Noninvasive early detection of colorectal cancer by hypermethylation of the LINC00473 promoter in plasma cell-free DNA

Abstract

Background: Current noninvasive assays have limitations in the early detection of colorectal cancer. We evaluated the clinical utility of promoter methylation of the long noncoding RNA LINC00473 as a noninvasive biomarker to detect colorectal cancer and associated precancerous lesions.

Methods: We evaluated the epigenetic regulation of LINC00473 through promoter hypermethylation in colorectal cancer cell lines using bisulfite genomic sequencing and expression analyses. DNA methylation of LINC00473 was analyzed in primary colorectal tumors using 450K arrays and RNA-seq from The Cancer Genome Atlas (TCGA). Tissue-based findings were validated in several independent cohorts of colorectal cancer and advanced colorectal polyp patients by pyrosequencing. We explored the clinical utility of LINC00473 methylation for the early detection of colorectal cancer in plasma cell-free DNA by quantitative methylation-specific PCR and droplet digital PCR.

Results: LINC00473 showed transcriptionally silencing due to promoter hypermethylation in colorectal cancer cell lines and primary tumors. Methylation of the LINC00473 promoter accurately detected primary colorectal tumors in two independent clinical cohorts, with areas under the receiver operating characteristic curves (AUCs) of 0.94 and 0.89. This biomarker also identified advanced colorectal polyps from two other tissue-based clinical cohorts with high diagnostic accuracy (AUCs of 0.99 and 0.78). Finally, methylation analysis of the LINC00473 promoter in plasma cell-free DNA accurately identified patients with colorectal cancer and advanced colorectal polyps (AUCs of 0.88 and 0.84, respectively), which was confirmed in an independent cohort of patients.

Conclusions: Hypermethylation of the LINC00473 promoter is a new promising biomarker for noninvasive early detection of colorectal cancer and related precancerous lesions.

Keywords: Cell-free DNA; Colorectal cancer; DNA methylation; Early detection; LINC00473.

Fig. 1

Epigenetic silencing of the LINC00473 in colorectal cancer cells. A Bisulfite genomic sequencing analysis of LINC00473 promoter CpG island in the colorectal cancer cell line HCT-116 and normal tissue. Locations of CpG dinucleotides (vertical lines) and the TSS (long black arrow) are shown. Ten single clones are represented for each sample. The presence of unmethylated and methylated CpGs is indicated by white and black squares, respectively. B DNA methylation-associated transcriptional silencing of LINC00473 in the colorectal cancer cell line HCT-116. Expression levels of LINC00473 were determined by qRT-PCR in the methylated cancer cell line HCT-116 and in colorectal normal tissues (N = 3). C Restored LINC00473 expression in the methylated cancer cell line HCT-116 after AZA treatment analyzed by qRT-PCR. Values were determined from triplicates and are expressed as the mean ± SEM. D Methylation and expression analysis of LINC00473 in common colorectal cancer cell lines and normal colon mucosa using 450K array and RNA-seq public datasets. Expression levels obtained by RNA-seq were expressed as transcripts per million (TPMs). TSS, transcription start site; NC; normal colon mucosa

Fig. 2

Methylation of LINC00473 in colorectal cancer tissues. Methylation analysis of LINC00473 promoter in tissues from primary CRC and matched normal colorectal mucosa (controls) of two independent cohorts analyzed by 450K array (Cohort 1) and pyrosequencing (Cohort 2). A, B Methylation status of LINC00473 in (A) all CRC patients (N = 293) and (B) in those CRC patients (N = 283) with available clinical information on their TNM tumor stage (Cohort 1). C ROC curve analysis evaluating the methylation of LINC00473 for the detection of CRC in tissue samples in Cohort 1. D, E Validation of the methylation status of LINC00473 in (A) all CRC patients (N = 180) and (B) in those CRC patients (N = 178) with available clinical information on their TNM tumor stage in Cohort 2. F ROC curve analysis to validate the methylation of LINC00473 for the detection of colorectal tumors in Cohort 2. Horizontal lines represent mean methylation levels of LINC00473. P, p value analyzed by Mann–Whitney U test or ROC curve; AUC, area under the ROC curve; Ctrl, controls; CRC, colorectal cancer

Fig. 3

Methylation levels of LINC00473 in tissue precancerous colorectal lesions. A Methylation levels of LINC00473 promoter in tissues from premalignant colorectal polyps, CRC and matched normal colorectal mucosa (controls) by pyrosequencing (Cohort 3). B, C Methylation levels of LINC00473 promoter in tissues from N-ACP, ACP and CRC (Cohort 3) analyzed by pyrosequencing. D ROC curve analysis evaluating the methylation of LINC00473 promoter for the detection of ACPs with respect to the combination of controls and N-ACPs (Cohort 3). E ROC curve to evaluate the methylation of LINC00473 promoter for the detection of AN with respect to the combination of controls and N-ACPs (Cohort 3). Horizontal lines represent mean methylation levels of LINC00473. P, p value analyzed by Mann–Whitney U test or ROC curve; AUC, area under the ROC curve; Ctrl, controls; P, polyps; CRC, colorectal cancer; N-ACP, non-advanced colorectal polyps; ACP, advanced colorectal polyps; AN, advanced neoplasia

Fig. 4

Methylation of LINC00473 in cfDNA from patients with advanced colorectal polyps and colorectal cancer. A Methylation status of LINC00473 promoter in plasma cfDNA of CRC patients analyzed by qMSP (Cohort 5). B ROC curve analysis evaluating the methylation of LINC00473 for the detection of CRC in plasma cfDNA (Cohort 5). C, Methylation status of LINC00473 promoter in plasma cfDNA of patients with ACP analyzed by qMSP (Cohort 6). D ROC curve analysis evaluating the methylation of LINC00473 for the detection of ACP in plasma cfDNA (Cohort 6). E Validation of the methylation status of LINC00473 promoter in plasma cfDNA of patients with ACP by ddPCR analysis (Cohort 7). F, ROC curve analysis to validate the methylation of LINC00473 for the detection of ACP and CRC in plasma cfDNA (Cohort 7). Horizontal lines represent mean methylation levels of LINC00473. P, p value analyzed by Mann–Whitney U test or ROC curve; AUC, area under the ROC curve; Ctrl, controls. ACP, advanced colorectal polyps; CRC, colorectal cancer.