pharmacogenetics, Jesi, Italy). Blue line indicates positive control, purple line indicates negative control; yellow line indicates, BRAF V600E–specific probe; and red line indicates, threshold. Analysis of the MNNG HOS Transforming gene (MET) (E and F). Fluorescence in situ hybridization (FISH) negative for MET amplification with a gene copy number of 2.1 in the diagnostic tissue biopsy sample (E), whereas in the postosimertinib histological sample FISH analysis showed a cluster MET amplification (F) (MET signals ≥ 20, chromosome 7 signals = 3; MET-to–chromosome 7 ratio ≥5). Bars indicate 10μm. 4′,6-Diamino-2-phenylindole staining was reported at ×1000 original magnification. MET amplification was assessed by FISH by using a Poseidon Repeat Free MET (7q/31) and SE7 probe (Kreatech Diagnostics, Amsterdam, the Netherlands).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Immunohistochemistry (IHC) for MNNG HOS Transforming gene (MET) and BRAF V600E. Double IHC for MET and BRAF V600E was performed. After coimmunostaining, the BRAF V600E mutated clone was stained brown (C), whereas the MET amplified clone was stained red (E). In the BRAF V600E–mutated clone, the inflammatory cells in the alveolar space (e.g., macrophages) were stained red (C). Hematoxylin and eosin staining results were reported at ×2 original magnification (A) and ×200 original magnification (B and D). The red arrows indicate the tissue sections that were shown at higher magnification in panel B and D, respectively. Primary antibodies: BRAF V600E (clone VE1, Roche, Basel, Switzerland) and MET (clone SP44, Roche). The sections were immunostained for BRAF primary antibody with horseradish peroxidase polymer (Optiview DAB IHC Detection kit, Roche) and for the MET primary antibody with alkaline phosphatase polymer (Ultraview Universal Alkaline Phosphatase Red Detection Kit, Roche) in accordance with the manufacturer’s specifications.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Actually little is known about resistance mechanisms after osimertinib as first-line treatment.2Ramalingam S.S. Yang J.C. Lee C.K. et al.Osimertinib as first-line treatment of EGFR mutation-positive advanced non-small-cell lung cancer.J Clin Oncol. 2018; 36: 841-849Google Scholar BRAF V600E has recently been associated with resistance to first-line treatment with another third-generation EGFR tyrosine kinase inhibitor,3Bearz A. De Carlo E. Doliana R. Schiappacassi M. Acquired BRAF V600E mutation as resistant mechanism after treatment with third-generation EGFR tyrosine kinase inhibitor.J Thorac Oncol. 2017; 12: e181-e182Google Scholar and MET amplification has been already described as a distinct resistance mechanism after administration of osimertinib in first- and second-line treatment.2Ramalingam S.S. Yang J.C. Lee C.K. et al.Osimertinib as first-line treatment of EGFR mutation-positive advanced non-small-cell lung cancer.J Clin Oncol. 2018; 36: 841-849Google Scholar, 4Minari R. Bordi P. Tiseo M. Third-generation epidermal growth factor receptor-tyrosine kinase inhibitors in T790M-positive non-small cell lung cancer: review on emerged mechanisms of resistance.Transl Lung Cancer Res. 2016; 5: 695-708Google Scholar Here, we have presented what to our knowledge is the first report of concurrent acquired BRAF V600E mutation and MET amplification detected from a tissue biopsy of a patient who progressed in response to first-line osimertinib. Double immunochemistry performed with MET and BRAF V600E antibodies revealed a potential clonality of these mechanisms. Analysis of EGFR in DNA from BRAF V600E and MET amplification clones revealed that both shared a common tumor origin, supporting the heterogeneous scenario of acquired mechanisms of resistance.5Chabon J.J. Simmons A.D. Lovejoy A.F. et al.Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients.Nat Commun. 2016; 7: 11815Google Scholar Our report shows a new example of heterogeneity in resistance mechanisms presenting a concurrent MET amplification and BRAF mutation that has never been described before either in first-line or in second-line treatment with osimertinib, opening the possibility of future combinations to overcome resistance. This work was supported by AIRC (Italian Association for Cancer Research), Milan (grant IG2017-20074 to the principal investigator [Dr. Tiseo]).