A retrospective observational study of clinicopathological features of KRAS, NRAS, BRAF and PIK3CA mutations in Japanese patients with metastatic colorectal cancer

Background The mutation in KRAS exon 2 is a validated biomarker of resistance to anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer (mCRC). Several reports have confirmed associations of other RAS mutations with resistance to anti-EGFR therapy. However, the impact of BRAF and PIK3CA mutations on the efficacy of anti-EGFR therapy remains controversial. Little is known about the frequencies and clinicopathological features of these mutations, as well as the therapeutic effects of anti-EGFR therapy in mCRC patients with these mutations, especially in the Asian population. Methods In this retrospective observational study, frequencies and clinicopathological features of KRAS, NRAS, BRAF and PIK3CA mutations were evaluated in patients with mCRC. Among patients treated with anti-EGFR therapy, objective response, progression-free survival (PFS), and overall survival (OS) were evaluated according to gene status. Results Among 264 patients, mutations in KRAS exon 2, KRAS exons 3 or 4, NRAS, BRAF and PIK3CA were detected in 34.1%, 3.8%, 4.2%, 5.4% and 6.4%, respectively. Thus, a total of 12.1% of patients without KRAS exon 2 mutations had other RAS mutations. Primary rectal tumors tended to be more frequently observed in RAS mutant tumors. BRAF mutations were more frequently observed with right-sided colon, poorly differentiated or mucinous adenocarcinoma, and peritoneal metastasis. Among the 66 patients with KRAS exon 2 wild-type tumors treated with anti-EGFR agents, PFS (5.8 vs. 2.2 months) and OS (17.7 vs. 5.2 months) were significantly better in patients with all wild-type tumors (n = 56) than in those with any of the mutations (n = 10). The response rate also tended to be better with all wild-type tumors (26.8 vs. 0%). Conclusion Other RAS and BRAF mutations were observed in KRAS exon 2 wild-type tumors, which were associated with some clinicopathological features and resistance to anti-EGFR therapy in our patient cohort. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1276-z) contains supplementary material, which is available to authorized users.


Background
Colorectal cancer was the third most common cancer in men (746,000 cases, 10.0% of the total) and the second in women (614,000 cases, 9.2% of the total) worldwide in 2012 [1]. Mutations in KRAS exon 2 occur in~35% of all metastatic colorectal cancers (mCRCs) [2,3], and constitutively activate the mitogen-activated protein kinase (MAPK) pathway [4,5]. These mutations are validated biomarkers for resistance to anti-epidermal growth factor receptor (EGFR) therapy in patients with mCRC [6][7][8][9][10][11]. Although conventional KRAS tests are useful to exclude patients without benefit from anti-EGFR therapy, response rates and disease control rates to anti-EGFR antibody monotherapy among patients with KRAS exon 2 wild-type tumors are only 13-17% and 51%, respectively [6,7]. Therefore, more accurate patient selection requires identification of other predictive factors to improve the riskbenefit profile of anti-EGFR therapy.
BRAF mutations were detected in 5-10% of patients with mCRC with V600E as a hot spot. BRAF is a downstream molecule of KRAS and the clinical data suggest that BRAF V600E mutations are associated with poor prognosis in patients with mCRC [11,12,[19][20][21][22][23][24]. However, the relationship between BRAF mutations and the efficacy of anti-EGFR therapy remains controversial [19][20][21][22]. Besides the KRAS-BRAF pathway, the other major downstream signaling pathway activated by EGFR is the PI3K-AKT signaling pathway. PIK3CA mutations, most of which were in exons 9 and 20, were detected in 10-15% of patients with mCRC. According to a European Consortium report [19], PIK3CA mutations in exon 20 but not in exon 9 were associated with resistance to anti-EGFR therapy for mCRC. However, in other studies, no clear correlation between PIK3CA mutations and the efficacy of anti-EGFR therapy has been observed [21,22]. Meanwhile, targeting agents for these mutations are under development.
We previously reported that a multi-gene cancer panel with Luminex technology (GENOSEARCH Mu-PACK, MBL, Japan) is useful for detection of 36 mutations in KRAS exons 3 or 4, NRAS, BRAF and PIK3CA in a single reaction using 50-ng template DNA from formalin-fixed, paraffin-embedded (FFPE) specimens [25]. Importantly, the analysis of 82 samples was fully concordant with conventional direct sequencing. However, information about the frequencies and clinicopathological features of these mutations in clinical practice, including the relationship between mutation status and the efficacy of anti-EGFR therapy, especially among Asian populations, is still limited.
In the present study, we evaluated the frequencies andclinicopathological features of KRAS, NRAS, BRAF and PIK3CA mutations in Japanese mCRC patients, and assessed their corresponding effects on the efficacy of anti-EGFR therapy.

