A study published in Clinical Laboratory, demonstrates that a multitarget (FIT and DNA) assay in stool (ColoAlert) detects colorectal cancer (CRC) with a significantly better performance than other non-invasive screening options.
There is an urgent need to develop non-invasive convenient and sensitive testing strategies to decrease mortality of CRC says the study´s lead author, Matthias Dollinger, MD, and professor at Department of Medicine at Klinikum Landshut in Bavaria, Germany. Dollinger states, that by detecting tumor DNA, the ColoAlert test effectively minimizes the analytical gap between the colonoscopy and other stool tests.
As the colonoscopy participation rates are fairly low under the invasive screening procedures, sensitive and non-invasive screening alternatives are urgently needed.
Epidemiology of colorectal cancer
Colorectal cancer is the third most common form of cancer worldwide in terms of incidence and the second in terms of mortality with 1.9 million new cases and 930.000 deaths reported in 2020. Corresponding numbers for US are 150.000 and 53.000, respectively. Although the majority of CRCs in the US are in adults aged 50 and older, there has recently been a considerable rise in early-onset CRC, so that 17.930 (12 % of total cases) will be diagnosed in individuals younger than age 50, the equivalent of 49 new cases per day.
Persons with an increased risk of developing CRC in comparison to the normal population are assigned to the following risk groups: 1. Patients with a hereditary increased risk of CRC (patients with adenomas, first-degree relatives of patients with CRC or adenomas); 2. Proven or possible genetic carriers of hereditary CRC: familial adenomatous polyposis [FAP], attenuated FAP, hereditary nonpolyposis colorectal cancer [HNPCC], patients with hamartomata’s polyposis syndromes such as Peutz-Jegers syndrome [PJS] and juvenile polyposis coli [JPC]; 3. Patients with chronic inflammatory bowel disease (CIBD: ulcerative colitis, Crohn’s disease); 4. A heterogenous group of risk factors including diet, smoking, alcohol consumption, obesity, sedentary lifestyle, increased use of antibiotics and endogenous factors, such as oxidative stress, chronic inflammation, or intestinal dysbiosis, which lead to the transformation of benign adenomatous polyps into a dysplastic polyp, favoring cumulative mutations, which finally lead to the development of CRC.
Colorectal mucosa cell biology
The colon is an extraordinary organ with many unique and fascinating characteristics. Completely unfolded, it has the size of a soccer field and the multibillion cells of the lining mucosa have a life span between one day or a few days, depending on their location. Many billion cells are thus expelled into the colonic lumen every day and mixed with other components of stool. This fast renewal of cells makes it possible to analyze DNA signatures in stool and distinguish between signatures from normal cells, from precancerous cells, and from cancer cells. Any lesion starts at the beginning on a molecular level.
Colorectal cancer (most of them belong to a histological group named adenocarcinomas) develops over time from the normal intestinal mucosa to benign precancerous advanced adenomas, then to a carcinoma and finally to an aggressive metastatic cancer. This process is slow, normally spans over years and, in many cases, more than 10 years. This transition is characterized by several genetic and epigenetic changes. The original classical pathway (adenoma-carcinoma sequence) is characterized by adenomatosis polyposis (APC) gene mutations or deletions leading to chromosomal instability driving the development of CRC. Activating mutations of the KRAS and BRAF oncogenes and inactivating mutations of the TP53 tumor suppressor gene further promote adenoma-carcinoma progression. Also, it is known that uncontrolled cell growth is mediated by a molecular switch by somatic mutations in the RAS and RAF genes, resulting in uncontrolled cell growth. Under normal circumstances, those genes encode proteins involved in cell signaling pathways that control cell growth, cell maturation, and cell death. Having said that, those genes are interesting candidates as biomarkers for early detection of any lesions in the gut.
Colorectal carcinomas: Molecular pathways
Recent studies have shown a much more complex (compared to adenoma-carcinoma sequence) and diversified molecular progression towards CRC and there exist at least 4 consensus molecular subtypes (CMS), each subtype demonstrating both independent prognostic value as well as unique clinical and biological features of the tumors. CMS1 (MSI immune) has a high rate of mutations and a low number of somatic copy number alterations. CMS2 (canonical) has a high number of somatic copy number alterations whereas CMS3 (metabolic) shows low levels of hypermethylation and a high incidence of KRAS mutations. Finally, CMS4 (mesenchymal) show extremely high levels of somatic copy number alterations. Each of the 4 subtypes have unique genetic and/or epigenetic signatures that can be analyzed in stool samples.
