Introduction

BRAF mutation leads to downstream phosphorylation of MEK and ERK, resulting in constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway, which drives tumor cell proliferation and differentiation [1]. Approximately 90% of BRAF mutations occur at the level of the T1799 transversion in exon 15, which results in the substitution of valine for glutamic acid (V600E) and increases BRAF activity by approximately tenfold compared to wild type [2]. Nearly 30% of BRAF-V600E mCRC tumors have MSI, and most MSI BRAF tumors are caused by hypermethylation of the MLH1 promoter [3]. The BRAF V600E mutation occurs in approximately 10% of patients with metastatic colorectal cancer (mCRC). BRAF V600E mutations are more common in women and the elderly, and the majority of BRAF V600E-mutated mCRCs occur in the right colon, tend to be mucinous in histology, and have a microsatellite instability (MSI)-high phenotype [4]. The prognosis of mCRC with BRAF V600E mutation is extremely poor with median overall survival (OS) of 10–17 months [5, 6]. And the risk of death in mCRC patients with the BRAF V600E mutation was twice that of BRAF wild-type patients [7,8,9].

National and international guidelines recommend two- or three-drug chemotherapy (based on fluorouracil in combination with platinum or/and irinotecan) with bevacizumab as first-line treatment for mCRC patients with BRAF V600E mutation [10]. The randomized phase 3 TRIBE study demonstrated that FOLFOXIRI (folinic acid, 5-fluorouracil, oxaliplatin, and irinotecan) plus bevacizumab was associated with longer OS (29.8 vs. 25.8months, p = 0.030) compared to FOLFIRI plus bevacizumab in mCRC patients.TRIBE subgroup analysis suggested that FOLFOXIRI plus bevacizumab also improved OS (19.1 vs. 10.8 months) and progression-free survival (PFS) (7.5 vs. 5.5 months) in patients with BRAF V600E-mutated mCRC [11]. The subgroup analysis of the subsequent TRIBE2 study [12], comparing FOLFOXIRI plus bevacizumab to FOLFOX plus bevacizumab, did not confirm the increased benefit among BRAF V600E-mutated mCRC. The individual patient data meta-analysis by Cremolini et al. [13] went along with it. As expected, the intensified regimen increases the incidence of grade 3 and 4 adverse events (diarrhea, stomatitis, and neutropenia) [12]. Therefore, the efficacy of first-line FOLFOXIRI or doublet chemotherapy combined with bevacizumab in patients with BRAF 600E-mutated mCRC remains controversial.

In our study, we retrospectively collected clinical data of patients with BRAF V600E-mutated mCRC from multi-center real-world data and analyzed their clinicopathological characteristics. We also conducted a comparison between FOLFOXIRI regimens plus bevacizumab and doublet chemotherapy plus bevacizumab as first-line therapy in patients with BRAF V600E-mutated mCRC to explore the efficacy of these two regimens.

Materials and methods

Study design and patients

This retrospective study consecutively collected patients diagnosed with BRAF V600E-mutated mCRC from the 3 July 2017 to 24 December 2022. All patients were collected from the database of three institutions, including West China Hospital of Sichuan University, Yunnan Cancer Hospital, and The First Affiliated Hospital of Chongqing Medical University. Patients were included if they met the following criteria: (1) pathologically confirmed metastatic colorectal adenocarcinoma with at least one measurable disease according to the Response Evaluation Criteria in Solid Tumors (v1.1); [14]; (2) BRAF V600E mutation confirmed by gene detection; (3) age older than 18 years; Patients were excluded if they lacked complete clinical information. Reporting of this study conforms to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement [15].

Variables

The demographic features [including age, gender, Eastern Cooperative Oncology Group performance status (ECOG PS)], histological type, primary tumor location, metastatic pattern, and metastatic organ, treatment modalities (surgery, chemotherapy regimen, and targeted therapy), and survival data were obtained by medical record review. Age was dichotomized into two groups: ≤60 years and > 60 years according to the age segmentation between middle-aged people and elderly people in China. Primary tumor location was dichotomized into three groups: the right-sided colon (including the ileocecal junction, cecum, ascending colon, hepatic flexure, and transverse colon), left-sided colon (including the splenic flexure, descending colon, and sigmoid colon), and rectum.

