Clinicopathologic characteristics and outcomes of Chinese patients with non‐small‐cell lung cancer and BRAF mutation

Abstract BRAF mutation is one of the important driver oncogene in non‐small‐cell lung cancer (NSCLC). Data on Chinese patients with BRAF‐mutant NSCLC are inadequate. Hence, we conducted this study to investigate the clinicopathologic features and outcomes of Chinese patients with NSCLC and BRAF mutations. We identified patients with BRAF‐mutant NSCLC between January 2012 and April 2016. Patient characteristics and treatment outcomes were analyzed. In total, 1680 patients were included. Twenty‐eight (1.7%) patients harbored BRAF mutations. Compared to patients with non‐BRAF mutation, patients with BRAF mutations were associated with adenocarcinomas (89.3% vs. 70.6%, P = 0.048) and never smokers (78.6% vs. 56.7%, P = 0.019). There were no significant differences in the age, gender distribution, metastasis, or stage at first diagnosis between two groups. Response rates and progression‐free survival (PFS) were similar between patient with BRAF mutations and EGFR (5.6 vs. 5.8 months; P = 0.277) or KRAS (5.6 vs. 4.7 months; P = 0.741) mutations to first‐line chemotherapy. Compared to patients with non‐V600E mutations, patients with V600E‐mutated tumors had a shorter PFS to first‐line chemotherapy, although this did not reach statistical significance (5.2 vs. 6.4 months; P = 0.561). In multivariate analyses, only ECOG PS remained the independent predictor of overall survival (HR = 0.208; P = 0.004). In conclusion, BRAF mutation in Chinese patients with NSCLC was rare. BRAF mutation is more likely to be associated with adenocarcinoma and never smokers. BRAF mutations are not associated with enhanced chemosensitivity and novel and effective drugs inhibiting the BRAF pathway are in urgent need.

Introduction BRAF, one of the serine/threonine protein kinase, belongs to the RAF kinase family in the RAS-RAF-MEK-ERK signaling pathway [10,11]. When activated by mutations, BRAF activates MEK and this leads to the activation of the ERK signaling pathway to promote cell growth, proliferation, and survival [12]. The most common mutation in BRAF is the valine (V) to glutamic acid (E) substitution at residue 600 (BRAF V600E), which results in a mutant BRAF protein that no longer requires dimerization for its activity and is constitutively active and transforming in vitro [13][14][15][16]. Somatic mutations in BRAF are found in several kinds of cancers, including melanoma, ovarian carcinomas, colorectal cancers, papillary thyroid cancers, and lung cancers. BRAF mutations are most commonly seen in melanoma, where BRAF V600E is the driver mutation that can be effectively targeted with selective BRAF and/or MEK inhibitors [17][18][19][20]. BRAF mutations are also observed in 1-3% of NSCLC [21][22][23][24][25]. Studies on lung cancers, in which BRAF mutations were observed have generated considerable interest because these mutations may be associated with increased sensitivity to agents directly targeting BRAF or BRAF-mediated downstream signaling pathways [26,27]. Hence, several previous reports have begun to define the prevalence, distribution, and prognosis of BRAF mutations in patients with NSCLC [21][22][23][24][25]28]. But there are several limitations of the published articles: (1) the enrolled patients were from Europe and America and little study included Chinese patients with NSCLC. As is known, the genetic background between Caucasians and Asians with NSCLC is totally different. (2) limited by relatively small numbers of patients, few study reported the effect of first-line chemotherapy in NSCLC patients with BRAF mutations; (3) they also did not compare the effect of first-line chemotherapy in BRAF-mutant patients with patients who harbored other activating mutations such as EGFR and KRAS. We therefore conducted this study with the aim of clarifying the clinicopathologic characteristics and effect of chemotherapy in Chinese patients with BRAF-mutant NSCLC.
Toward this aim, we analyzed arguably the largest cohorts to describe the clinicopathologic characteristics of Chinese patients with BRAF-mutant NSCLC in this study. Meanwhile, we assessed the effect of first-line chemotherapy in patients with NSCLC and BRAF mutations. In addition, we also compared the therapeutic effect of chemotherapy in NSCLC patients who harbored BRAF mutations with those who harbored EGFR or KRAS mutations.