Signatures in stool from gut microbes
Colorectal cancers have long been known to arise due to environmental exposures such as unhealthy diet or smoking. Recent studies have shown that gut microbes play an important role in the development of these types of colorectal cancer. Researchers from EMBL, the University of Trento in Italy, and their international collaborators have now shown unique disease-specific microbiome changes which are globally robust – consistent across seven countries on three continents – despite differences in environment, diet and lifestyle for colorectal cancer.
Signatures in stool from host mRNA molecules
Also, host mRNA stool testing recently showed to be more than a valid option, particularly to detect advanced adenomas as well as colorectal cancer, both with high sensitivity and specificity values, being superior to FIT alone. Based on the significant exfoliation of dysplastic cells from colorectal lesions into the lumen, host mRNA and corresponding upregulated expression profiles recently served as another potential signature. Herring et al. from the University
Sherbrooke in Canada, reduced a candidate mRNA biomarker panel from initially 27 candidate genes down to 5. Once those 5 genes, in particular called CECAM5, ITGA6, MACC1, PTGS2, S100A4 are combined with a FIT score under a score-based algorithm, advanced adenoma and colorectal cancer sensitivities represented as 75% and 95 % respectively, at a high specificity of 95%. Those data show, that the combination of host mRNA together with a DNA based test like ColoAlert is a desirable feature.
Screening recommendations for the asymptomatic population
The chance of early diagnosis through targeted screening are very favorable with CRC due to the long latency period of several years required for the carcinoma to develop from a premalignant preliminary stage, the advanced adenoma. A complete colonoscopy has the greatest degree of sensitivity (95 %) and specificity (100 %) in diagnosing CRC or an advanced adenoma, and it is the only procedure to offer the option of linking diagnostics and therapy. For secondary prevention purposes, the adenoma-carcinoma sequence can be interrupted through the removal of adenomas, and the development of carcinomas can be prevented.
In the US, regular screening is recommended, beginning at age 45, and proven an important key to preventing colorectal cancer and finding it early. The US. Preventive Services Task Force (USPSTF) recommends that adults aged 45 to 75 are screened for colorectal cancer. The Task Force recommends that adults aged 76 to 85 ask their doctor if they should be screened.
The Task Force recommends several colorectal cancers screening strategies, including stool tests, flexible sigmoidoscopy, colonoscopy, and CT colonography (virtual colonoscopy).
Unfortunately, the population’s compliance with screening methods for early diagnosis of bowel cancer has been limited in the past; only 40 % of women and 18-20 % of men currently undergo testing.
Stool-based screening test
There are currently three types of screening for detecting CRC: stool-based, imaging, and endoscopic tests. Stool-based tests (fecal occult-based tests, FOBT) shows the presence of heme (gFOBT) or human globin (FIT) of hemoglobin in stool samples. The gFOBT has a long history and consists of a colorimetric assay which uses the guaiac reaction. FIT is an immunochemical test, which exploits a specific antibody. It has replaced gFOBT because it is more sensitive and accurate at detecting CRC (sensitivity 69–95 % vs. 25–38 %) and does not require dietary restrictions and targets the species specific the human hemoglobin only.
Patients with positive FIT tests are referred for a colonoscopy for further investigations. To respond to the best cost–benefit strategy, numerous studies have tried to fix the optimal cut-off of FIT. The most used value currently appears to be 100 ng/mL, corresponding to 10 μg of Hb per gram of stool. However, there are country specific variations in that cut-off value. Drawbacks of FIT are related to variability of test performance, the less-than-optimal level of enrolment in screening programs, the high number of false positive results (15–30 %), the poor ability to detect serrated polyps, and the low sensitivity for adenomas.
In recent years, new test strategies have been developed to optimize CRC screening and diagnosis. Several studies on KRAS oncogene mutations, DNA alterations in stools have been investigated, as well as proteins and microRNAs/mRNAs.
Following the first studies on DNA and the feasibility of the prototype of a multitarget panel assay in stools, Imperiale et al. further developed and tested the multitarget stool DNA ColoGuard (MT-sDNA; Exact Sciences Corporation) in a large screening setting. This test is an alternative stool test, which has been approved by the Food and Drug Administration (FDA) and is currently used in the USA. The MT-sDNA test, ColoGuard, combining stool DNA markers (methylated BMP3 and NDRG4 promoter regions, mutant KRAS, quantification of human DNA in stool) with the results of FIT, undoubtedly represented a milestone of stool-based molecular testing in CRC screening. It was tested in nearly 10,000 people, showing a significantly better sensitivity than FIT alone in predicting any stage of CRC (92.3 vs. 73.8 %, respectively) and advanced adenomas (42.2 vs. 23.8 %), and retaining a high specificity (89.9 vs. 96.4 %).
