The prognostic significance of KRAS and BRAF mutation status in Korean colorectal cancer patients

We retrospectively reviewed specimens from 1096 consecutive patients who underwent surgical CRC resection at Seoul St. Mary’s Hospital, The Catholic University of Korea, between July 2010 and September 2013. CRC cases with tissue blocks eligible for the KRAS and BRAF mutation testing were included in this study. Two gastrointestinal pathologists reviewed and classified CRC slides according to World Health Organization classification. Clinicopathological parameters were obtained from patient medical records and pathology reports at our institution. Adjuvant chemotherapy was recommended to high-risk (cancer obstruction, perforation, poor differentiation, or lymphovascular/perineural invasion) stage II or stage III CRC patients. According to the BRAF and KRAS mutational status, patients were offered targeted agents as an adjunct to systemic chemotherapy. However, due to insurance coverage issues, only 3 patients received anti-EGFR and only 12 received anti-vascular endothelial growth factor therapy during the study period. Approval for this study was acquired from the Institutional Review Board of the Catholic University of Korea, College of Medicine (KC16RISI0011).

For DNA isolation, 10-μm-thick sections from formalin-fixed paraffin-embedded (FFPE) tissue samples were used for each case. Hematoxylin & eosin sections were used as a reference and the largest tumor area was scraped off with a scalpel under a dissecting microscope. Genomic DNA was extracted using the QIAamp DNA FFPE tissue kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s recommendations. Sanger sequencing was performed using an ABI 3730 automated sequencer (Applied Biosystems, Inc., Foster City, CA), to detect the presence of KRAS exon 2 mutations with previously reported primers [ 21]. Exon 15 of the BRAF gene was amplified by polymerase chain reaction (PCR) using the following forward primer (5′-AATGCTTGCTCTGATAGGAAAAT-3′) and reverse primer (5′-TAATCAGTGGAAAAATAGCCTC-3′), resulting in a 209 base pair PCR product. The resultant PCR products were purified using the QIAquick PCR Purification Kit (Qiagen Inc., Valencia, CA) and the appropriate protocol on the QIAcube robotic workstation. Each chromatogram was visually inspected for abnormalities.

Five microsatellite markers (BAT-25, BAT-26, D2S123, D5S346, and D17S250) recommended by a National Cancer Institute workshop on MSI determined the microsatellite status [ 22]. PCR analyses were performed and the shift of PCR products from tumor DNA was compared to normal DNA. Tumors with at least 2 of the 5 microsatellite markers displaying shifted alleles were classified as MSI-H, whereas tumors with only 1 marker exhibiting a novel band were classified as MSI-L. Samples in which all microsatellite markers displayed the same patterns in tumor and normal tissues were classified as MSS; subsequently, MSS and MSI-L tumors were grouped for analyses based on genetic implications [ 22].

Continuous variables were analyzed by student’s t or Mann-Whitney U test, expressed as the mean ±SD. For categorical variables, χ ²-test analysis or Fisher’s exact test was used. Survival analysis was performed by the Kaplan-Meier method. Statistical analysis was performed with SPSS software version 18 (SPSS Inc., Chicago, IL) and the R programing language (R Core Team 2015, A language and environment for statistical computing, R Foundation for Statistical Computing, Vienna, Austria, URL http://​www.​r-project.​org/​). A P-value of <0.05 was considered significant.

The present study included 1092 patients with KRAS and 1096 patients with BRAF mutation data. Tables 1 and 2 summarize the clinicopathological characteristics of patients. A total of 401 patients (36.7%) had KRAS mutations. KRAS mutated CRCs were significantly associated with females (45.1% vs 34.6% with wild-type KRAS; P = 0.001), right sided tumors (32.4% vs 21.0%; P < 0.001), higher T stage (T4, 15.3% vs 11.0%; P = 0.005), well to moderate differentiation (98.7% vs 94.7%; P = 0.002), and mucinous adenocarcinoma (9.2% vs 4.9%; P = 0.002). BRAF mutations were detected in 44 patients (4.0%). The proportion of BRAF mutation was higher in tumors located in the right colon (56.8% vs 23.9% with wild-type BRAF; P = 0.001), with an advanced tumor stage (T4, 29.5% vs 11.9%; P = 0.005), with lymph node metastasis (N2, 38.6% vs 20.5%; P = 0.015), and with lymphatic invasion (65.9% vs 44.0%; P = 0.007). BRAF mutated tumors trended toward poorly differentiated histology (10.0% vs 3.6%, P = 0.099) and an infiltrative growth pattern (22.7% vs 15.2%; P = 0.065) compared to wild-type BRAF tumors, but these were not statistically significant. In addition, gender distribution according to KRAS mutation status did not differ significantly, showing a bimodal distribution pattern along the colorectum. Distributions with respect to tumor sites for all three tumor subgroups ( KRAS-mutated, BRAF-mutated and null CRCs), stratified for gender, are shown in Fig. 1a–c.

