To the best of our knowledge, this is the first large-scale study in which a KRAS mutational analysis was performed between primary tumors and corresponding metastases in Asian MRCRC patients. Overall, a high concordance rate of KRAS status was observed in Korean MRCRC patients, as had been previously reported in Western populations. We observed significant differences in initial metastatic patterns according to the KRAS mutational status. MT KRAS tumors developed lung metastases more frequently as the initial metastatic site; however, liver and distant LN were more frequently involved as the initial metastatic sites in WT KRAS tumors. Additionally, the degree of concordance in KRAS mutational status was significantly different according to the sites of related metastatic organs, where the lung was the most frequent metastatic site showing the discordance of KRAS status.
Our study demonstrated that the clinical presentation of CRC varied according to the KRAS mutational status. KRAS mutational status was shown to affect the presenting pattern of distant metastasis in MRCRC patients. Recurrent cases after curative treatment for localized diseases were more common in MT KRAS patients; however, systemic metastasis was more frequent at the time of initial CRC diagnosis in WT KRAS patients (Table ). One previous study showed that the risk of recurrence was significantly higher for MT KRAS than WT KRAS tumors in patients with localized CRC [10
]. If localized CRC patients with MT KRAS had more chances of recurrence, then the MT KRAS cases would be selected and thus a higher frequency of MT KRAS in recurrent CRC patients would be expected than in patients with stage IV disease at the time of initial CRC diagnosis. However, as our patient cohort is relatively small, our assumption needs to be further investigated in future large studies.
In the present study, organs initially involved by distant metastasis were shown to be different according to the KRAS mutational status. Patients with MT KRAS had an initial lung metastasis more frequently than patients with WT KRAS. In contrast, the WT KRAS patients had liver or distant LN metastases more frequently as the initial metastatic sites. However, other metastatic sites such as the peritoneum were not affected by KRAS status (Table ). When the analyses were conducted on patients with initial distant metastasis confined to a single organ, the results were similar except for distant LN metastasis (Table ). However, since the number of cases with LN-only metastasis was small (N = 6), the difference of percentages (7.7% vs. 3.3%) might not have reached statistical significance. Although the MT KRAS tumors showed a trend for more frequent development of peritoneum-only metastasis (13.5% vs. 27.9%; P
= 0.062), it was not statistically significant (Table ). In the multivariate analysis, KRAS status, primary tumor site, and clinical situations for the development of systemic metastasis were significant predictors for liver-only and/or lung-only metastases (Table ). The reason why the clinical situations of developing systemic metastasis influenced the initially involved metastatic organs (liver or lung) is not clear; this may be related to the process of patient enrollment to this study as patients with available tissues from both primary and paired metastatic sites were only included. During this process, recurred CRC patients with tissue-available lung metastasis might be selectively included. However, even after adjusting for these clinical variables, the KRAS mutational status was an independent predictive factor for both liver-only and lung-only metastases in our study. A previous study, which analyzed the KRAS status in primary tumors of CRC patients, showed that there were more MT KRAS tumors in patients with lung metastasis than in patients with liver metastasis [14
]. Based on this finding, they suggest that KRAS-mutated primary CRC tumors can recur with lung metastasis more frequently than with liver metastasis. Although the KRAS mutational status was regarded as positive if KRAS was mutated in any place of primary tumors or related metastases in the present report, our results also support their suggestion. Furthermore, when the analysis was conducted based on the KRAS status of primary tumor, the result of our study was also the same as above. Our work along with previous studies strongly suggests that the sequence of organs involved by systemic metastasis is influenced by KRAS mutational status in CRC patients.
