Non-Invasive Blood and Stool CRC Screening Tests: Available Modalities and Their Clinical Application
Introduction
Colorectal cancer (CRC) screening significantly reduces CRC incidence and mortality, but only 65% of eligible individuals report being up-to-date with screening.1 Colonoscopy is the most widely used opportunistic screening method in the United States and is associated with many barriers to uptake. Providing patients a choice of colonoscopy and/or stool-based tests, improves screening adherence in randomized controlled trials.2,3 Non-invasive screening options have expanded from stool occult blood and multi-target DNA tests, to multi-target stool RNA tests, and novel blood-based tests, the latter only U.S. Food and Drug Administration (FDA) approved for patients who refuse colonoscopy and stool-based tests.
Stool Occult Blood Tests
Guaiac-based fecal occult blood testing (gFOBT) significantly reduces CRC mortality by 33%-35% when implemented on an annual or biennial basis.4,5 Fecal immunochemical testing (FIT) has supplanted gFOBT with advantages including independence from dietary restriction and medication-related interference, use of antibodies specific to human globin, and the need for only a single stool sample.
The most common threshold for a positive FIT in the U.S. is ≥ 20 micrograms (μg) of hemoglobin per gram (g) of stool. FIT is approved by the FDA as a qualitative positive or negative result based on a threshold value.6 A meta-analysis summarized test characteristics of commercially available FITs at various detection thresholds.7 The CRC sensitivity and specificity was 75% and 95% for ≥ 20 ug hemoglobin/g stool, and 91% and 90% for 10 ug hemoglobin/g stool, respectively. The sensitivity for advanced adenomas ranged from 25% at 20 μg/g to 40% at a 10 μg/g. Programmatic use of FIT in adults ages ≥ 50 years at 20 ug/g of stool, in cohort and case control studies, has been shown to significantly reduce CRC mortality by 33%-40% and advanced stage CRC by 34%.8,9
Over 57,000 average-risk individuals ages 50–69 years were randomized to biennial FIT or one-time colonoscopy and followed for 10 years.10 CRC mortality and incidence was similar between the groups: 0.22% with FIT vs. 0.24% with colonoscopy and 1.13% with FIT vs. 1.22% with colonoscopy, respectively. Thus, confirming biennial FIT screening is non-inferior to one-time colonoscopy in important CRC-related outcomes.
Multi-Target Stool Tests
Two multitarget stool DNA tests (mt-sDNA) known as Cologuard™ and Cologuard Plus™ have been approved by the FDA. Both tests include a FIT (with a positivity threshold of 20 μg hemoglobin per gram of stool) combined with DNA methylation markers. The test result is qualitative, reported as a positive or negative. Cologuard™ markers include methylated BMP3, NDRG4, and mutant KRAS while Cologuard Plus™ assesses methylated LASS4, LRRC4, and PPP2R5C. The respective mt-sDNA tests were studied in 9989 of 12,776 and 20,176 of 26,758 average-risk individuals undergoing colonoscopy and the results were compared to a commercially available FIT (with a positivity threshold of 20 μg hemoglobin/gram of stool).11,12 In both trials, the sensitivity for CRC and advanced precancerous lesions was higher with the mt-sDNA tests compared to FIT but had a significantly lower specificity for advanced precancerous lesions versus FIT (see Table 1). An age-related decline in specificity was noted in both trials with mt-sDNA, a trend not observed with FIT. This reduction may be attributed to age-related DNA methylation.
Multi-Target Stool RNA Test
A multi-target stool RNA test (mt-sRNA) commercially available as ColoSense™ is FDA-approved. It combines FIT (at a positivity threshold of 20 μg hemoglobin/gram of stool) with RNA-based stool markers. The combined results of the RNA markers, FIT, and smoking status provide a qualitative single test result. In the trial, 8,920 adults aged ≥45 underwent the mt-sRNA test and FIT followed by colonoscopy (13). The mt-sRNA showed higher sensitivity for CRC than FIT (94.4% versus 77.8%) and advanced adenomas (45.9% versus 28.9%) but lower CRC specificity (84.7% vs 94.7%) (Table 1). Unlike mt-sDNA-based tests, mt-sRNA showed consistent performance across age groups, addressing concerns about age-related declines in specificity attributed to DNA methylation.