Patients
We have conducted a retrospective observational study in our institution to evaluate the frequencies and clinicopathological features of KRAS, NRAS, BRAF and PIK3CA mutations in Japanese mCRC patients. Principal inclusion criteria were as follows: histologically confirmed adenocarcinoma of the colon or rectum; and presence of unresectable metastatic disease.
Between January 2013 and June 2014, we analyzed 264 patients with mCRC who met the inclusion criteria. The study was conducted with the approval of the National Cancer Center Institutional Review Board. Written informed consent was obtained from as many patients as possible. For the deceased patients and their relatives, we also disclosed the study design at the website of National Cancer Center and gave them the opportunity to express their wills in accordance with the Epidemiological Study Guideline of Ministry of Health, Labour and Welfare in Japan.
Patient characteristics, including age, sex, site of primary lesion, histology, site of metastases, and treatment results, were collected from medical records. Sites of primary lesions were divided into right colon, left colon, and rectum. Right-sided tumors were defined as those arising anywhere from the cecum to the transverse colon, and left-sided tumors as those arising anywhere from the splenic flexure to the rectosigmoid junction. The efficacy of anti-EGFR therapy was evaluated according to gene status in patients who met the following inclusion criteria: Eastern Cooperative Oncology Group performance status (ECOG PS) score ≤ 2, KRAS exon 2 wild type, at least one prior chemotherapy regimen, treatment with anti-EGFR either as monotherapy or in combination with irinotecan or FOLFIRI (5-FU, Lleucovorin and irinotecan), baseline computed tomography (CT) performed within 28 days of anti-EGFR therapy, initial evaluation of treatment effect via CT scan within 3 months of initial anti-EGFR therapy and adequate hematological, hepatic and renal function.

Statistical methods
Gene mutation frequencies and associations of RAS or BRAF mutations with clinicopathological features were estimated in mCRC patients.
Response rate (RR) and disease control rate (DCR; including complete or partial response and stable disease) were evaluated for anti-EGFR therapy according to the Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1). Progression-free survival (PFS) was defined as the time from initial administration of anti-EGFR regimens until the first objective evidence of disease progression or death from any cause. Overall survival (OS) was defined as the time from initial administration of anti-EGFR regimens until death from any cause. For PFS or OS, patients were censored at the time of their last follow-up if they were free of disease progression or alive, respectively. PFS and OS rates were estimated using the Kaplan-Meier method, and differences among the groups according to KRAS, NRAS, BRAF and PIK3CA gene status were identified by univariate and multivariate analyses using Cox proportional hazards models and presented as hazard ratios (HRs) with 95% confidence intervals (CIs). Confounders in univariate and multivariate analyses included ECOG PS (0 vs. 1 and 2), numbers of metastatic sites (1 vs. ≥ 2), treatment line of anti-EGFR regimens (2nd vs. 3rd) and types of anti-EGFR regimens (monotherapy vs. combination therapy).
The χ 2 test, Fisher's exact test, Mann-Whitney U test, or Kruskal-Wallis test was used to compare patient characteristics and treatment response, as appropriate. Statistical analyses were performed using IBM SPSS Statistics version 21 (IBM Corporation, Armonk, NY, USA). All tests were two-sided, and differences were considered significant when P was < 0.05.  of all samples using HGVS nomenclature are shown in Additional file 1. All mutations were mutually exclusive except for those in PIK3CA, and 21 (12.1%) patients without KRAS mutations in exon 2 had other RAS mutations in either KRAS exons 3 or 4 or NRAS exons 2 or 3 ( Figure 1).

