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Cited by 37MicroRNA in disease regulationColorectal Cancer Screening and DetectionMolecular Biology Techniques and Applications

Novel Approach to Fecal Occult Blood Testing by Assay of Erythrocyte-Specific microRNA Markers

Open access
Wu, Chung Wah, Cao, Xiaoming, Berger, Calise K., Foote, Patrick H., Mahoney, Douglas W., Simonson, Julie A., Anderson, Bradley W., Yab, Tracy C. +4 more·2017·Digestive Diseases and Sciences
DOI 10.1007/s10620-017-4627-6PMC PMC6021130
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Abstract

BackgroundFecal occult blood testing (FOBT) has historically relied on methods to detect hemoglobin with no fundamental innovations in decades.AimTo examine microRNA (miRNA) as a new marker class for FOBT.MethodsCandidate miRNA markers were identified by small RNA sequencing of human whole blood compared to colorectal epithelia. Markers were tested in human blood cell subsets and blood from non-human species. We assessed assay linearity in blood spiking and marker stability in stool over incubation experiments. Levels of candidate erythrocyte markers were explored in stools from colorectal cancer (CRC) cases and controls.ResultsBased on small RNA sequencing and validation RT-qPCR, expression level of each of the top blood-enriched markers (hsa-miR-144-3p, 144-5p, 451a, 486-5p, 363-3p, 20b-5p) could perfectly discriminate blood from colorectal epithelia. All six markers arose from and showed specificity to human erythrocytes. Marker levels increased linearly with erythrocyte concentration in saline or stool and demonstrated a broader dynamic range than did immunochemical test for hemoglobin. Degradation of markers occurred in stool but was reduced with preservative buffers. Erythrocyte marker candidates for stool testing were selected in an exploratory set of stools (20 CRC, 40 normal). Candidates were then further tested in a feasibility set (29 CRC, 31 advanced adenoma, and 115 normal); a miRNA panel (hsa-miR-451a, 144-5p, and 200b-3p as normalizer) yielded an AUC of 0.89 (95% CI 0.82–0.95, P < .0001) for CRC.ConclusionsA novel miRNA-based approach accurately quantifies fecal blood levels over a broad, clinically relevant range.

AI snapshot

Written from the paper’s full text.

TL;DR

This study identifies erythrocyte-specific microRNAs as a new marker class for fecal occult blood testing, with candidate markers showing linear quantification across clinically relevant blood concentrations. In an independent feasibility set, a three-miRNA panel detected colorectal cancer with an AUC of 0.89.

Key findings

  • Small RNA sequencing of 120 libraries found 19 blood-enriched miRNAs with AUC 1.0 for discriminating blood from colorectal epithelia; six had >20-fold blood-over-epithelia change: hsa-miR-144-3p, 451a, 144-5p, 20b-5p, 486-5p, and 363-3p. All six arose from erythrocytes with no cross-expression in white blood cell types or epithelia.
  • In saline spike-in dilutions, all six miRNAs were linearly associated with erythrocyte concentration (R² = 0.984–1.000, P < .0001 each), whereas FIT linearity was lower (R² = 0.899, P = .0039). In stool spike-ins across a clinical range, FIT and hsa-miR-363-3p, 451a, and 144-5p correlated highly with erythrocyte concentration (R² = 0.99–0.999, P < .0001 each).
  • In the feasibility set (29 CRC, 31 advanced adenoma, 115 normal), a panel using hsa-miR-451a, 144-5p, and 200b-3p as normalizer yielded an AUC of 0.89 (95% CI 0.82–0.95, P < .0001) for CRC; at 95% specificity, sensitivity was 66% for all cancers, 92% for distal cancers versus 48% for proximal cancers (P = .0084). Advanced adenoma detection was poor (AUC = 0.58, 95% CI 0.45–0.69, P = .244).
  • Erythrocyte miRNA markers degraded in stool over 14 days at room temperature, but degradation was reduced with preservative buffers; DNA/RNA Shield was most stable, and none returned to baseline even in non-preservative saline.