Patients cohort
Data of patients with pathologically confirmed lung cancer who received EGFR, KRAS, and BARF mutation test at the Thoracic Cancer Institute, Tongji University from January 2012 to April 2016 were retrospectively reviewed. The major clinicopathologic characteristics including sex, age, smoking history, Eastern Cooperative Oncology Group performance status (ECOG PS), lung cancer histology (WHO classification) [29], EGFR, KRAS, and BARF mutation status, metastases and stage were all collected. A never smoker was defined as a person who had smoked <100 cigarettes during his/ her lifetime. Age, smoking status, and ECOG PS were documented at first diagnosis. Thoracic Cancer Institute, Tongji University School of Medicine established requirements for clinical information on patient follow-up under treatment, including response to treatment and survival. Patients were followed from the date of cancer diagnosis until date of death or last available follow-up. Tumor response was evaluated according to the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1), including complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). The treatment response was evaluated 1 month after the initiation of therapy and then every 2 months. This study was approved by Shanghai Pulmonary Hospital Ethics Committee and a written informed consent was obtained from each patient to use the clinical data for research before the medical intervention started.

Molecular analysis
All mutational analyses were conducted at the Thoracic Cancer Institute, Tongji University Medical School, Shanghai. Briefly, DNA from tissue was extracted using the DNeasy Blood and Tissue Kit or the QIAamp DNA FFPE Tissue Kit (both from Qiagen, Hilden, Germany). EGFR, BRAF, and KRAS mutations were tested by amplification refractory mutation system (ARMS) as described in our previous studies (Amoy Diagnostics Co. Ltd., Xiamen, China) [30][31][32][33]. BRAF mutations were further confirmed by direct sequencing.

Statistical analysis
The categorical variables were analyzed by chi-square tests, or Fisher's exact tests when needed. The continuous variable was compared using ANOVA and Tukey's multiple comparison tests. Kaplan-Meier curve and two-sided logrank test were used for univariate survival analyses. Cox proportional hazards model was used for uni-and multivariate survival analyses to calculate the hazard ratios (HR) and corresponding 95% confidence intervals (CI). Overall survival (OS) was calculated from the date of lung cancer diagnosis to death from any reasons or was censored at the last follow-up date. Progression-free survival (PFS) was defined as the time from the date of first-line treatment initiation to the date of systemic progression or death and was censored at the date of last tumor assessment. Disease progression was defined in accordance with the RECIST version 1.1. P values were considered significant if less than 0.05 (two-sided). All statistical analyses were performed using the SPSS statistical software, version 22.0 (SPSS Inc., Chicago, IL).

Clinicopathologic characteristics associated with BRAF-mutant NSCLC
BRAF mutations were present in 16 women and 12 men with an average age of 64 years (range, 37-78 years). Twenty-two patients (78.6%) were never smokers.
Histopathologic stage varied and included IIIB (n = 17) and IV (n = 11). Twenty-five tumors were adenocarcinomas and three tumors were non-adenocarcinoma. Six patients had distant metastasis at time of diagnosis (21.4%). Most of them (n = 20) had the good performance score (0-1). More details of patients' BARF mutations are listed in Supplemental Table S1. Compared to total patients with non-BRAF mutation, patients with BRAF-mutant tumors were more likely to be never smokers (78.6% vs. 56.7%, P = 0.019). Patients with BRAF mutation were associated with adenocarcinoma than those with non-BRAF mutation (89.3% vs. 70.6%, P = 0.048). There were no significant differences in the age, sex distribution, metastasis, or stage at time of diagnosis between patients with BRAF-mutant and BRAF wild-type tumors ( Table 1).  Table 2). There were no significant differences in objective response rate (ORR) and disease control rate (DCR) between patients with BRAF mutation and EGFR or KRAS mutation ( Table 2). Figure 1 shows the survival data in these patients. Briefly, median PFS of patients with BRAF mutation who received first-line chemotherapy was 5.6 months (Fig. 1A) compared with 5.3 months for wild-type patients (P = 0.693; Fig. 1C), and median OS was 14.7 months (Fig. 1B) in patients with BRAF mutation. Within BRAF-mutant patients, the median PFS was shorter in patients with V600E mutation compared with non-V600E mutations, but did not achieve statistical significance (5.2 vs. 6.4 months; HR = 0.74, 95% CI: 0.29-1.94, P = 0.561; Fig. 1D). Compared to patients with EGFR mutation, median PFS was similar in patients with BRAF mutation who received first-line chemotherapy (median PFS: 5.6 vs. 5.8 months; HR = 1.25, 95% CI: 0.82-1.99, P = 0.277; Fig. 2A). The median PFS of first-line chemotherapy was also similar between patients with BRAF mutation versus patients with KRAS mutation (median PFS: 5.6 vs. 4.7 months; HR = 0.93, 95% CI: 0.63-1.39, P = 0.741; Fig. 2B).

