Biomarkers in Colorectal Cancer

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Colorectal Cancer Diagnostics and monitoring

CRC diagnostics and monitoring follows two pronged strategy

  1. Diagnostic tools such as
    • Colonoscopy: Specific, Can Remove Polyps and Take Biopsies. Adverse: Can cause perforation, Can miss small adenomas
    • Flexible sigmoidoscopy: Specific, Can Remove Polyps and Take Biopsies. Adverse: Reduced risk of perforation
    • Double-contrast barium enema: Specific Can miss some Adenomas
    • Computer tomographic colonography
  2. Lab Diagnosis using Biomarkers such as
    • Fecal Occult Blood Test: Cheap for screening, Non Specific
    • Stool DNA test: Expensive, Tough to perform
    • Biochemical markers:
    • Genetic markers

Biomarkers of cancer  include a broad range of biochemical entities, such as nucleic acids, proteins, sugars, lipids, and small metabolites, cytogenetic and cytokinetic parameters as well as whole tumour cells found in the body fluid. Biomarkers in Cancer can be broadly classified as genetic, cancer antigens, metabolic biomarkers.

 Biomarker Classification

Various options of CRC Diagnostic and Prognostic Biomarkers (Starting from the oldest and most commonly deployed to the more modern strategies for CRC):

Stool-Based Tests

Fecal Occult Blood Test; The gFOBTs are based on the detection of the pseudoperoxidase activity of  heme in stool samples resulting from bleeding in adenomatous or neoplastic lesions therefore detecting the presence of occult blood.

One of the disadvantages of this method is that since colorectal bleeding might be intermittent, this test has to be performed on multiple occasions in order for it to be sensitive. In addition, gFOBTs are not specific for human pseudoperoxidase and may detect bleeding from any site in the gastrointestinal tract. Prior to testing, patients need to adhere to a three-day diet that eliminates meats and NSAIDs because ingestion of certain foods and drugs may cause false-positive results.

DNA-Based Tests. The detection of CRC-specific DNA markers in stool has been studied extensively. These types of markers should have higher specificity since they are directly derived from tumor cells. This test is based on the detection of vimentin methylation, which is found in 53–83% of colorectal tumors the sensitivity and sensitivity for colorectal cancer range from 72.5–83% and 53–86.9%, respectively

Clinical Relevance of Genetic Biomarkers for CRC

Commonly Available Blood-Based Tests

Carcinoembryogenic Antigen. Extensive research has been performed to identify CRC-specific antigens in blood. However, there only two blood-based biomarkers available to monitor CRC patients, CEA and carbohydrate antigen 19-9 (CA19-9). CEA, a high molecular weight glycoprotein, is found in embryonic tissue and colorectal malignancies. It was discovered in 1965 and is the only acceptable tumor marker to monitor CRC recurrence to date. Elevated CEA levels are considered a poor prognostic factor for resectable CRC and correlate with cancer progression

Genetic Biomarkers for CRC

Background: There are three major molecular mechanisms that cause aberrant gene expression resulting in colon carcinogenesis: microsatellite instability (MSI), chromosomal instability (CIN), and the CpG island methylator phenotype (CIMP). These pathways lead to a transition in lesion pathology and progression to malignancy, which is accompanied by deregulated gene expression of tumor suppressor genes and oncogenes. These cytogenetic alterations have been considered as potential CRC molecular markers because they can provide the clinician with diagnostic, prognostic, and predictive treatment response information.

Microsatellite Instability (MSI). Mutations and/or epimutations in genes involved in the DNA mismatch repair system (MMR), MLH1, MSH2, MSH6, and PMS2, result in alterations in highly repeated DNA sequences (microsatellites).MSI is a hallmark of Lynch Syndrome, an inherited CRC syndrome, and is used as a diagnostic marker for this disease. In sporadic CRC, 10–15% of tumors display MSI although somatic mutations in MMR genes are rarely found. Methylation induced silencing of MLH1 is responsible for the majority of sporadic CRC with MSI.

Accumulating evidence supports that MSI status may predict responsiveness to adjuvant chemotherapy. Reports from clinical trials, retrospective case series, and metaanalysis have reported that patients with MSI tumors do not benefit from 5-fluorouracil (5-FU) adjuvant chemotherapy compared to patients with microsatellite stable tumors (MSS)

KRAS Gene Mutations. Mutations in genes associated with chemoresistance to particular compounds are currently used as predictive markers in CRC in order to identify the best treatment regime for patients. Detection of KRAS mutations is currently the most utilized predictive marker for response to the anti-EGFR (epidermal growth factor receptor) antibody-based therapies, cetuximab and panitumumab. However, recent studies have reported compelling evidence that, in addition to KRAS, mutations in NRAS predict nonresponse to anti-EGFR therapy. These studies support the use of extended RAS (KRAS and NRAS) mutational analyses as negative predictive markers for anti-EGFR therapy in metastatic CRC (mCRC).

