Approximately 2% of non-small cell lung cancer (NSCLC) harbors a BRAF mutation, and BRAF V600E mutation accounts for 50–70% of BRAF mutated lung adenocarcinomas. Recently, the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) approved the combination of dabrafenib with trametinib as an effective treatment for BRAF V600E mutated patients. Another study reported an objective response rate (ORR) of 44.9% and a median progression-free survival (PFS) of 5.2 months in 100 BRAF V600 mutated NSCLC patients treated with Vemurafenib (1).

However, the question of the therapeutic options beyond BRAF inhibitor (BRAFi) remains a critical issue. Recently, FDA approved immune checkpoint inhibitor (ICPi) atezolizumab for BRAF V600 unresectable or metastatic melanoma based on the result of IMspire150 trial (NCT02908672) (2). NSCLC patients who had high programmed death ligand-1 (PD-L1) expression without EGFR/ALK/ROS1 drivers and had benefited from ICPi have been reported, while, the benefit in BRAF V600E NSCLC is still unclear. IMMUNOTARGET registry study is to investigate the activity of ICPi across NSCLC harboring oncogenic alterations in 24 centers from 10 countries, the result showing that ORR was 24% and median PFS was 3.1 months for 43 BRAF NSCLC patients, with the data of 4.1 months for smokers and 1.9 months for never smokers,1.8 months for V600E and 4.1 months for other BRAF mutations; however there were only around 5% patients in the first line setting (3). The BRAF V600E non-smoking NSCLC patients left unsolved the question of the place of immunotherapy, especially in first line setting. In this study, we report a TKI naïve non-smoker female with a lung adenocarcinoma driven by BRAF V600E mutation concomitant with high PD-L1 expression treated with atezolizumab in combination with chemotherapy followed by BRAFi Vemurafenib, which could open a new perspective of treatment for non-smoking BRAF mutated lung cancer.

The tissue biopsy at diagnosis was detected by BRAF V600E/EGFR/ALK/ROS1 Mutations Detection Kit (AmoyDx^®). PD-L1 expression was confirmed by immunohistochemical (IHC) assay using E1L3N (Cell Signaling Technology, Danvers, MA). The cell-free DNA (cfDNA) at diagnosis and the tumor DNA and cfDNA at progression on immunotherapy and BRAFi were analyzed by next-generation sequencing (NGS) including 457 genes (BerryOncology Inc.). Objective tumor response was measured by computed tomography (CT) using RECIST v1.1criteria. The patient provided written informed consent authorizing publication of clinical case.

A 62-year-old female non-smoker experienced a month-lasting fatigue, and PET/CT demonstrated an enlarged lung nodule on right lower lobe, bilateral supraclavicular and mediastinal lymphadenectases and multiple bony metastases confirming stage IV-T1bN3M1c ( Figure 1A ). Endobronchial ultrasound-guided transbronchial biopsy on subcarinal lymph node on 2018/03/28 revealed poorly differentiated lung adenocarcinoma with co-expressing tumors of BRAF V600E and PD-L1 ≥50% ( Figure 1B ). The concomitant liquid biopsy using NGS confirmed BRAF V600E mutation ( Supplementary Table 1 ).

The patient enrolled in the trial IMpower132 (NCT02657434) and randomly assigned into the combination group was given atezolizumab/cisplatin/pemetrexed every three weeks during four-induction cycle period before atezolizumab/pemetrexed maintenance. The first cycle of atezolizumab (1,200 mg)/cisplatin/pemetrexed in the induction period was commenced on 2018/04/18. The patient experienced pyrexia (40°C) and grade III gastrointestinal toxicity, leading to a 25% dose reduction of chemotherapy from the second cycle, but without dose interruption or reduction of atezolizumab per protocol. A partial response (PR) of 57.3% including 60% of primary lung and 56.0% of metastatic lymph nodes was observed on first CT-scan evaluation after two cycles of atezolizumab in combination with cisplatin/pemetrexed treatment ( Figure 2 ), and a continued PR was achieved for the following 14 cycles of atezolizumab/pemetrexed maintenance. Patient received atezolizumab maintenance from the 19^th cycle on 2019/05/09 considering grade II AST and ALT elevation. Unfortunately, disease progressed with the new pericardial effusion on 2019/12/17 after eighth cycles of atezolizumab maintenance, with a total of 20-months PFS of first-line treatment ( Figure 2 ). The patient was treated with second-line treatment of BRAFi Vemurafenib (960 mg bid po) on 2019/12/20 and disease showed stable disease (SD) after two cycles. Unfortunately, the disease continued to progress with the new left supraclavicular lymphadenectasis but still with the controllable primary lung after 5.5 months. NGS showed the blood-based tumor mutation burden (bTMB) was decreased from 9.63/Mb at diagnosis to 3.50/Mb at post-ICPi-progression and bounced off to 10.51/Mb at post-BRAFi-progression ( Figure 3A and Supplementary Table 1 ). The variant allele fraction (VAF) of mutation clusters in liquid biopsy was dropped at post-ICPi-progression compared with pre-ICPi; conversely, it drastically increased (excluding cluster 4) and new mutations occurred at post-BRAFi-progression compared with pre-/post-ICPi ( Figure 3B ).