Blood-Based Tests
In 2014, the first blood-based (BBT) CRC screening test known as Epi proColon™ was FDA but not Centers for Medicare & Medicaid Services (CMS) approved for average-risk adults ≥50 years of age who are offered and refused other U.S Preventive Services Task Force (USPSTF) endorsed CRC screening tests. It is a qualitative test for detection of circulating methylated Septin 9 (mSeptin9). The accuracy of mSeptin9 to detect CRC was assessed in a subset of 7941 asymptomatic average risk adults undergoing screening colonoscopy.14 The sensitivity and specificity for CRC were 48% and 91.5%, respectively. The sensitivity for advanced adenomas was 11.2%. An increase in sensitivity to 63.9% and reduction in specificity to 88.4% for CRC was demonstrated in a sub-analysis of available samples where an additional (third) polymerase chain replicate was performed. Epi proColon™ is not currently reimbursed by Medicare and not endorsed in the latest USPSTF guidelines.
Technologic advancements have improved the detection of circulating tumor markers in the blood. The Shield™ BBT approved by the FDA in 2024 for average risk adults ≥ 45 years integrates three types of cfDNA data (epigenetic changes resulting in the aberrant methylation or fragmentation patterns, and genomic changes resulting in somatic mutations) into a positive or negative test result. In the trial, 22,877 average-risk, asymptomatic individuals ages 45–84 were enrolled and clinical validation was performed in 7,861 of the participants.15 The sensitivity for CRC was 83.1% which decreased to 55% for stage I tumors (see Table 1). CRC specificity was 89.6% and the sensitivity for advanced adenomas and large sessile serrated lesions was 13.2%.
Another BBT SimpleScreen™, which is not yet FDA-approved, analyzed circulating, cell-free DNA methylation patterns in 27,010 evaluable average-risk, asymptomatic adults ages 45–85 years undergoing screening colonoscopy.16 The sensitivity and specificity for CRC was 79.2% and 91.5%, respectively. Similar to Shield, the sensitivity for stage I CRC was low at 57.1%. The sensitivity for advanced precancerous lesions, a secondary endpoint, was 12.5% which did not meet the prespecified study criteria.
Effectiveness and Cost Effectiveness
Modeling studies have evaluated novel noninvasive CRC screening tests compared to FIT and colonoscopy.17-20 One compared a hypothetical BBT performed every 3 years that meets the minimum CMS threshold CRC sensitivity and specificity of 74% and 90%, respectively, to other established CRC screening tests beginning at age 45.17 Every 3-year BBT reduced CRC incidence and mortality by 40% and 52%, respectively compared to no screening. However, the reductions were much lower than yearly FIT (72% and 76%, respectively), every 10 year colonoscopy (79% and 81%, respectively), and triennial mt-sDNA (68% and 73%, respectively). The BBT resulted in fewer quality-adjusted life-years per person compared to the alternatives.
Additionally, FIT, colonoscopy, and mt-sDNA were less costly and more effective. Advanced precancerous lesion detection was a key measure for a test’s effectiveness. BBT characteristics would require a CRC sensitivity and specificity of >90% and 90%, respectively, and 80% sensitivity for advanced precancerous lesions at a cost of ≤$120–$140 to be cost-effective compared to FIT at comparable participation rates.
Another analysis simulated colorectal neoplasia progression and compared clinical effectiveness and cost between annual FIT, every 3 year stool mt-sRNA, every 3 year stool mt-sDNA tests, every 3 year stool Shield™; these outcomes were compared to colonoscopy every 10 years and no screening in adults ≥ age 45 over different adherence rates.19 At real-world adherence rates of 60%, colonoscopy prevented most CRC cases and associated deaths. FIT was the most cost-effective strategy at all adherence levels. Between the multi-target stool tests and Shield™, mt-sRNA was the most cost-effective. Compared to FIT, mt-sRNA reduced CRC cases and deaths by 1% and 14%.