Treatment regimens and evaluation

The Doublet-drug arm received cytotoxic doublets, including XELOX (Oxaliplatin 130mg/m2, ivgtt, d1 + Capecitabine 1000mg/m2,po, bid, d1-14, q3w), mFOLFOX6 (Oxaliplatin 85mg/m2, ivgtt, d1 + leucovorin 400mg/m2, ivgtt, d1 + Fluorouracil 400mg/m2, ivgtt, d1 + Fluorouracil 1200mg/m2.d, ivgtt, d1-d2, q2w), or FOLFIRI (Irinotecan 180mg/m2, ivgtt, d1 + leucovorin 400mg/m2, ivgtt, d1 + Fluorouracil 400mg/m2, ivgtt, d1 + Fluorouracil 1200mg/m2.d, ivgtt, d1-d2, q2w) plus bevacizumab (5 mg/kg, ivgtt, d1, q2w), and triplet-drug group received a modified FOLFOXIRI regimen (Oxaliplatin 85mg/m2, ivgtt, d1 + Irinotecan 165mg/m2, ivgtt, d1 + leucovorin 400mg/m2, ivgtt, d1 + Fluorouracil 1200mg/m2.d, ivgtt, d1-d2, q2w) plus bevacizumab (5 mg/kg, ivgtt, d1, q2w). The chemotherapy alone group received chemotherapy without bevacizumab. Tumor assessment by computed tomography was performed based on the RECIST version 1.1.

Survival outcomes and follows-up

The primary objective of the study was to evaluate OS according to the treatment regimen. The secondary endpoints were PFS, objective response rate (ORR), and disease control rate (DCR) in the first-line treatment and to estimate the difference in terms of efficacy between the doublet-drug and triplet-drug regimen as first-line therapy. OS was defined as the time from the diagnosis of metastatic disease to death from any cause. PFS was measured from the start of treatment to the date of disease progression or death from any cause. ORR was defined as complete response (CR) or partial response (PR), while DCR was defined as CR, PR, and stable disease (SD). Follows-up started when patients were diagnosed with mCRC and was conducted by telephone every 2 months.

PSM method

We used a 1:1 propensity score matching (PSM) strategy to match different patients with doublet-drug group and triplet-drug group to create well-matched cohorts and reduce potential confounding effects. We included the variables with potential impact on prognosis, including age, sex, grade, primary tumor location, liver metastasis, peritoneal metastasis, distant lymph node metastasis, and number metastatic organs in this propensity score model. The nearest neighbor matching algorithm without replacement was applied to ensure adequate matches with a caliper of 0.02 standard deviation of the logit of the propensity score.

Statistical analysis

Statistical analysis was performed using SPSS version 25.0 and R version 4.2.2. Univariate and multivariate analysis was performed using a Cox proportional hazards model. Kaplan-Meier analyses were performed to analyze survival, including OS and PFS. The hazard ratio (HR) with 95% confidence intervals (CIs) was used to compare the risk of the two regimens. p-value less than 0.05 in a two-tailed test was used to define statistical significance.

Results

Patients and baseline characteristics

A total of 125 patients were enrolled, 28 in the chemotherapy alone group (22.4%), 25 in the triplet-drug cohort (20.0%) and 67 in the doublet-drug cohort (53.6%) and 5 patients received single-agent chemotherapy or immunotherapy. The patient selection flowchart is shown in Fig. 1. The characteristics of all enrolled patients diagnosed with BRAF V600E-mutated metastatic colorectal cancer are summarized in Table 1. After 1:1 PSM, 25 patients of each group were obtained and no significant differences were discovered between the two cohorts (Supplement Table 1).

Fig. 1
Fig. 1
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Flow diagram of patient selection

Table 1 Characteristics of all enrolled patients diagnosed with BRAF V600E-mutated metastatic colorectal cancers
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The overall survival and quartile analysis

For the 125 patients with BRAF V600E-mutated mCRC, the median follow-up time was 22.4 months (95% CI 19.6–25.1months), the median OS was 14.9 months (95% CI 12.6–18.9 months), and the median PFS of first-line therapy was 6.10 months (95%CI 5.23–7.70 months) (Fig. 2). We divided the OS of the enrolled patients by quartile method, and the quartile interval was 1.5–7.15 months, 7.15–10.73 months, 10.75–17.1 months, and 17.1-69.23 months, respectively. Patients with OS less than 7.15 months were included into the poor prognosis group, and patients with OS more than 17.1 months were included into the good prognosis group. The median OS of the two groups was 29.2 months and 6.0 months, respectively. The difference in baseline variables between the two groups showed that the poor prognosis group was associated with poor differentiation, multiple organ metastasis and liver metastasis (p < 0.050).

Fig. 2
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Kaplan–Meier survival curves for all enrolled BRAF V600E-mutated mCRC patients: (a) overall survival of patients, (b) progression-free survival of patients

Univariate and multivariate analysis

In univariate analysis of OS, the following factors, including age, ECOG PS score, grade, liver metastasis, and first-line therapy regimen were associated with OS (p < 0.050). The above variables were included in the multivariate analysis. Based on multivariate analysis, variables such as age>60, poor differentiation, liver metastasis, and first-line doublet regimen were associated with worse OS in patients with BRAF V600E-mutated mCRC (Table 2). Combined with quartile analysis, we further verified that poor differentiation and liver metastasis were independent predictors of OS in patients with BRAF V600E-mutated mCRC.

