Mass Spectrometry-Based Analysis for the Discovery and Validation of Potential Colorectal Cancer Stool Biomarkers

Abstract

Colorectal cancer (CRC) is the third leading cause of cancer mortality for both men and women, and the second leading cause of cancer death for men and women combined. If detected early, before metastasis has occurred, survival following surgical resection of the tumor is > 90%. Early detection is therefore critical for effective disease surveillance. Unfortunately, current biomarker assays lack the necessary sensitivity and specificity for reliable early disease detection. Development of new robust, non- or minimally invasive specific and sensitive biomarkers or panels with improved compliance and performance is therefore urgently required. The use of fecal samples offers several advantages over other clinical biospecimens (e.g., plasma or serum) as a source of CRC biomarkers, including: collection is noninvasive, the test can be performed at home, one is not sample limited, and the stool effectively samples the entire length of the inner bowel wall contents (including tumor) as it passes down the gastrointestinal tract. Recent advances in mass spectrometry now facilitate both the targeted discovery and validation of potential CRC biomarkers. We describe, herein, detailed protocols that can be used to mine deeply into the fecal proteome to reveal candidate proteins, identify proteotypic/unitypic peptides (i.e., peptides found in only a single known human protein that serve to identify that protein) suitable for sensitive and specific quantitative multiplexed analysis, and undertake high-throughput analysis of clinical samples. Finally, we discuss future directions that may further position this technology to support the current switch in translation research toward personalized medicine.

Introduction

In spite of extensive translational research efforts, colorectal cancer (CRC) remains the third leading cause of cancer mortality for both men and women, and the second leading cause of cancer death for men and women combined (http://www.cancer.org/cancer/colonandrectumcancer/detailedguide/colorectal-cancer-key-statistics). It accounts for around 600,000 deaths worldwide annually and there is a lifetime risk of developing this disease of about 1 in 21 (4.7%) for men and 1 in 23 (4.4%) for women. The 5-year survival rate (Dukes Stage A) for localized stage CRC following surgical resection is above 90%. However, if the cancer has spread to surrounding tissues or organs and/or regional lymph nodes, the 5-year survival rate drops to around 70%. Sadly, once cancer has metastasised to distant parts of the body (Dukes Stage D) like the liver, the 5-year survival rate falls precipitously to only 13%. Detection of CRC at its earliest stage combined with surgical resection provides the greatest chance of alleviating these statistics and improving patient survival. Despite this, at present only 9% of CRC patients are diagnosed early enough for surgery to be near curative, with about 60% of patients displaying metastases at initial clinical presentation.

There are recognized molecular pathways of disease progression in CRC, associated with key genetic/genomic changes. There early adenomas develop into intermediate and late adenomas, then into carcinomas accompanied by key gene mutations, activation of oncogenes, loss and gain of chromosomes or their constituents, and/or chromosome amplifications (Fearon & Vogelstein, 1990). This process typically takes from 10 to 15 years, with transition from carcinoma to metastatic CRC taking only an additional 2–3 years. There is, therefore, an excellent window of opportunity for early detection before the cancer has spread if and when suitable and reliable methods for screening can be discovered.

The fecal occult blood test (FOBT), which screens for the presence of blood in patients’ stools, is currently the most widely used, noninvasive, primary screening tool for CRC. However, although the FOBT is a simple and cost-effective procedure for screening for CRC, it has relatively poor selectivity and sensitivity (87.6 ± 11.4% and 45.7 ± 26.5% (Allameh, Davari, & Emami, 2011)) resulting in an unacceptable number of both false positives and false negatives. Because of these clinical specificity/sensitivity reasons, all positive FOBTs are typically followed up with colonoscopy. While colonoscopy has excellent selectivity and sensitivity (99.8 ± 0.2% and 94.7 ± 4.6%, respectively (Allameh et al., 2011)), it is expensive, invasive, needs trained staff to perform the procedure, does not completely examine all the gastrointestinal tract (i.e., colonoscopy looks at colon and lower part of the small intestine while sigmoidoscopy examines only rectum and lower part of the colon), requires an unpleasant bowel preparation prior to the test, and has some risk of morbidity and mortality. Both the FOBT and colonoscopy have relatively poor patient uptake, and education is urgently required to convince patients to accept any squeamishness handling their own stool biospecimens for more compliant screening using FOBT (Gimeno Garcia, 2012). Development of new robust, non- or minimally invasive specific and sensitive biomarkers or biomarker panels with improved performance is therefore urgently required. This would immediately not only reduce the number of unnecessary follow-up colonoscopies that are currently undertaken due to false-positive results from the FOBT, but, with the help of surgical resection when the tumor is still localized (stage A/B or I/II), would result in improved survival, with enormous savings to the global health budget.