The third study evaluated CRC incidence and mortality, quality-adjusted life-years and costs with annual FIT, colonoscopy every 10 years, mt- sDNA tests, mt-sRNA test, and BBTs.20 The latest mt-sDNA (Colguard plus™) and mt-sRNA achieved benefits approaching FIT but the Shield™ test was substantially less effective. The authors hypothesized that if 15% of the population substituted Shield™ for current effective CRC screening strategies, an increase in CRC deaths would occur and require 9-10% of the unscreened population to uptake screening with Shield to avert the increases in CRC deaths due to the substitution effect.
Clinical Implications
The effectiveness of non-invasive screening strategies depends on their diagnostic performance, adherence, and ensuring a timely colonoscopy after a positive test. Two claims-based studies found 47.9% and 49% of patients underwent follow-up colonoscopy within 6 months of an abnormal stool or BBT CRC screening test, respectively.21-22
Conclusions
Non-invasive stool mt-sDNA and mt-sRNA have higher effectiveness than the new BBTs. BBTs can lead to increased CRC mortality if substituted for the FDA and CMS-approved, USPSTF-endorsed, CRC screening modalities. If future BBTs increase their sensitivity for CRC (including early-stage CRC) and advanced precancerous lesions and decrease their cost, they may prove to have similar cost-effectiveness to stool-based tests. Currently, BBTs are not a substitute for colonoscopy or other stool tests and should be offered to patients who refuse other CRC screening modalities. A personalized, risk-adapted approach, paired with improved adherence and follow-up are essential to optimize the population-level impact of CRC screening and ensure equitable, effective cancer prevention.
Dr. Gupta is based at the Division of Gastroenterology and Hepatology, Department of Medicine, University of Maryland School of Medicine, Baltimore. Dr. Burke and Dr. Macaron are based at the Department of Gastroenterology, Hepatology, and Nutrition, Cleveland Clinic, Cleveland, Ohio. Dr. Gupta and Dr. Macaron declared no conflicts of interest in regard to this article. Dr. Burke declared research support from Emtora Biosciences. She is a current consultant for Lumabridge, and has been a consultant for Sebela and Almirall. She also disclosed support from Myriad, Genzyme, Ferring, Merck, Sharp and Dohme, Abbvie, Salix, and Natera.
References
1. Benavidez GA, Sedani AE, Felder TM, Asare M, Rogers CR. Rural-urban disparities and trends in cancer screening: an analysis of Behavioral Risk Factor Surveillance System data (2018-2022). JNCI Cancer Spectr. 2024 Nov 1;8(6):pkae113
2. Galoosian A, Dai H, Croymans D, et al. Population Health Colorectal Cancer Screening Strategies in Adults Aged 45 to 49 Years: A Randomized Clinical Trial. JAMA. 2025 Aug 4:e2512049. doi: 10.1001/jama.2025.12049. Epub ahead of print.
3. Pilonis ND, Bugajski M, Wieszczy P, et al. Participation in Competing Strategies for Colorectal Cancer Screening: A Randomized Health Services Study (PICCOLINO Study). Gastroenterology. 2021 Mar;160(4):1097-1105.
4. Shaukat A, Mongin SJ, Geisser MS, et al. Long-term mortality after screening for colorectal cancer. N Engl J Med. 2013;369(12):1106–1114.
5. Kronborg O, Fenger C, Olsen J, Jørgensen OD, Søndergaard O. Randomised study of screening for colorectal cancer with faecal-occult-blood test. Lancet. 1996 Nov 30;348(9040):1467-71. doi: 10.1016/S0140-6736(96)03430-7. PMID: 8942774.
6. Burke CA, Lieberman D, Feuerstein JD. AGA Clinical Practice Update on Approach to the Use of Noninvasive Colorectal Cancer Screening Options: Commentary. Gastroenterology. 2022 Mar;162(3):952-956. doi: 10.1053/j.gastro.2021.09.075. Epub 2022 Jan 28. PMID: 35094786.