A KRAS codon 12 mutation was observed in 296 patients. A KRAS codon 13 mutation was observed in 98 patients. Seven other patients had either KRAS codon 14 or 30 mutations. The most frequent amino acid change was Gly12Asp, which accounted for 36.9% of KRAS mutations (148/401). The second most frequent mutation was Gly13Asp (24.2%, 97/401), and the third was Gly12Val (21.9%, 88/401). Table 3 lists detailed nucleotide and codon changes . Regarding BRAF mutations, Val600Glu in exon 15 showed the highest frequency (97.7%, 43/44) (Table 4). In addition, our data revealed 3 KRAS and BRAF co-mutated cases. Among these 3 cases, 2 had Gly13Asp KRAS mutations, 1 had a Gly12Asp mutation, and all BRAF mutations were Val600Glu. All 3 cases had lymph node metastasis and were included in stage III; however, no recurrences or deaths were observed.

After a median follow-up of 29 months, the 5-year disease free survival rate of the study population was 81%. There was no significant difference according to KRAS mutation status; however, DFS trended toward being shorter in patients with KRAS mutations than those with wild-type KRAS ( P = 0.0548). DFS was also significantly worse in patients with BRAF mutated cancers compared to wild-type BRAF by both univariate (HR 1.98, P = 0.0252) and multivariate analyses (HR 2.222) (Fig. 2a and b).

Regarding OS, the 5-year rate was 80%. No significant difference in OS according to KRAS mutation status was revealed ( P = 0.108). OS was significantly shorter for patients with BRAF mutations than those with wild-type BRAF by univariate analysis (HR 3.46, 95% CI 1.9–6.3, P < 0.0001). In the multivariate analysis, BRAF mutations also had a negative impact on OS (HR 4.037, 95% CI 2.172–7.506, P < 0.0001) (Fig. 2c and d). In addition, we assessed whether the detrimental effect of KRAS mutations was different according to mutation subtypes and showed that there were no significant differences in DFS ( P = 0.931) or OS ( P = 0.816) (Additional file 1: Fig. S1A and B).

Considering KRAS and BRAF mutations together, DFS and OS were significantly more favorable in patients with wild-type KRAS and BRAF compared to patients with mutations in both genes (HR 1.540, 95% CI 1.140–2.080, P = 0.0049) and OS (HR 1.860, 95% CI 1.280–2.720, P = 0.0010) (Fig. 3a and b).

In stage I colorectal cancer, BRAF mutations had a negative impact on both DFS (HR 3.936, 95% CI 2.120–7.306, P < 0.0001) and OS (HR 4.037, 95% CI 2.172–7.506, P < 0.0001). However, KRAS mutations did not demonstrate a significant effect on DFS (HR 1.539, 95% CI 1.039–2.279, P = 0.112) or OS (HR 1.555, 95% CI 1.048–2.305, P = 0.107) (Fig. 4a and b). In stage II and III colorectal cancer, BRAF mutations had a negative impact on DFS (HR 1.940, 95% CI 1.050–3.570, P = 0.0322) and OS (HR 3.320, 95% CI 1.820–6.070, P < 0.0001). However, KRAS mutations did not demonstrate a significant effect on DFS (HR 1.250, 95% CI 0.910–1.720, P = 0.169) or OS (HR 1.400, 95% CI 0.950–2.070, P = 0.0917) (Fig. 4c and d). In stage IV CRC, BRAF mutation status did not show a significant effect on DFS (HR 1.180, 95% CI 0.290–4.870, P = 0.82) or OS (HR 2.660, 95% CI 0.950–7.450, P = 0.0548). KRAS mutation status also did not demonstrate a significant effect on DFS (HR 1.140, 95% CI 0.670–1.930, P = 0.627) or OS (1.410, 95% CI 0.790–2.520, P = 0.247) (Fig. 4e and f).

MSI test data were available in 83 patients. Univariate analysis was performed according to clinicopathologic factors and MSI status. A significant difference was noted in CRC location ( P = 0.037). MSH-H had a higher frequency in colon cancers of the right side (66.7% vs 23.4%). MSS/MSI-L CRCs were more prevalent on the left (50.6% vs 16.7%). Regarding histological differentiation, a significant difference was noted ( P = 0.012). MSI-H had higher number of poorly differentiated CRC (1.4% vs 25.0%). Mucinous CRC was observed more frequently in the MSI-H group (6.5% vs 83.3%, P < 0.001) (Table 5).

We compared DFS and OS between MSS/MSI-L and MSI-H groups to evaluate the value of MSI status as a prognostic marker. MSI status did not show a significant difference in DFS ( P = 0.294) or OS ( P = 0.557) (Fig. 5a and b).