Table 2 Univariate and multivariate analysis for the prognostic variables of overall survival for patients diagnosed with BRAF V600E-mutated metastatic colorectal cancer
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First-line therapy efficacy and survival analysis

The overall response of all the enrolled patients before and after PSM is summarized in Table 3. No significant differences in ORR and DCR were found in the doublet group and triplet group before and after PSM (Table 3).

Table 3 The overall response of all the enrolled patients before and after propensity score matching
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Regarding survival, the median OS was significantly improved in the triplet group compared to that in doublet-drug group (17.4 vs. 13.4 months, p = 0.022). While median PFS was improved without significant differences (8.5 vs. 6.5 months, p = 0.140) (Fig. 3). After PSM, the trend for OS remained and became more obvious (17.4 vs. 10.4 months, p = 0.004). A similar prognostic trend for PFS was observed in the matched cohort (Fig. 3).

Fig. 3
Fig. 3
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Kaplan–Meier survival curves for BRAF V600E-mutated patients before and after matching: (a) overall survival of patients before matching, (b) progression-free survival of patients before matching, (c) overall survival of patients in the matched cohort, and (d) progression-free survival of patients in the matched cohort

Stratified analysis

Patients in the triplet group had a better prognosis with the following beneficial factors: younger age (< 60 years old), better ECOG PS score (0–1), multiple metastatic organs, and presence of liver metastases (Fig. 4).

Fig. 4
Fig. 4
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Stratified analysis for overall survival in BRAF V600E-mutated mCRC patients

Discussion

In our study, BRAF V600E-mutated mCRC were more likely to be located in the right colon and more common in older female. We also found that BRAF V600E-mutated mCRC was associated with aggressive behaviors: the majority of patients had synchronous metastases, and multiple organ involvement, affecting more frequently distant lymph node, liver, and peritoneum and less frequently lungs, which was consistent with previous studies [8, 16]. Notably, only 4.8% of the BRAF V600E mutated tumors in our study were dMMR. This proportion is obviously lower than that in previous studies in European countries (37.2%) [17] and in Sweden (50.0%) [18]. In another retrospective study conducted from Sun Yat-sen University in China [19], the incidence of dMMR in BRAF V600E-mutated mCRC patients was only 1.9%. Therefore, the difference between countries may be due to racial differences.

Many studies have investigated prognostic factors in BRAF V600E-mutated mCRC. One study found that MMR, primary tumor resection, and metastasis resection were positively associated with survival, and they also reported that the neutrophil-to-lymphocyte ratio ≥ 3, the serum albumin levels ≥ 3.6 g/dL, and systemic inflammation score of 1–3 were associated with significantly improved OS [20]. While synchronous metastases, and absence of curative surgery, were statistically associated with poor survival in patients with BRAF V600E mutation [21]. In our study, according to multivariate analysis and quartile analysis, liver metastasis and poor differentiation were identified as negative independent predictors of OS in patients with BRAF V600E-mutated mCRC.

In terms of treatment efficacy, the ORR and DCR of patients receiving triplet therapy were numerically higher than those in doublet group, without reaching statistical significance before and after PSM. The results were consistent with the TRIBE, TRIBE-2 and the individual patient data meta-analysis of FOLFOXIRI plus bevacizumab [11,12,13]. After PSM, the OS of patients treated with first-line triplet was still significantly longer than that of doublet patients (p = 0.004) and there was still no difference in median PFS (p = 0.120). We further performed subgroup analysis of OS and found that the triplet-drug regimen appears to be associated with a better prognosis in patients with better baseline status and higher tumor burden.

The study had the following limitations: first, it was a retrospective study with a small sample size, and the treatment regimen was chosen by different investigators, which may have led to potential selection bias. Second, all the patients enrolled in our study were Chinese. Nevertheless, this will help to explore the characteristics of the Asian population with BRAF V600E-mutated mCRC. Further prospective clinical trials in larger cohorts are warranted to validate the results of this study.

Future perspectives

Our study found that liver metastasis was the negative independent predictor of OS in patients with BRAF V600E-mutated mCRC. Clinically, BRAF V600E-mutated mCRC patients with liver metastasis had a worse prognosis, which may be attributed to the local microenvironment of liver metastasis. In the future, basic experiments are needed to explore the relationship between the microenvironment of liver metastases and tumor progression.

Conclusion

BRAF V600E-mutated mCRC was associated with a poor prognosis. The proportion of patients treated with three-drug combined bevacizumab is relatively low. Poor differentiation and liver metastases were negative independent prognostic factors for OS. The first-line triplet chemotherapy combined with bevacizumab group was associated with better OS, especially in patients with good physical condition and high tumor burden.