Methylation levels of LINE-1 in primary lesion and matched metastatic lesions of colorectal cancer

Abstract

Background: LINE-1 methylation level is a surrogate marker of global DNA methylation. LINE-1 methylation in primary colorectal cancers (CRCs) is highly variable and strongly associated with a poor prognosis. However, no study has examined LINE-1 methylation levels of metastatic CRCs in relation to prognosis or assessed the heterogeneity of LINE-1 methylation level within the primary CRCs.

Methods: Pyrosequencing was used to quantify LINE-1 methylation level in 42 liver metastases, 26 matched primary tumours, and 6 matched lymph node (LN) metastases. KRAS, BRAF, and PIK3CA mutation status and microsatellite instability (MSI) status were also examined.

Results: The distribution of LINE-1 methylation level in liver metastases was as follows: mean, 67.3; range, 37.1-90.1. Primary tumours showed LINE-1 methylation levels similar to those of matched liver and LN metastases. The difference in LINE-1 methylation level between superficial areas and invasive front areas was within 7.0 in all six cases evaluated. Prognostic impact of LINE-1 hypomethylation in liver metastases on overall survival was not observed. The concordance rate was 94% for KRAS, 100% for BRAF, 88% for PIK3CA, and 97% for MSI.

Conclusion: Alteration of LINE-1 methylation level may occur in early CRC tumorigenesis, and the LINE-1 methylation level is relatively stable during CRC progression.

Figure 1

(A) Flow chart of the study population. (B) In this study, we examined the LINE-1 methylation level in 26 primary tumours, 26 matched liver metastases, and 6 matched LN metastases. (C) Macrodissection to separate the superficial area from the invasive front area.

Figure 2

Correlation between the LINE-1 methylation level in primary tumours and that in liver metastases. The LINE-1 methylation level in liver metastases was significantly associated with that in matched primary tumours (n=26; Pearson's correlation coefficient r=0.70; P=0.0006).

Figure 3

LINE-1 methylation level in superficial and invasive areas of primary tumours, matched LN metastases, and matched liver metastases (n=6). (A) In each case, the LINE-1 methylation level of the LN and liver metastases was similar to that in the matched primary tumours. (B)The LINE-1 methylation level in superficial areas was significantly associated with that in the matched invasive areas within primary tumours (n=6; Pearson's correlation coefficient, r=0.95; P=0.0048).

Figure 4

Gene mutation analysis in 26 paired primary CRCs and liver metastases and 6 matched LN metastases. The wild type is shown in blue, and the mutational type is shown in red. The white square denotes the lack of a specimen. (A) KRAS mutation status. Six primary tumours showed the KRAS mutation type. Thirty liver and LN metastases showed a KRAS status identical to that of the matched primary tumours. The KRAS status was confirmed by Scorpion Amplified Refractory Mutation System (ARMS) technology and supported the reliability of the pyrosequencing assay for KRAS mutation detection in six cases. (B) PIK3CA mutation status. Seven primary tumours showed the PIK3CA mutation type. Twenty-eight liver and LN metastases showed a PIK3CA status identical to that of the matched primary tumours. (C) Microsatellite instability status. Three primary tumours showed MSI. Thirty-one liver and LN metastases showed an MSI status identical to that of the matched primary tumours.

Figure 5

Kaplan–Meier curves for overall survival in the two groups of LINE-1 methylation in metastatic CRC tumours. The hypomethylation (37.1–68.5%, n=21) group experienced an overall mortality similar to that of the hypermethylation group (⩾69.0%, n=21).