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

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]-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]-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]-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]-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]-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]-22]. Previous trials suggest that intensive combination chemotherapy with FOLFOXIRI (5-FU,
L-leucovorin, 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.