Methods

  • Prospective sequential study: marker discovery by small RNA sequencing of human whole blood (n = 25) and colorectal epithelia (normal n = 20, neoplastic n = 75), followed by RT-qPCR validation, blood cell subset origin testing, species specificity testing, spike-in linearity assessment, stool stability experiments, and blinded clinical stool studies.
  • Clinical stool testing used an exploratory set and an independent feasibility set. The exploratory set included 20 CRC, 20 pancreatic cancer disease controls, and 20 healthy controls per Methods, with results comparing CRC (n = 20) to normal controls (n = 40); the feasibility set included 29 CRC, 31 advanced adenomas, and 115 normal colonoscopy controls.
  • Stool miRNA levels were measured by RT-qPCR from EDTA-buffered stool, with hsa-miR-200b-3p as an epithelial normalizer. Analyses included Mann–Whitney tests, generalized additive modeling, and ROC AUC estimation.

Limitations

  • Stated: CRC cases in both cohorts were recruited from symptomatic patients in the referral setting, so performance may not represent a screening population. Sensitivity for advanced adenomas was relatively low, and the predominantly proximal location of advanced adenomas may have contributed. Definitive studies are needed for upper gastrointestinal bleeding, and formal comparisons with established FOBTs are premature; further work is needed to prove non-inferiority to FIT.
  • Stated: modeling was not performed in the exploratory set to avoid over-fitting in a small sample size.
  • Evident from the text: the clinical evaluation used small pilot cohorts, and the exploratory set had only 20 CRC cases; the paper reports no formal head-to-head FIT comparison in the feasibility set.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1007/s10620-017-4627-6

Cites

27 of 27 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

1 of 32 cited sources checked · 0 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    All six markers demonstrated no significant difference in expression across colorectal epithelial subgroups at tissue level.

    “All six markers demonstrated no significant difference in expression across colorectal epithelial subgroups at the tissue level.”
    Quote in the paperSubstantial claim
  • Findingown data

    Degradation of markers was reduced by variable degrees in different preservative buffers.

    “Degradation was reduced by variable degrees in different preservative buffers”
    Quote in the paperSubstantial claim
  • Findingown data

    Sensitivity for advanced adenomas was relatively much lower than for CRC in this study.

    “Sensitivity for advanced adenomas (conventional adenomas and SSAs combined) was relatively much lower than for CRC in this study.”
    Quote in the paperSubstantial claim
  • Findingown data

    None of the markers returned to baseline even in stools homogenized with non-preservative saline and incubated for 14 days at room temperature.

    “None of the markers returned to baseline even in stools homogenized with a non-preservative saline solution and incubated for 14 days at room temperature.”
    Quote in the paperSubstantial claim
  • Findingown data

    FIT and hsa-miR-363-3p, 451a, and 144-5p correlated highly with erythrocyte concentration in stool (R2=0.99-0.999, P<.0001 for each).

    “FIT and hsa-miR-363-3p, 451a, and 144-5p correlated highly with erythrocyte concentration (R2 = 0.99–0.999 levels, P < .0001 for each).”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 23
  • May combine two claims. 21

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    hsa-miR-144-3p/5p and hsa-miR-451a are members of the hsa-miR-144/451 cluster located on chromosome 11.

    “It is known that both hsa-miR-144-3p/5p and hsa-miR-451a are members of the hsa-miR-144/451 cluster located on chromosome 11.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [24] Dore LC, Amigo JD, Dos Santos CO, et al. A GATA-1-regulated microRNA locus essential for erythropoiesis. Proc Natl Acad Sci USA. 2008;105:3333–3338. source
    • cites [25] Rasmussen KD, Simmini S, Abreu-Goodger C, et al. The miR-144/451 locus is required for erythroid homeostasis. J Exp Med. 2010;207:1351–1358. source

      The Results and Discussion section identifies miR-144 and miR-451 as members of the miR-144/451 cluster. The passages in our copy do not establish that the human cluster is on chromosome 11 or identify the specific hsa-miR-144-3p/5p and hsa-miR-451a forms. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Several miRNA markers were highly discriminant for colorectal epithelium and absent in whole blood.

    “Several miRNA markers were identified as highly discriminant for colorectal epithelium and absent in whole blood.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Top epithelial markers achieved AUC of 1.0 with >1000-fold differential expression over blood.

    “Top epithelial markers achieved AUC of 1.0 with >1000-fold differential expression over blood.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cross-reactivity was observed with hsa-miR-451a in blood from pig and with hsa-miR-486-5p in blood from sheep.