Association of RAS and BRAF mutations with clinicopathological features
We analyzed the correlation between RAS or BRAF genotypes and the clinicopathological features of mCRC. Primary rectal tumor tended to be more frequently observed in KRAS exon 2 and other RAS mutant tumors than in RAS wild-type tumors (60.0 vs. 61.9 vs. 48.3%, P = 0.08) ( Table 1), although this was not statistically significant.

Efficacy of anti-EGFR therapies according to gene status Patient characteristics
Between January 2013 and June 2014, 66 patients who met the inclusion criteria were treated with second-and third-line regimens containing anti-EGFR agents. Fifty-six patients had tumors with no mutations (all wild-type tumors) and 10 had tumors with mutation in either KRAS codons 61 or 146, NRAS, BRAF, or PIK3CA (any of the mutations). Among the 10 patients with any of the mutations, three had KRAS codon 146 mutations, two had NRAS mutations, two had BRAF mutations, two had PIK3CA mutations (1 in exon 9 and 1 in exon 20), and one had BRAF and PIK3CA exon 9 mutations (Table 4). Patients with any of the mutations were more likely to have worse PS and to be treated with anti-EGFR monotherapy than combination in comparison with all wild-type tumors. No other significant difference was seen between the two groups ( Table 4).
Among the 10 patients with mutations, three were treated with second-line anti-EGFR-containing regimens and seven were treated with third-line regimens. All three patients treated with second-line anti-EGFR therapy were  irinotecan-naïve and had stable disease. Among these, one was treated with irinotecan plus panitumumab and showed stable disease after > 3 months. In contrast, all seven patients treated with third-line anti-EGFR therapy were irinotecan refractory and only two had stable disease at < 3 months.