7. Imperiale TF, Gruber RN Stump TE, et al. Performance characteristics of fecal immunochemical tests for colorectal cancer and advanced adenomatous polyps: a systematic review and meta-analysis. Ann Intern Med 2019; 170(5):319-329
8. Doubeni CA, Corley DA, Jensen CD, et al. Fecal Immunochemical Test Screening and Risk of Colorectal Cancer Death. JAMA Netw Open. 2024 Jul 1;7(7):e2423671. doi: 10.1001/jamanetworkopen.2024.23671.
9. Chiu HM, Jen GH, Wang YW, et al. Long-term effectiveness of faecal immunochemical test screening for proximal and distal colorectal cancers. Gut. 2021 Dec;70(12):2321-2329. doi: 10.1136/gutjnl-2020-322545. Epub 2021 Jan 25.
10. Castells A, Quintero E, Bujanda L, et al; COLONPREV study investigators. Effect of invitation to colonoscopy versus fecal immunochemical test screening on colorectal cancer mortality (COLONPREV): a pragmatic, randomised, controlled, non-inferiority trial. Lancet. 2025;405(10486):1231–1239
11. Imperiale TF, Ransohoff DF, Itzkowitz SH, et al. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med. 2014;370(14):1287-1297
12. Imperiale TF, Porter K, Zella J, et al. Next-Generation Multitarget Stool DNA Test for Colorectal Cancer Screening. N Engl J Med. 2024 Mar 14;390(11):984-993
13. Barnell EK, Wurtzler EM, La Rocca J, et al. Multitarget Stool RNA Test for Colorectal Cancer Screening. JAMA. 2023 Nov 14;330(18):1760-1768.
14. Church TR, Wandell M, Lofton-Day C, et al. Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer. Gut 2014; 63:317–325.
15. Chung DC, Gray DM 2nd, Singh H, et al. A Cell-free DNA Blood-Based Test for Colorectal Cancer Screening. N Engl J Med. 2024 Mar 14;390(11):973-983.
16. Shaukat A, Burke CA, Chan AT, et al. Clinical Validation of a Circulating Tumor DNA-Based Blood Test to Screen for Colorectal Cancer. JAMA. 2025 Jul 1;334(1):56-63.
17. Ladabaum U, Mannalithara A, Weng Y, et al. Comparative Effectiveness and Cost-Effectiveness of Colorectal Cancer Screening with Blood-Based Biomarkers (Liquid Biopsy) vs Fecal Tests or Colonoscopy. Gastroenterology. 2024 Jul;167(2):378-391.
18. van den Puttelaar R, Nascimento de Lima P, Knudsen AB, et al. Effectiveness and cost-effectiveness of colorectal cancer screening with a blood test that meets the Centers for Medicare & Medicaid Services coverage decision. Gastroenterology 2024;167:368–377.
19. Shaukat A, Levin TR, Liang PS. Cost-effectiveness of Novel Noninvasive Screening Tests for Colorectal Neoplasia. Clin Gastroenterol Hepatol. 2025 Jun 23:S1542-3565(25)00525-7. doi: 10.1016/j.cgh.2025.06.006. Epub ahead of print. PMID: 40562290.
20. Ladabaum U, Mannalithara A, Schoen RE, Dominitz JA, Lieberman D. Projected Impact and Cost-Effectiveness of Novel Molecular Blood-Based or Stool-Based Screening Tests for Colorectal Cancer. Ann Intern Med. 2024 Dec;177(12):1610-1620.
20. Ciemins EL, Mohl JT, Moreno CA, Colangelo F, Smith RA, Barton M. Development of a Follow-Up Measure to Ensure Complete Screening for Colorectal Cancer. JAMA Netw Open. 2024 Mar 4;7(3):e242693. doi: 10.1001/jamanetworkopen.2024.2693.
21. Zaki TA, Zhang NJ, Forbes SP, Raymond VM, Das AK, May FP. Colonoscopic Follow-up After Abnormal Blood-Based Colorectal Cancer Screening Results. Gastroenterology. 2025 Jul 21:S0016-5085(25)05775-0. doi: 10.1053/j.gastro.2025.07.019. Epub ahead of print. PMID: 40744392.