Abstract IA16: Current and emerging molecular diagnostic assays for colorectal cancer
Authors: Grady William M. 
Published: March 2017

Abstract
Introduction: In the last 20 years, advances in our understanding of the molecular genetics and epigenetics of colorectal cancer (CRC) have led to the discovery and assessment of a large number of cancer specific molecular alterations as biomarkers for CRC. The majority of these biomarkers have not evolved into clinical assays for a variety of reasons, but a small number of them are currently in use in the clinic and a larger number of emerging biomarkers are under active investigation for their use in early detection, prognosis and prediction for treatment response.
Early detection assays: 1. Stool-based biomarkers: Since the initial discovery of mutant KRAS in fecal specimens from patients with CRC(1), numerous studies have assessed stool DNA biomarkers for early CRC detection. Biomarkers assessed to date include mutant genes, methylated genes, microRNAs, and mRNA. Methylated DNA and mutant DNA biomarkers are now in the clinic.
A large number of Phase I and II biomarker studies have identified stool-based methylation biomarkers for early CRC detection.(2) Studies of methylated SFRP2, SFRP5, PGR, CALCA, and IGFBP2, in fecal DNA identified methylated SFRP2 as a diagnostic biomarker for CRC detection with high sensitivity (77-90%) and specificity (77%).(3) A second study of stool DNA markers among subjects with colorectal adenomas demonstrated that methylated SFRP2 can also identify patients with precancerous colonic polyps.(4) Another well-studied fecal DNA biomarker for CRC detection is methylated VIM, the gene for vimentin. Methylated VIM specifically occurs in adenoma and CRC tissues and is detected in fecal DNA with reasonably high sensitivity (46%) and specificity (90%).(5) Studies demonstrating the potential for using methylated VIM as a biomarker for the early detection of CRC led to the development of an assay that detects methylated VIM as one of the first commercial fecal-DNA screening tests for CRC (ColoSureTM, Lab Corp, Burlington, NC). Other hypermethylated genes (APC, ATM, BMP3, CDKN2A, SFRP2, GATA4, GSTP1, HLTF, MLH1, MGMT, NDRG4, RASSF2A, TFPI2, VIM, and WIF1) have been analyzed in fecal DNA for the early detection of CRC.(5-13)
Efforts to develop an accurate noninvasive biomarker assay for CRC and colon polyp detection have culminated in the development of an FDA-approved, clinically available stool-based CRC screening test—Cologuard® (Exact Sciences Corporation, Madison, WI), which combines both the stool DNA test and the OC Auto FIT. This stool DNA based assay, which detects methylated BMP3, methylated NDRG4, and mutant KRAS, was recently compared to FIT and had better sensitivity than FIT for both CRC and advanced adenomas, but lower specificity for both endpoints.(14,15) The ColoGuard® assay is approved for use by the FDA and in the European Union; however, its feasibility in clinical settings is unknown. Of note, the Cologuard assay has been included as an acceptable screening method in the 2016 USPTF screening guidelines and the 2014 American Cancer Society's colorectal cancer prevention and early detection guidelines.
