LINE-1 methylation shows little intra-patient heterogeneity in primary and synchronous metastatic colorectal cancer

Abstract

BackgroundLong interspersed nucleotide element 1 (LINE-1) hypomethylation is suggested to play a role in the progression of colorectal cancer (CRC). To assess intra-patient heterogeneity of LINE-1 methylation in CRC and to understand its biological relevance in invasion and metastasis, we evaluated the LINE-1 methylation at multiple tumor sites. In addition, the influence of stromal cell content on the measurement of LINE-1 methylation in tumor tissue was analyzed.MethodsFormalin-fixed paraffin-embedded primary tumor tissue was obtained from 48 CRC patients. Matched adjacent normal colon tissue, lymph node metastases and distant metastases were obtained from 12, 18 and 7 of these patients, respectively. Three different areas were microdissected from each primary tumor and included the tumor center and invasive front. Normal mucosal and stromal cells were also microdissected for comparison with the tumor cells. The microdissected samples were compared in LINE-1 methylation level measured by multicolor MethyLight assay. The assay results were also compared between microdissected and macrodissected tissue samples.ResultsLINE-1 methylation within primary tumors showed no significant intra-tumoral heterogeneity, with the tumor center and invasive front showing identical methylation levels. Moreover, no difference in LINE-1 methylation was observed between the primary tumor and lymph node and distant metastases from the same patient. Tumor cells showed significantly less LINE-1 methylation compared to adjacent stromal and normal mucosal epithelial cells. Consequently, LINE-1 methylation was significantly lower in microdissected samples compared to macrodissected samples. A trend for less LINE-1 methylation was also observed in more advanced stages of CRC.ConclusionsLINE-1 methylation shows little intra-patient tumor heterogeneity, indicating the suitability of its use for molecular diagnosis in CRC. The methylation is relatively stable during CRC progression, leading us to propose a new concept for the association between LINE-1 methylation and disease stage.

Prognostic and predictive significance of long interspersed nucleotide element-1 methylation in advanced-stage colorectal cancer

“…Tumor LINE-1 hypomethylation has consistently been associated with worse survival in CRC patients [19–21]. We also reported earlier that LINE-1 hypomethylation was predictive of good response to oral fluoropyrimidines in the adjuvant setting [22] and that LINE-1 methylation levels in primary tumors correlated well with those of metastatic lesions from the same patient [23, 24]. Recently, the predictive and prognostic significance of LINE-1 hypomethylation has been studied by several independent groups in metastatic (Stage IV) CRC using large patient cohorts [25, 26].…”

“…LINE-1 methylation in CRC cell lines was quantified using methylation-specific real-time PCR [32] with the ABI-PRISM 7900 Sequence Detection System (Applied Biosystems, Osaka, Japan) and Premix Ex Taq (TaKaRa Bio, Otsu, Japan). The primers and probes used for LINE-1 methylation analyses [23, 32] were shown in Additional file 1: Table S1 and Additional file 2: Figure S1. The level of LINE-1 methylation was expressed as a median value with 25th–75th percentile.…”

Clonal intratumor heterogeneity of promoter hypermethylation in breast cancer by MS-MLPA

“…4,7,8,11,13 The marked interindividual heterogeneity challenges 'one-size-fits-all' approaches for cancer management as well as biomarker development and validation, and highlights the need for personalized medicine approaches. The intratumor heterogeneity can lead to sampling bias and thereby hamper accurate prognostication and treatment decisions.…”

Genetic alterations and epigenetic alterations of cancer-associated fibroblasts

“…In addition to hypermethylation, DNA promoter hypomethylation may also exist in CAFs. Activation of long interspersed nucleotide element-1 in tumorigenesis-related CRC is due to its hypomethylated promoter (119). Gene methylation profiles are associated with the status of receptors.…”