Our results are generally consistent with previous studies that have reported a high concordance rate of KRAS mutation (about 90%) between primary and metastatic tumors [14
]. Paired metastatic tissues in previous studies were mostly derived from the liver [24
] because these tissues were easily available from hepatic metastasectomy [33
]. However, reports on the degree of KRAS concordance of other metastatic organs other than the liver have been very limited. Our study evaluated the KRAS status of metastatic tissues from various organs besides liver. The degree of KRAS mutational concordance was different according to the related metastatic sites, with a significantly higher rate of discrepancy in lung metastases (32.4%) when compared with other metastatic organs (12.3%) or liver (10.6%) (P
-values < 0.05; Table ). Although some studies have recently demonstrated a relatively high degree of discordance, with a discordance rate of up to 50% [11
], there has been no report showing the site-specific KRAS discordance, as was shown in this study. The mechanism behind the discordant KRAS mutational status is still not exactly known [31
]. Sampling errors, heterogeneity within primary tumors, and the development of mutations during the process of metastasis may be the causes of this discordance. The reason why lung is the most frequent site where the KRAS discordance takes place is also unknown. In the present study, we analyzed the pattern of KRAS discordance (i.e. from WT in primary tumors to MT in related metastatic sites or vice versa); as the sample size was small (N = 25), the KRAS discordance pattern did not show any relation to clinical situations for the development of systemic metastasis (stage IV vs. recurred) or metastatic organs from which tumor specimens were obtained. Among 12 cases with KRAS-discordant lung metastasis, 6 cases (50%) had the change of KRAS status from WT in a primary tumor to MT in the lung and 6 cases (50%) had vice versa; there was no statistically significant difference in the discordance pattern between the lung and other paired metastatic organs (Table ). Therefore, the underlying causes of KRAS discordance need to be further evaluated in future large studies.
Our study has some limitations. First, this study was performed at a single institution and all MRCRC patients diagnosed at our institution were not included. Instead, only MRCRC patients with both primary and paired metastatic tissues were consecutively included. In such a process, unrecognized biases might have influenced our study. Second, a KRAS mutational analysis was not repetitively conducted in cases with discordant KRAS status partly because of insufficient remaining tissue specimens for further examination. This might raise concerns about the sensitivity of the KRAS mutation analysis. We actually used traditional sequencing (Sanger) method with relatively low sensitivity for KRAS mutation analysis [37
]. In addition, biopsied specimens of primary tumor showed a trend for higher discordance rate than resected specimens in this study (40.0% vs. 15.8%; P
= 0.073), although no significance was shown in multivariate analysis. However, we used tumor cell enrichment by microdissection under the supervision of experienced pathologists to increase the sensitivity of the sequencing method. In addition, biopsy of primary tumors in our study was all performed by endoscopy. Actually, a biopsy of distant metastasis may be more problematic than endoscopic biopsy of primary tumors in context of tumor cell percentage [38
]. In the present study, a small number of metastatic specimens (8/143) was obtained from needle biopsy and only 1 case had KRAS discordance [1/8 (12.5%) for biopsied metastatic specimens vs. 24/135 (17.8%) for resected metastatic specimens; P
= 1.000]. Moreover, the concordance rates observed between primary and metastatic tissues including the liver only (89.4%) or all metastatic organs other than lung (87.7%) were similar to the concordance rates reported in previous studies. All these findings suggest that the high KRAS discordant rate of lung metastasis (32.4%) had not simply resulted from types of tumor tissue specimens (biopsied vs. resected) or less sensitive analytic methods performed at our institute. Instead, the results from our study reflect the real situation of clinical fields as the traditional sequencing (Sanger) analysis is the most frequent method used in the real clinical practice setting. More sensitive methods, such as real-time PCR for KRAS mutation analysis, are only used in the investigational setting and not widely spread in the clinical practice.
Despite these limitations, our study provides some clinically meaningful suggestions. The present study demonstrated that the KRAS mutational status was an independently predictive factor for organs initially involved by distant metastasis. This observation implies that surveillance strategies after curative surgery might be tailored to individual CRC patients according to the KRAS mutational status. Postoperative surveillance might be more focused on lung metastasis (i.e., chest computed tomography) in patients with MT KRAS than in patients with WT KRAS, when considering the chance of performing metastasectomy after the early detection of pulmonary metastasis. Our study also raised the hypothesis that the discordant rates of KRAS mutational status might be metastatic site-specific in CRC. Using the sequencing method, we found different discordant rates according to the metastatic sites. A high KRAS discordant rate in patients with lung metastasis, observed in our study, warrants further large validation studies.