A number of potential new blood-based biomarkers have been proposed (e.g., CEA, CA 19-9, TIMP-1), but to date none of them have passed the test of time when applied to larger clinical cohorts (Drucker and Krapfenbauer, 2013, Fung et al., 2014), with many being useful only as late stage markers. It is now recognized that panels of biomarkers may well be needed, due to the heterogeneity of the disease, and indeed several CRC biomarker panels are currently under investigation (Fung et al., 2015, Garcia-Bilbao et al., 2012, Lind et al., 2011). While blood is a readily available and accepted medium for clinical analysis, it is a complex biological matrix containing a number of housekeeping proteins (e.g., serum albumin, immunoglobulins, α1-antitrypsin, transferrin, haptoglobin, fibrinogen, α2-macroglobulin, α1-acid glycoprotein, apolipoproteins, complement C3, and transthyretin) present at mg/mL concentrations. Additionally, plasma/serum contains an extremely large dynamic range (10¹²–10¹³) of overall protein abundances (Anderson and Anderson, 2002, Geyer et al., 2016). This complicates both MS analysis, due to the occupancy of available MS “discovery space” by peptides that are derived from far more abundant proteins and which cannot be separated from lower abundance species on chromatography, as well as antibody-based multiplexed analyses due to potential problems around antigen selectivity when analysing crude biological samples.

Stool protein detection offers a compelling and advantageous alternative to blood-based assays for CRC, as the stool biospecimen directly samples the entire length of the inner bowel wall contents. As the stool passes down the GI tract, it effectively constantly samples the cellular environment that is exposed to epithelial tissues as well as to the tumor and its attendant microenvironment. Protein tumor markers could conceivably be found in stools because of leakage, exfoliation, and/or secretion from the tumor. Leakage of markers into the intestinal lumen could also be due to the disturbance of blood and/or blood vessels by tumor growth or changes in intestinal permeability. These processes may not be continuous and might also be observed from nonneoplastic lesions. Thus, “leaked” markers may have limited sensitivity and specificity if used individually. Exfoliated and secreted markers may arise from both healthy and diseased colonocytes shed into the lumen of the gut. They are potentially highly specific because they arise directly from the tumor. Additionally, the relative concentrations of human proteins in stool may be higher than in blood (Kim et al., 2003) because lower levels of plasma proteins are able to reach the luminal contents across the bowel wall unless it is breached, facilitating ease of cancer protein detection. The added advantages of stool-based assays are they are a noninvasive procedure, do not require trained staff, can readily be done at home, do not require the unpleasant bowel preparation procedure required for colonoscopy, and are not sample limited. As is essential for all biomarker studies, strict SOPs need to be developed for sample collection and storage.

Besides the FOBT, other CRC stool-based assays have been proposed, including a next-generation multitarget stool DNA test that has been reported to accurately detect CRC and large adenomas (Sweetser & Ahlquist, 2016). PKM2 has been measured in stools and shows relatively high sensitivity for CRC diagnosis, with a reported sensitivity of over 90% in some studies (Koss, Maxton, & Jankowski, 2008). The human microbiome is becoming a growing area of research as it appears to be related to human health and disease. In a recent example, shotgun metagenomic sequencing was performed on fecal samples from a CRC case–control study and compared to a previously published 16S rRNA study on the same samples (Vogtmann et al., 2016). Associations were found between certain fecal bacteria and CRC.

Our own studies have focussed on the development of fecal proteomics for the detection and surveillance of CRC (Ang and Nice, 2010, Ang et al., 2010, Ang et al., 2011). Initially, proof of principal studies were developed using a mouse model system (Ang et al., 2010) that carries a mutant allele of the murine APC (adenomatous polyposis coli) gene, encoding for a nonsense mutation at codon 850 that causes protein truncation. Similar to humans with germline APC mutations, the APC^min mice are predisposed to developing intestinal adenomas. Success with the murine model prompted us to develop a human fecal proteomics library of proteotypic peptides from which to develop sensitive and specific quantitative multiplex multiple reaction monitoring (MRM) assays to probe for panels of potential biomarkers (Ang and Nice, 2010, Ang et al., 2011). Key methods arising from these studies are presented later.