Discussion
To the best of our knowledge, this study was arguably the first large-scale retrospective study to investigate the clinicopathologic characteristics and outcomes of Chinese patients with NSCLC and BRAF mutation. We enrolled 1680 NSCLC patients and 28 of them had BRAF mutations. The rate of BRAF mutations was 1.7%, which was similar to those reported in Asian populations but lower than those reported in Caucasian populations [22,25,28,34]. The relative paucity of BRAF mutations in the Chinese patients may be associated with ethnic differences and the high frequency of EGFR mutations in Chinese NSCLC patients. Our findings also indicated that NSCLC with BRAF mutations are associated with unique clinicopathologic features compared with BRAF wild type and other genomic subtypes. In our study, BRAF mutations are more likely in never smokers; this is similar to patients with activated EGFR and ALK alterations, who are also associated with never smokers. In contrast, several previous studies suggested that BRAF mutations occurred most often in former/ current smokers [25,28]. The possible reason may include that the distribution of BRAF mutation types was uneven. In our cohort, 85.7% of patients harbored BRAF V600E and only 50-60% of included patients harbored BRAF V600E in the previous studies. This was demonstrated by another study, which included 36 lung adenocarcinomas that harbored BRAF mutation and showed that BRAF V600E was significantly more frequent in never smokers and in female patients, whereas all non-V600E mutations were detected in smokers [34]. Also another two studies based on Chinese population demonstrated that BRAF V600E was markedly associated with never smoking and female sex [35,36]. Furthermore, a recent meta-analysis, which included 10 studies, indicated that there was no significant difference in BRAF mutation frequency in former/current smokers versus never smokers (OR = 0.95, 95% CI: = 0.45-2.02), but the difference was significant between former or current smokers and never smokers in patients with BRAF V600E (OR = 0.14, 95% CI: = 0.05-0.42) [37]. Taken together, we can conclude that BRAF V600E  mutation is more likely in never smokers, and BRAF non-V600E mutations occur most often in former or current smokers. Specifically, we did not observe an association between gender, age, number of metastases, or stage at time of diagnosis of NSCLC and BRAF mutations.
The survival outcomes of patients with BRAF-mutant NSCLC to first-line chemotherapy closely resembled those with wild-type tumors. This result was consistent with a previous report that median PFS of NSCLC patients with BRAF mutations received platinum-based combination chemotherapy was similar to patients with BRAF wild type (5.2 vs. 6.7 months, P = 0.622) [28]. Moreover, the median PFS was similar between patients with BRAF mutation and EGFR/KRAS mutations. These results suggested that BRAF mutations are not associated with enhanced chemosensitivity. Compared to patients with non-V600E mutations, patients with V600E mutations had shorter PFS, although these differences did not reach statistical significance because of low power due to the limited sample sizes. The differences did not seem to be related to imbalances among the subgroups in terms of type of chemotherapy received. Our findings are consistent with three previous reports that showed poor outcomes among patients with BRAF V600E mutations compared with BRAF wild type [28,34,35]. Likewise, authors have reported that V600E mutation was frequently related to a more aggressive histotype characterized by micropapillary features [34]. Cardarella and colleagues also reported that the median PFS was shorter in patients with V600E mutation compared with non-V600E mutations, but did not achieve statistical significance (4.1 vs. 8.9 months; P = 0.297) [28]. In our study, we did not collect the histological details and we therefore cannot determine the association between micropapillary histology and BRAF V600E mutations. To clarify this relationship, future research with large number of cases is warranted.