Discussion
We elucidated the prevalence of KRAS, NRAS, BRAF and PIK3CA mutations in Japanese mCRC patients, and clarified the relationship between gene status and clinicopathological features, including the efficacy of anti-EGFR therapy. To date, clinical evidence about these mutations in mCRC has been based on clinical studies in western countries. The present study is believed to be the first to provide information on frequency and type of KRAS, NRAS, BRAF and PIK3CA mutations in Japanese patients with mCRC. In addition, the clinical feasibility of the present novel multiplex kit was demonstrated. In our patient cohort, the frequency of patients with KRAS exon 2 (34.1%) mutant tumors was similar to that in previous studies [2][3][4]. A total of 12.1% of patients without KRAS exon 2 mutations had other RAS mutations, which was lower than that in recent studies from western countries, which showed 15-26% of these mutations [12][13][14][15][16][17][18]. Another previous study from Japan showed that other RAS mutations were detected in seven (12.7%) of 55 samples without KRAS exon 2 mutations with 3-13% sensitivity [26], which was similar to our result. Several possible explanations for the relatively lower frequency of other RAS mutations in our study compared with western studies might be considered. First, there were some differences in detectable RAS mutations by multiplex kit between our study and western studies. In our study, we did not analyze KRAS codons 59 and 117 and NRAS codons 59, 117 and 146, while these codons were analyzed in most western studies. Although the frequencies of these mutations are considered to be low, it might be one of the causes of the lower frequency in our patient cohort. Second, the sensitivity of RAS mutation analysis may vary among studies. In the present study, all mutations were detectable with 5-10% sensitivity. In contrast, Surveyor Scan Kits, BEAMing technology and pyrosequencing were used in pivotal studies, and RAS mutations were detected with 1-10% sensitivity [12][13][14][15][16][17][18]. A recent multicenter study in Japan, including our institution, showed that other RAS mutations were detected in 15% of patients with KRAS exon 2 wild type, using a newer multiplex kit (MEBGEN RASKET Kit) [27]. This method detected 48 RAS mutations in exon 2 (codons 12 and 13), exon 3 (codons 59 and 61) and exon 4 (codons 117 and 146), with 1-5% sensitivity in a single reaction using 50-100-ng DNA from FFPE tissue without manual dissection. Given these methodological differences, further studies are required to confirm differences in the prevalence of other RAS mutations between Asian and western populations. In this study, we detected BRAF mutations in 5.4% of patients. The prevalence of BRAF mutation might be dependent on the patient population studied. mCRC patients with BRAF mutant tumors have a poor prognosis, so the prevalence of BRAF mutant populations may decline in  pretreated patients compared with chemonaïve patients. The prevalence of BRAF mutations in our patient cohort was similar to that of previous studies of pretreated patients with mCRC [11,12,[19][20][21][22][23][24]. We also investigated the clinicopathological features of mCRC patients with respect to RAS and BRAF mutations. Primary rectal tumor tends to be more frequently observed in KRAS exon 2 and other RAS mutant tumors rather than RAS wild-type tumors, although this was not statistically significant. Previous studies showed that KRAS exon 2 mutation was significantly higher in the right colon [28,29], in disagreement with our analysis. No significant differences in other clinicopathological features such as age, sex, primary lesion, histology, and site of metastasis were observed between KRAS exon 2 and other RAS mutant tumors, which is similar to previous studies [30]. Regardless of these clinicopathological features, it is reported that other gene expression profiles based on The Cancer Genome Atlas appear to be similar in patients with KRAS and NRAS mutant mCRC, suggesting that treatment selection based on molecular profile is important [30]. In accordance with previous reports [23,24], BRAF mutant tumors are more likely to develop in the right colon, and to have poorly differentiated or mucinous adenocarcinoma, and peritoneal metastasis in comparison with BRAF wild-type tumors.
In agreement with previous studies [19,25], mutations in KRAS exons 3 or 4, NRAS, BRAF or PIK3CA were not associated with clinical benefits from anti-EGFR therapy in the present cohort. On the basis of recent prospective and retrospective randomized trials of anti-EGFR therapy [12][13][14][15][16][17][18], the National Comprehensive Cancer Network (NCCN) recommends anti-EGFR therapy for mCRC patients without other RAS mutant tumors or KRAS exon 2 mutant tumors [31]. The Japanese Society of Medical Oncology (JSMO) also recommends testing for all RAS mutations in patients with mCRC before anti-EGFR therapy. In contrast, whether BRAF and PIK3CA mutations are predictive of the efficacy of anti-EGFR therapy remains controversial [19][20][21][22]. Previous trials suggest that intensive combination chemotherapy with FOLFOXIRI (5-FU, Lleucovorin, irinotecan, and oxaliplatin) and bevacizumab might be especially effective for BRAF mutant mCRC [32]. Recently, the combination of BRAF inhibitors and anti-EGFR monoclonal antibodies, with or without PI3K inhibitors or MEK inhibitors, has shown promising results in phase I trials in patients with BRAF mutant CRC [33,34]. Patients with BRAF mutant CRC are often refractory to systematic chemotherapy and have poor prognosis, therefore, screening for BRAF mutations is important during recruitment of patients for these clinical trials. Accordingly, we conducted a multi-institutional screening (GI-SCREEN) study using the present multiplex kit to elucidate the nationwide prevalence of these targetable mutations.
There were several methodological limitations to the present study. First, not all of the patients in our study period were evaluated for their RAS gene status. Thus, the analysis may have been subject to some selection bias. Second, the small sample size and single-center population were other major limitations. Owing to the overall small number of patients with KRAS exon 3 or 4, NRAS, BRAF or PIK3CA mutations, we could not evaluate the impact of each gene mutation on the efficacy of anti-EGFR therapy. In addition, our analyses were explorative and hypothesis generating. This issue should be analyzed in a larger cohort.

Conclusions
Other RAS and BRAF mutations have been observed in KRAS exon 2 wild-type tumors, which were associated with some clinicopathological features and resistance to anti-EGFR therapy in our patient cohort. Importantly, because there are a certain number of mCRC patients with molecular alteration other than KRAS exon 2, further refinement of tumor-specific genetic markers is needed to improve the efficacy of anti-EGFR therapy.