Published OnlineFirst May 31, 2019; DOI: 10.1158/1055-9965.EPI-18-1291 Research Article Cancer Epidemiology, Biomarkers Urinary Metabolomics to Identify a Unique & Prevention Biomarker Panel for Detecting Colorectal Cancer: A Multicenter Study Lu Deng1, Kathleen Ismond1,2, Zhengjun Liu3, Jeremy Constable4, Haili Wang2, Olusegun I. Alatise5, Martin R. Weiser4, T.P. Kingham4, and David Chang1,2 Abstract Background: Population-based screening programs are paring the metabolomic profiles from colorectal cancer versus credited with earlier colorectal cancer diagnoses and treat- controls. Multiple models were constructed leading to a good ment initiation, which reduce mortality rates and improve separation of colorectal cancer from controls. patient health outcomes. However, recommended screen- Results: A panel of 17 metabolites was identified as possible ing methods are unsatisfactory as they are invasive, are biomarkers for colorectal cancer. Using only two of the select- resource intensive, suffer from low uptake, or have poor ed metabolites, namely diacetylspermine and kynurenine, a diagnostic performance. Our goal was to identify a urine predictor for detecting colorectal cancer was developed with an metabolomic-based biomarker panel for the detection of AUC of 0.864, a specificity of 80.0%, and a sensitivity of colorectal cancer that has the potential for global popula- 80.0%. tion-based screening. Conclusions: We present a potentially "universal" metabo- Methods: Prospective urine samples were collected from lomic biomarker panel for colorectal cancer independent of study participants. Based upon colonoscopy and histopathol- cohort clinical features based on a North American popula- ogy results, 342 participants (colorectal cancer, 171; healthy tion. Further research is needed to confirm the utility of the controls, 171) from two study sites (Canada, United States) profile in a prospective, population-based colorectal cancer were included in the analyses. Targeted liquid chromatogra- screening trial. phy-mass spectrometry (LC-MS) was performed to quantify Impact: A urinary metabolomic biomarker panel was iden- 140 highly valuable metabolites in each urine sample. Poten- tified for colorectal cancer with the potential of clinical tial biomarkers for colorectal cancer were identified by com- application. Introduction programmatic rather than opportunistic to ensure a high rate of compliance (2). Such programs have been instituted nationally or Colorectal cancer is the third most commonly diagnosed regionally within many countries in Europe (e.g., UK, Ireland, malignancy and the fourth leading cause of cancer-related deaths Germany, France), United States, Japan, and Australia as reviewed in the world. On the basis of 2018 estimates, the 2040 incidence by Navarro and colleagues (3). rates for colorectal cancer are projected to increase by 72% to 3.1 The most commonly used population-based screening modal- million new cases, while mortality rates will increase by 82% to ities are the fecal immunochemical test (FIT) and colonoscopy (4). 1.5 million deaths (https://gco.iarc.fr/tomorrow). Mortalities due FIT detects hidden blood in stool that occurs mostly in the later to colorectal cancer are largely preventable through regular screen- stages of cancer and has low sensitivity for detecting the precursors ing and early detection using fecal-based tests and colonosco- to colorectal cancer, adenomatous polyps (9). A new fecal DNA py (1). To be effective, population-based screening must be test detects DNA mutations in addition to hidden blood in stool with improved sensitivity (5), but it is costly and only available in a few countries. To date, fecal-based tests are limited to colorectal 1Metabolomic Technologies Inc., Edmonton, Alberta, Canada. 2Department of cancer detection not prevention, and have low adherence rates 3 Medicine, University of Alberta, Edmonton, Alberta, Canada. Department of due to the need for stool collection and manipulation (6–10). Mathematical and Statistical Sciences, University of Alberta, Edmonton, Alberta, Colonoscopy has a superior sensitivity and specificity to nonin- Canada. 4Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York. 5Department of Surgery, Obafemi Awolowo University and vasive screening tests, but is costly in terms of direct and indirect Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria. health care dollars, has a higher risk of procedural-related com- plications, and, like fecal-based tests, has low rates of screening Note: Supplementary data for this article are available at Cancer Epidemiology, Biomarkers & Prevention Online (http://cebp.aacrjournals.org/). compliance (11). To increase screening compliance rates, programs have largely Corresponding Author: Lu Deng, Metabolomic Technologies Inc., 132, 9650 focused on colorectal cancer education and sending reminders to 20th Avenue, Edmonton, Alberta T6N 1G1, Canada. Phone: 587-772-1684; Fax: 780-492-8121; E-mail: [email protected] eligible participants (12, 13). An alternative approach for improv- ing colorectal cancer screening rates is to use a biosample other Cancer Epidemiol Biomarkers Prev 2019;28:1283–91 than stool (14). A blood-based screening test has been shown to doi: 10.1158/1055-9965.EPI-18-1291 have higher patient uptake than FIT (15), but its cost-effectiveness Ó2019 American Association for Cancer Research. is debatable for population-based screening (16). Urine is www.aacrjournals.org 1283 Downloaded from cebp.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst May 31, 2019; DOI: 10.1158/1055-9965.EPI-18-1291 Deng et al. commonly used for many clinical tests, can be readily collected, Ethics approval was obtained from the Health Research and is more acceptable to patients (17, 18). Recently, putative Ethics Boards at the University of Alberta (Pro0000514 and biomarkers of colorectal cancer were identified in urine in the Pro00074045) and MSKCC (IRB catalog nos. 06-107 and 15-209). forms of volatile organic compounds (19), modified cytosine nucleosides (20), and polyamines (21, 22). As well, we have Metabolite analysis reported a urine-based screening test specific for colorectal ade- Targeted liquid chromatography-mass spectrometry (LC-MS) nomatous polyps (23, 24) developed in a Canadian population was performed to quantify urinary metabolites in each sample and its subsequent validation in a homogenous Asian cohort to using the LC-MS kit TMIC00UJ designed and prepared by The demonstrate its clinical relevance transcending both diet and Metabolomics Innovation Centre (TMIC) at the University of ethnicity (25). Alberta in Edmonton, Alberta. Calibration solutions (Cal 1–Cal In the current multicenter study, the potential utility of urine- 7), isotopically labeled standard mix, quality control solutions based metabolomics for detecting colorectal cancer was investi- (QC 1–QC 3), LC-MS methods, and standard operating proce- gated. This was done by analyzing metabolites in urine samples dures were provided by TMIC. The TMIC00UJ kit was a combi- from colonoscopy- and histopathology-confirmed cases of colo- nation of three assays to identify 140 unique urinary metabolites rectal cancer and healthy controls (e.g., polyp- and colorectal (see Supplementary Table S1) indexed by the Human Metabo- cancer-free). Our findings highlight the predictive potential of lome Database (www.hmdb.ca). The phenyl isothiocyanate urinary metabolites for colorectal cancer and we discuss the (PITC) assay quantified 47 biologic amines in the LC mode while clinical relevance of a proposed screening test. 75 lipids were semiquantified in the flow injection analysis (FIA) mode. The organic acid assay quantified 17 compounds while ascorbic acid was quantified independently. The TMIC00UJ kit components were run on an API4000 Qtrap Materials and Methods tandem mass spectrometry instrument (AB Sciex) coupled with a Study participants and sample collection Waters UPLC system (Waters Limited). Urine samples were Adult patients with newly diagnosed colorectal cancer (based thawed on ice, vortexed, then centrifuged at 13,000 Â g. Each on preoperative imaging, colonoscopies, and pathology reports plate contained 82 unique urine samples as well as 1 solvent blank of biopsies) were eligible for study inclusion provided they had solution, 3 matrix solutions, 7 calibration solutions (Cal 1–Cal 7), not received colorectal cancer–related treatment. Canadian and 3 quality control (QC) samples. PBS (1Â, pH 7.4) was used as recruitment (October 2008–2010) was conducted at four tertiary the matrix solution. Metabolite quantification was achieved using hospitals in the Edmonton region (Grey Nuns Hospital, Miser- the AB Sciex Analyst software, version 1.6.2. During quantifica- icordia Hospital, University of Alberta Hospital, and the Royal tion, each metabolite was identified using the internal standard Alexandra Hospital) and included patients from across the and compared against the established calibration curve. The lower prairie provinces (i.e., CRC-CADcohort).Americanpatients limits of detection (LLOD) were calculated as three times the value were recruited (February–July 2018) from the Memorial Sloan of the matrix solutions. The upper limit of detection was not Kettering Cancer Center (MSKCC) in New York City, New York reached for any metabolite. (i.e., CRC-MSKCC cohort). Patients diagnosed with colorectal cancer provided a urine Statistical analysis sample prior to any