In our cohort, the co-occurring driver rate among patients with BRAF-mutant NSCLC was 14.3%. The cooccurrence of BRAF mutations with EGFR and KRAS mutations has previously been reported in NSCLC, including two patients in the series by Marchetti et al. with concurrent BRAF V600E plus EGFR mutations and one patient with BRAF V600E plus PIK3CA mutation and two patients with BRAF G464 plus KRAS mutations in the series by Cardarella et al. [28,34]. In a study, which enrolled Asian populations, five non-V600E mutations (four G469A and one G464E/G466R) exhibited concomitant EGFR mutations [22]. Li et al. reported that five out of eight Chinese patients with lung adenocarcinoma and BRAF V600E mutation had concomitant EGFR mutations [35]. Lung Cancer Mutation Consortium (LCMC) also reported that double-mutation rate among patients with BRAF-mutant NSCLC was 16% [25]. This emphasizes the role of multiplexed genotyping or next generation sequencing in NSCLC genotype because more than one targetable driver mutation may exist within one patient.
To date, two popular second-generation BRAF inhibitors, dabrafenib and vemurafenib, have shown the promising efficacy in patients with BRAF V600E-mutant NSCLC. In a histology-independent phase 2 "basket" study, patients with BRAF V600 mutation received vemurafenib [38]. In the cohort with NSCLC, the ORR was 42% and median PFS was 7.3 months. This is the first time where the efficacy of vemurafenib in NSCLC patients with BRAF V600E mutation in the clinical trial has been demonstrated. Then, a phase 2, multicenter, nonrandomized, open-label study assessed the clinical activity of dabrafenib in patients with NSCLC and BRAF V600E mutation [27]. The investigatorassessed ORR was 33% in previously treated patients and 66.7% in previously untreated patients. Furthermore, another recent phase 2, multicenter, nonrandomized, open-label study investigated the antitumor activity and safety of dabrafenib plus trametinib in patients with BRAF V600E-mutant NSCLC [39]. The result showed that combination therapy could achieve a high ORR of 63.2% in previously treated patients. This result indicated that dabrafenib plus trametinib could become a new targeted therapy with robust antitumor activity in these patients. With the publication of these clinical trials, the effectiveness of these BRAF targeted agents would be extensively demonstrated in patients with NSCLC and BRAF V600E mutation. In view of the high response rate with dabrafenib plus trametinib in patients with BRAF V600E-mutant NSCLC, future research will investigate the position of dabrafenib plus trametinib as an early treatment option compared with platinum-based chemotherapy or immunotherapy options.
Our study has several limitations that should be acknowledged. Firstly, despite the initial cohort being large, the number of patients who entered the final analysis was relatively small. Secondly, BRAF mutations were detected using ARMS that identified only a limited number of BRAF point mutations. We note that other BRAF mutations in NSCLC have been identified including mutations in amino acids 421, 439, 459, 466, 471, 595, 597, 604, and 606. Thirdly, although we performed the subgroup analysis of treatment outcomes according to molecular mutations including BRAF, EGFR, and KRAS mutations, compared the BRAF-mutated patients with more specified subgroup. Finally, this study is a retrospective study, which might have induced selection bias. Therefore, the findings in this study need to be validated in prospective trials with large scale.
In summary, this study identified BRAF mutations in 1.7% of Chinese patients with NSCLC. BRAF mutation is associated with adenocarcinoma, and BRAF V600E mutation is more likely in never smokers. BRAF mutations are not associated with enhanced chemosensitivity. This indicates that new and effective drugs targeting the BRAF pathway are in urgent need. In addition, NSCLC patients with BRAF mutations had the high co-occurring driver rate. This emphasizes the significance of comprehensive genomic profiling in assessing patients with NSCLC, especially BRAF-mutant NSCLC.