2. Blood-based biomarkers: Due to accessibility and high patient acceptance, blood is invariably the most ideal analyte for cancer biomarkers. With the development of massively parallel sequencing methods, there have been renewed efforts to determine whether blood-based diagnostic assays based on circulating methylated DNA can be used for colon polyp or CRC detection. Okugawa et al recently summarized aberrantly methylated genes discovered in the plasma or serum of CRC patients, which, consequently are candidate biomarkers.(11)
Following the initial reports of methylated CDKN2A in circulating DNA in a variety of human cancers in 1999(16-18), a growing number of studies have examined the potential of methylated genes to be blood-based biomarkers for CRC patients. Currently, the most established methylated DNA blood biomarker is methylated Septin 9 (SEPT9). Lofton-Day and colleagues identified methylated SEPT9 as a non-invasive diagnostic biomarker for CRC with 69% sensitivity and 86% specificity for distinguishing CRC patients from healthy individuals.(19) Upon further validation of methylated SEPT9 as a potential biomarker for CRC screening, it is now commercially-offered as a blood-based screening test in various assays including Epi proColon® 1.0 (Epigenomics, Seattle, WA), ColoVantage® (Quest Diagnostics, Madison, NJ) and RealTime mS9 (Abbott Laboratories, Des Plaines, IL). A recent prospective clinical trial (PRESEPT) demonstrated equivalent sensitivity and specificity of methylated SEPT9 versus FOBT for CRC, confirming its potential usefulness as a blood-based biomarker for CRC.(20) This test has been recently approved by the US FDA for use as a CRC screening assay. However, methylated SEPT9 has a limited sensitivity for the detection of advanced adenomas (11%), underscoring the need for further improvement of this test for implementation for population-based screening of colorectal neoplasia. A recent study demonstrated that the methylated SEPT9 assay was superior to FIT at detecting CRC, but both approaches were suboptimal for diagnosing patients with advanced adenomas.(21) To date, several other blood-based diagnostic methylation biomarkers have been identified for CRC detection, including ALX4(22), APC(8), CDKN2A(12), HLTF(23), HPP1(24), hMLH1(23), MGMT(8), NEUROG1(25), NGFR, RASSF2A(8), SFRP2, VIM(26), and WIF1.(8) Unfortunately, there are no Phase III biomarker studies of mutant DNA based biomarker assays for CRC detection in a screening setting at this time. Thus, while a robust biomarker panel of methylated genes may be developed into a clinically accurate CRC screening method in the near future, currently there are no accurate blood based biomarker assays for colon polyp detection.
Prognostic and predictive biomarker assays: Recognition of the molecular heterogeneity of colorectal cancer (CRC) has led to the classification of CRC based on a variety of clinical and molecular characteristics. Although the clinical significance of the majority of these molecular alterations is still being ascertained, it is widely anticipated that these characteristics will improve the accuracy of our ability to determine the prognosis and therapeutic response of CRC patients. This interest in markers has led to an intense effort to identify predictive molecular markers for chemotherapy effectiveness both in the adjuvant and metastatic settings. Candidate biomarkers that have been heavily scrutinized include mutant TP53, thymidylate synthase (TS) expression, and amplified ERCC1, among others (27-29). Unfortunately, none of these markers has been established as a predictive marker that is ready to be used clinically. However, despite the lack of success thus far, recent data has shown that microsatellite instability (MSI) is a robust prognostic marker for survival and is also a promising marker for 5-FU responsiveness and potentially PD-1 checkpoint inhibitors (30-33). The CpG island methylator phenotype (CIMP) is showing promise as a predictive marker for cytotoxic chemotherapy, and the newly described Consensus Molecular Subtypes (CMS) 1-4 have high potential as prognostic and predictive biomarkers (34). There is clear lack of benefit from EGFR targeted therapies in individuals with metastatic CRC harboring KRAS exon 2 mutations. In addition, mutations in KRAS exons 3 and 4, NRAS exons 2-4, PIK3CA exon 20, and possibly TP53 are predictive of lack of benefit. No clinically useful predictive biomarkers for anti-angiogenic therapy have been identified. Inhibitors of PI3K, MEK, MET, HER2, and other newer targets are under active investigation but are not yet ready for application to clinical practice.
Multiple new actionable drug targets are being identified on a regular basis, but most are not ready for clinical use at this time. It is widely anticipated that the next era of CRC treatment trials will focus on tumor genetic and epigenetic signatures and molecular subtypes, rather than on single gene alterations. The increasing complexity of mutation profiling for therapies targeting EGFR and other endpoints suggests the need for greater molecular characterization of tumors before, and possibly after, starting treatment. Future trials will need to better adapt eligibility to who is predicted to respond to targeted therapy. In addition, combination targeted therapies and/or dual downstream inhibition of resistance mechanisms may prove to be more successful.
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Citation Format: William M. Grady. Current and emerging molecular diagnostic assays for colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr IA16.