Stanley R. Hamilton

Determining the microsatellite instability (MSI) status of colorectal carcinoma (CRC) and other tumor types has recently increased in importance for patient management and prevention strategies.^1,2 High-level MSI (MSI-H)/deficient mismatch repair (dMMR) (widely used alternative terminologies) has long been known to be a hallmark of Lynch syndrome. The identification of this small subset of patients (approximately 3% of patients with CRC) has major implications for prevention in blood relatives and for avoiding metachronous tumors in patients who have a first Lynch-associated tumor. Preoperative decisions can be made about prophylactic surgery at the initial treatment, including abdominal colectomy and hysterectomy with bilateral salpingo-oophorectomy after childbearing is completed.

The molecular characteristics of colorectal cancer (CRC) have been studied extensively since the 1980s, but translation of the remarkable increase in genomic knowledge into clinically used biomarkers has been distressingly slow. The Cancer Genome Atlas for CRC was published in mid-2012 ( 1 ), and molecular and pathologic findings including genetic and epigenetic abnormalities have now been incorporated into classification systems that have been reported to have implications for the clinical management of patients ( 2–4 ). Numerous individual molecular biomarkers with potential applications have been published, but few have achieved levels and breadth of evidence to become standard of care. Difficulty in convincing payers of the value of biomarkers and fiscal constraints have impeded adequate reimbursement for testing and disincentivized their clinical use.

Molecular pathology as applied to neoplasia is a rapidly expanding component of the discipline of pathology that uses molecular biology tools in addition to conventional morphologic, immunohistochemical and chemical analyses of abnormalities in tissues and cells to understand the etiology and pathogenesis of tumors, establish their diagnosis, and contribute to prognostication and therapeutic decisions for cancer patient care. Biomarkers are a fundamental component of personalized cancer care, and the discipline of molecular pathology therefore contributes throughout the continuum from biomarker research to use in standard‐of‐care personalized cancer therapy. This brief review addresses some of the specific roles of molecular pathology in that continuum.

Purpose: MicroRNAs are short noncoding RNAs that regulate gene expression and are over- or underexpressed in most tumors, including colorectal adenocarcinoma. MicroRNAs are potential biomarkers and therapeutic targets and agents, but limited information on microRNAome alterations during progression in the well-known adenoma-adenocarcinoma sequence is available to guide their usage. Experimental Design: We profiled 866 human microRNAs by microarray analysis in 69 matched specimens of microsatellite-stable adenocarcinomas, adjoining precursor adenomas including areas of high- and low-grade dysplasia, and nonneoplastic mucosa. Results: We found 230 microRNAs that were significantly differentially expressed during progression, including 19 not reported previously. Altered microRNAs clustered into two major patterns of early (type I) and late (type II) differential expression. The largest number (n = 108) was altered at the earliest step from mucosa to low-grade dysplasia (subtype IA) prior to major nuclear localization of β-catenin, including 36 microRNAs that had persistent differential expression throughout the entire sequence to adenocarcinoma. Twenty microRNAs were intermittently altered (subtype IB), and six were transiently altered (subtype IC). In contrast, 33 microRNAs were altered late in high-grade dysplasia and adenocarcinoma (subtype IIA), and 63 in adenocarcinoma only (subtype IIB). Predicted targets in 12 molecular pathways were identified for highly altered microRNAs, including the Wnt signaling pathway leading to low-grade dysplasia. β-catenin expression correlated with downregulated microRNAs. Conclusions: Our findings suggest that numerous microRNAs play roles in the sequence of molecular events, especially early events, resulting in colorectal adenocarcinoma. The temporal patterns and complexity of microRNAome alterations during progression will influence the efficacy of microRNAs for clinical purposes. Clin Cancer Res; 17(23); 7283–93. ©2011 AACR.

Colorectal cancer (CRC) remains one of the major cancer types and cancer related death worldwide. Sensitive, non-invasive biomarkers that can facilitate disease detection, staging and prediction of therapeutic outcome are highly desirable to improve survival rate and help to determine optimized treatment for CRC. The small non-coding RNAs, microRNAs (miRNAs), have recently been identified as critical regulators for various diseases including cancer and may represent a novel class of cancer biomarkers. The purpose of this study was to identify and validate circulating microRNAs in human plasma for use as such biomarkers in colon cancer. By using quantitative reverse transcription-polymerase chain reaction, we found that circulating miR-141 was significantly associated with stage IV colon cancer in a cohort of 102 plasma samples. Receiver operating characteristic (ROC) analysis was used to evaluate the sensitivity and specificity of candidate plasma microRNA markers. We observed that combination of miR-141 and carcinoembryonic antigen (CEA), a widely used marker for CRC, further improved the accuracy of detection. These findings were validated in an independent cohort of 156 plasma samples collected at Tianjin, China. Furthermore, our analysis showed that high levels of plasma miR-141 predicted poor survival in both cohorts and that miR-141 was an independent prognostic factor for advanced colon cancer. We propose that plasma miR-141 may represent a novel biomarker that complements CEA in detecting colon cancer with distant metastasis and that high levels of miR-141 in plasma were associated with poor prognosis.

Purpose: Early events in colorectal tumorigenesis include mutation of the adenomatous polyposis coli (APC) gene and epigenetic hypermethylation with transcriptional silencing of the O⁶-methylguanine DNA methyltransferase (MGMT), human mut L homologue 1 (hMLH1), and P16/CDKN2A genes. Epigenetic alterations affect genetic events: Loss of MGMT via hypermethylation reportedly predisposes to guanine-to-adenine or cytosine-to-thymine (G:C→A:T) transition mutations in KRAS and P53, and silencing of hMLH1 leads to high levels of microsatellite instability (MSI-H)/mutator phenotype, suggesting that epigenetic-genetic subtypes exist. Experimental Design: We evaluated the relationships of aberrant methylation of APC, MGMT, hMLH1, P16, N33, and five MINTs to mutations in APC, KRAS, BRAF, and P53 in 208 colorectal carcinomas. Results: We found that APC hypermethylation was age related (P = 0.04), in contrast to the other genes, and did not cluster with CpG island methylator phenotype (CIMP) markers. Hypermethylation of APC concurrently with either MGMT or hMLH1 was strongly associated with occurrence of G-to-A transitions in APC [odds ratio (OR), 26.8; P < 0.0002 from multivariable logic regression model], but C-to-T transitions had no associations. There was no relationship of hypermethylation of any gene, including MGMT, with G-to-A or C-to-T transitions in KRAS or P53, although APC hypermethylation was associated with P53 mutation (P < 0.0002). CIMP with MSI-H due to hMLH1 hypermethylation, or CIMP with loss of MGMT expression in non–MSI-H tumors, was associated with BRAF mutation (OR, 4.5; P < 0.0002). CIMP was also associated with BRAF V600E T-to-A transversion (OR, 48.5; P < 0.0002). Conclusions: Our findings suggest that the heterogeneous epigenetic dysregulation of promoter methylation in various genes is interrelated with the occurrence of mutations, as manifested in epigenetic-genetic subgroups of tumors.