A similar combined stool test, ColoAlert, to be used in combination with FIT, was developed by Mainz Biomed N.V., and in the study with 734 patients the performance of ColoAlert was like that of ColoGuard with 85 % sensitivity and 92 % specificity for CRC. The ColoAlert test measures mutant KRAS, mutant BRAF and total human stool DNA in addition to FIT.
The availability of newly developed multitarget tests as well as combining them with signature analyses from microbiota, is a milestone in the development of new tests that have been shown to detect many precancers and early cancers in the colon, says Professor Dollinger. The combination of signatures from genetic alterations (early warning sign) with invisible blood (FIT, normally a later sign) gives these tests a performance characteristic that are close to that of a regular colonoscopy.
Considered that most people in the US prefer a multitarget DNA/FIT test compared to FIT alone or colonoscopy, Professor Dollinger claims that this type of multitargeted test shows the greatest promise in the future to reduce the many unnecessary deaths from colorectal cancer. The multitarget ColoAlert stool test could have in the future the potential, to be used as a triage to determine who will need a colonoscopy. This approach could reduce potentially the number of colonoscopies needed to find a precancer or a cancer in the colon.
Since persons with aberrant DNA signatures or blood in the stool are at high risk, there is also a need for improving the colonoscopy procedures. Using a flexible time to be able to detect all polyps as well as the emerging promise of artificial intelligence, will also be important contributors to reducing numbers of unnecessary CRC deaths, says he.
Professor Dollinger’s claims are supported by others that KRAS and BRAF mutations are early risk factor of developing CRC. Early polyps can be missed in colonoscopy, even if KRAS mutations are already detectable in tissue. Age increases prevalence of KRAS and BRAF mutations, also in normal mucosa.
This recent knowledge of genetic changes during colorectal carcinogenesis as well as the progress in analyzing these signatures in stool makes it possible to detect tumor DNA. Thus, the ColoAlert test effectively minimizes the analytical gap between the colonoscopy and other stool tests says professor Dollinger. Another important take home message from these studies is that patients with negative colonoscopies but positive mutational status should be controlled more closely and follow upped in a structured manner.
 Dollinger, M., Behl, S. and Fleig, W.E.: Early Detection of Colorectal Cancer: A Multi-Center Pre-Clinical Case Cohort Study for Validation of a Combined DNA Stool Test. Clin Lab 2018 Oct;64(10):1719-1730.
 Global Cancer Observatory. Online. Last accessed on September 21, 2022.
 Imperiale, T.F., Levin, T.R., Lidgard, G.P., Itzkowitz, S.H., Ahlquist, D.A., Ransohoff, D.F., Lavin, P. and Berger, B.M.: Multitarget stool DNA testing for colorectal-cancer screening. N. Engl. J. Med. 2014, 370, 1287–1297.
 Molina-Cerrillo, J., San Román, M., Pozas, J., Alonso-Gordoa, T., Pozas, M., Conde, E., Rosas, M., Grande, E., García-Bermejo, M.L. and Carrato, A.: BRAF Mutated Colorectal Cancer: New Treatment Approaches. Cancers (Basel) 2020 Jun; 12(6): 1571.
 Herring E, Tremblay É, McFadden N, Kanaoka S, Beaulieu J-F. Multitarget Stool mRNA Test for Detecting Colorectal Cancer Lesions Including Advanced Adenomas. Cancers. 2021; 13(6):1228.
 Makaroff, K.E., Shergill, J., Lauzon, M., Ahluwalia, S.C., Spiegel, B.M.R. and Almario, C.V.: Patient Preferences for Colorectal Cancer Screening Tests in Light of Lowering the Screening Age to 45 Years. Clinical Gastroenterology and Hepatology 2022, in press.
 Margetis, N., Kouloukoussa, M., Pavlou, K., Vrakas, S. and Mariolis-Sapsakos, T.: K-ras Mutations as the Earliest Driving Force in a Subset of Colorectal Carcinomas. In Vivo 2017 Jul-Aug;31(4):527-542.