Nowadays the therapeutic strategies of BRAF-mutated NSCLC are often introduced from melanoma treatment regimes. Due to the insufficiency of the study on BRAF-mutated subgroup in immunotherapy trials in NSCLC, which therapeutic strategy, immunotherapy, or targeted therapy was used as first-line remains an issue. Patients with BRAF V600E melanoma had longer PFS than those with BRAF V600K melanoma in targeted-therapy, and conversely the shorter PFS in the immunotherapy, providing the differences in gene expression and mutational load between V600E and V600K BRAF-mutant melanomas with the differences in response to BRAFi−/+ MEKi inhibitors and immunotherapy (4). Immuno-target therapy might work better for BRAF-mutant NSCLC smokers and seldom beneficial to non-smokers and light smokers (3). Indeed, never-smoking status with a targetable driver is usually accompanied with low TMB and low PD-L1 expression, which might partially explain the low efficacy of ICPi (5). While a retrospective study reported that all eight Asian lung cancer patients with BRAF V600E and PD-L1 ≥50% had co-expressing tumors, six were non-smokers (6), which is unlikely the same as that in Caucasian population. Co-expressing tumors account for 42% in NSCLC west Asian population (6) that is much higher compared to 28% in NSCLC with around 70% Caucasian population (7), which may partially explain the difference of immunotherapy efficacy in BRAF non-smoking NSCLCL. In ICPi/chemotherapy NSCLC clinical cohorts (8–13), combination groups demonstrated longer median PFS or OS than chemotherapy alone groups. Especially, the trials KEYNOTE-189, IMpower150, and IMpower130 which investigated the patients with previously untreated metastatic non-squamous NSCLC also displayed that the combination treatment was superior to single-chemotherapy. Furthermore, the magnitude of PFS and OS benefit with ICPi/chemotherapy combination correlated well with PD-L1 and was observed in these cohorts (8–13). The similarities of these cohorts and our case are that patients were EGFR/ALK negative, and some of them presented high PD-L1 expression. Here we reported the first-line combination of atezolizumab with chemotherapy improved PFS to 20 months in a non-smoking BRAF V600E-mutated NSCLS patient. The comparison of the result of the IMMUNOTARGET registry study showed median PFS was 1.8 months with ICPi treatment for V600E in the later line which is much shorter than that in the first line.

Interestingly, in our case, bTMB dropped at progression on immunotherapy, implying immunotherapy resistance occurred as the result of the loss of neoantigen (14). At progression, BRAF V600E mutation was both identified in pericardial effusion (new lesion) and plasma. A PARP1 mutation occurred in pericardial effusion lesion at progression on the combination of immunotherapy and chemotherapy, though no direct evidence has pointed out the relationship between PARP1 mutation and immunotherapy resistance yet. However, new finding shows that PARP inhibition can synergy with ICPi (15). PARP inhibition regulates the DNA damage response pathway and accumulates DNA injuries, which may lead to the accumulation of neoantigens for ICPi (16). In addition, the research of PARP-inhibitor-mediated upregulation of PD-L1 has been reported (17).

There was a median PFS of 5.2 months in 100 BRAF V600-mutated NCSLC patients treated with Vemurafenib found by J. Mazieres et al. (1) and our case with around 5.5 months. BRAFi therapy yields high ORR, while therapeutic duration is limited by diverse mechanisms of acquired resistance. Our result showed a rapid increase of VAF of all mutation clusters (excluding cluster 4) during BRAFi treatment to progression, and reactivation of the MAPK/Erk pathway is a major contributor to BRAFi treatment failure. A PDGFRα G853D mutation was found after BRAFi treatment in liquid biopsy; PDGFR is upstream of RAS in the MAPK pathway and strong autophosphorylation was observed in PDGFRα G853D mutation (18). Deregulation of the PI3K pathway which was found out by the presence of PI3KCA, AKT1, PTEN, or PPP2R1A mutations can cause resistance to BRAFi. Our case showed PI3KCA mutations after BRAFi progression. Furthermore, the genomic alterations in PIK3CA have been depicted in resistance to BRAFi in BRAF V600E melanoma and NSCLC (19).

This case shows the durable response to the combination of atezolizumab with chemotherapy and the prolonged PFS of 20 months in an untreated patient with non-smoking BRAF V600E-mutated lung adenocarcinoma, supporting the idea that combination immunotherapy may be an attractive option for BRAF V600E non-smoking NSCLC with high PD-L1 expression patients. This case highlights the need for further research to explore the efficacy of immuno-target therapy and ICPi in correlation with different molecular parameters in BRAF mutant NSCLC.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The studies involving human participants were reviewed and approved by Shanghai Chest Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual for the publication of any potentially identifiable images or data included in this article.

Design and supervision of this study: XN, SL, and XA. Data collection and analysis: XN, YS, LC, and JB. Manuscript writing: XN, DP, and LC. Final approval: XN, YS, DP, LC, JB, XA, and SL. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the National Natural Science Foundation of China (No. 81972187), Projects of the Committee of Shanghai Science and Technology (Nos. 19ZR1449800, 20Y11913700, 19411950500, 18DZ1910702), National Key Research and Development Program of China (No. 2016YFC1303300), Clinical Research Project of Shanghai Municipal Public Health Bureau (No. 201840122), Advanced Appropriate Technology Promotion Project of Shanghai Municipal Public Health Bureau (No. 2019SY048), Doctoral Innovation Fund of Shanghai Jiao Tong University School of Medicine (No. BXJ201952), and Interdisciplinary Program of Shanghai Jiao Tong University (No. YG2017MS80),Project of Shanghai Talent Development Fund (No. 2019074).

LC and JB were employed by the company Berry Oncology Corporation.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.