BRAF Gene. BRAF, aRAF gene family serine/threonine kinase, is the immediate downstream effector of KRAS in the Ras/Raf/MAPK signaling pathway. Mutations in the BRAF gene have been associated with CRC development and are present in 40–50% of sporadic MSI-high CRC. These are absent in Lynch syndrome patients, making BRAF mutation status a very useful diagnostic tool to distinguish between familial and sporadic CRC. A missense mutation resulting in a valine to glutamic acid substitution (V600E) is the most common mutation observed . KRAS and BRAF mutations are generally mutually exclusive in colorectal tumors . Recent studies suggest that BRAF mutations may also be used as predictive markers for EGFR-targeted therapy. Mutations in BRAF are associated with poor prognosis.

Innovative Tumor-Based Tests

CpG Island Methylator Phenotype. The molecular classification of tumors is evolving as we gain a comprehensive knowledge about the mechanisms and processes resulting in colorectal carcinogenesis. The epimutation status of tumors has gained importance since the discovery that methylation driven transcriptional regulation leads to colorectal carcinogenesis and that the CpG island methylator phenotype (CIMP) status correlates to a particular CRC subtype. CIMP high colorectal tumors are more prevalent in women and are associated with BRAF mutations.They display distinct characteristics which include: proximal tumor location, poor differentiation, mucinous histology, MSI, and low frequency of TP53 mutations. Larger studies using a consensus panel and sensitive, methylation detection techniques will resolve the discrepancies in this field and will likely yield a CRC-specific methylation signature that could be developed into a diagnostic tool in the future.

RNA Expression. Gene expression analyses between tumor and normal tissue have contributed to a better understanding on the interplay between overexpressed or under expressed genes and the affected pathways resulting in colorectal neoplasms. These efforts have resulted in a wealth of publicly available RNA expression data used to identify differentially expressed transcripts, which can be used to identify a CRC-specific signature. Expressed sequence tags (EST), serial analysis of gene expression (SAGE), and microarray data have identified numerous promising candidate tumor biomarkers. Validation of a 23-gene microarray based prognostic signature. reported a 67% relapse predictive value. A seven-gene panel based on this study was tested in a larger study with a better performance, but a robust multigene signature has not yet been defined

MicroRNAs. Accumulating evidence supporting that noncoding microRNAs (miRNAs) contribute to oncogenesis have resulted in multiple studies aiming at identifying a miRNA biomarker panel. Studies examining miRNA expression in CRC have shown a total of 362 differentially expressed miRNAs when compared to noncancerous tissue; 242 were upregulated and 120 were downregulated [81]. The use of different platforms for miRNA expression profiling has resulted in contradictory reports; nevertheless, 101 of the 362 miRNAs were consistently reported to be dysregulated in CRC. Further clinical and mechanistic studies are needed to elucidate the clinical utility of miRNAs (individual or panels of miRNAs) as diagnostic, prognostic, and/or possible therapeutic tools.

EGFR Pathway. Beyond KRAS testing, other studies have focused on identifying additional biomarkers to predict response to anti-EGFR treatment in patients with wild type KRAS (up to 70% are unresponsive to cetuximab or panitumumab . Expression of the EGFR ligands amphiregulin and epiregulin have been associated with increased response to cetuximab . These ligands are being explored as candidate predictive markers. EGFR signaling triggers two main intracellular cascades, one involving KRAS and BRAF leading to the activation of mitogen activated kinases and another resulting in the phosphorylation of AKT1 via interactions between PIK3CA and PTEN. Mutations in both PIK3CA and PTEN have also been evaluated as predictive markers for anti-EGFR therapies. Mutations within PIK3CA have been found to independently affect the response to both cetuximab and panitumumab. This gene is mutated in approximately 20% of CRC patients.

New and Innovative Stool-Based Tests

DNA-Based Tests. Shedding of neoplastic colonocytes is known to be higher than the shedding of normal healthy colonic cells in stool; therefore, CRC-specific biomarkers should have high specificity.

RNA-Based Tests. The detection of RNA markers in stool has not been as extensively studied as DNA biomarkers partly due to the fact that RNA is less stable than DNA in stool.

Deploying modern methods to targeting genetic material using cell free DNA and Next Gen Sequencing the above biomarkers can revolutionize the diagnosis and prognosis of CRC . There is a need for developing better diagnostic algorithms and deploying these solutions in a standardized manner. In case of prognostic monitoring more standardized strategies are needed from combined therapy as well as monitoring standpoint.



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Rajeev Kumria
Rajeev Kumria
RAS LSS Advisor, Sr. Leadership Team

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