    “Cross-reactivity was observed with hsa-miR-451a in blood from pig and with hsa-miR-486-5p in blood from sheep.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    Based on complete blood count, serial dilutions yielded calculated concentrations ranging from 4 to 3.6 × 10^5 erythrocytes per μL solution.

    “Based on complete blood count, serial dilutions yielded calculated concentrations ranging from 4 to 3.6 × 105 erythrocytes per µL solution.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At spike-in concentration of 25 μL blood per gram stool, erythrocyte miRNA markers were least stable in absence of preservative and most stable in DNA/RNA Shield.

    “At a spike-in concentration of 25 µL blood per gram of stool, erythrocyte miRNAs markers were least stable in the absence of preservative and most stable in DNA/RNA Shield.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    These top six markers each retained high discrimination in validation by RT-qPCR, and their expression in blood exceeded that in tissue by 119- to 3988-fold.

    “These top six markers each retained high discrimination in validation by RT-qPCR, and their expression in blood exceeded that in tissue by 119- to 3988-fold.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The degree of degradation varied by individual miRNA and could be reduced using preservative buffers.

    “The degree of such degradation varied by individual miRNA and could be reduced using preservative buffers.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    hsa-miR-144-3p, 486-5p, and 20b-5p correlated with erythrocyte concentration in stool at R2=0.757 (P=.0242), 0.952 (P=.0009), and 0.973 (P=.0003) respectively.

    “hsa-miR-144-3p, 486-5p, and 20b-5p correlated with erythrocyte concentration at R2 = 0.757 (P = .0242), 0.952 (P = .0009), and 0.973 (P = .0003) levels, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Nineteen miRNAs had average read counts >50 in blood and demonstrated perfect discrimination between blood and epithelia with AUCs of 1.0 at P<.0001.

    “Nineteen miRNAs had average read counts above 50 in blood and demonstrated perfect discrimination between blood and epithelia with AUCs of 1.0 at P values <.0001”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All six markers arose from erythrocytes with no cross-expression in white blood cell types or in epithelia.

    “All six markers were found to have arisen from erythrocytes with no cross-expression in white blood cell types or in epithelia”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Samples with cel-miR-39-3p recovery >20% were determined valid for analysis.

    “Samples with cel-miR-39-3p recovery >20% were determined valid for analysis.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    An effort was made to balance age and gender within each clinical set.

    “An effort was made to balance age and gender within each set.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Combining FIT with molecular stool markers compensates for intermittency or absence of bleeding from colorectal neoplasms while exploiting complementary value of FIT.

    “Such a combination compensates for the intermittency or absence of bleeding from colorectal neoplasms [12, 13], while exploiting the complementary value of FIT.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [12] Ahlquist DA, McGill DB, Fleming JL, et al. Patterns of occult bleeding in asymptomatic colorectal cancer. Cancer. 1989;63:1826–1830. source

      Not checked in the free run.

    • cites [13] Heigh RI, Yab TC, Taylor WR, et al. Detection of colorectal serrated polyps by stool DNA testing: comparison with fecal immunochemical testing for occult blood (FIT). PLoS ONE. 2014;9:e85659. source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Interpretationreasoning

    The panel was more sensitive to distal lesions than proximal lesions, potentially due to lesser extent of bleeding with proximal lesions and degradation of analyte during luminal transit.

    “The panel was more sensitive to distal lesions than proximal lesions, potentially due to the lesser extent of bleeding with proximal lesions and degradation of analyte during luminal transit, a phenomenon that is also seen with stool assays for hemoglobin [9].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [9] Imperiale TF, Ransohoff DF, Itzkowitz SH, et al. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med. 2014;370:1287–1297. source

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    There has been no fundamental innovation around the targeted biomarkers for FOBT in recent decades.

    “While technical refinements with conventional FOBTs continue, there has been no fundamental innovation around the targeted biomarkers themselves in recent decades.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    FOBT approaches have historically relied on detecting components of hemoglobin.

    “Historically, FOBT approaches have relied on detecting components of hemoglobin, each with distinct advantages and limitations [3].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [3] Ahlquist DA, Young GP. Approach to the patient with occult gastrointestinal bleeding. In: Yamada T, Alpers DH, eds. Principles of clinical gastroenterology. New York: Wiley; 2008:152–169. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The predominantly proximal location of advanced adenomas in our study might have also contributed to the low marker sensitivity.

    “The predominantly proximal location of advanced adenomas in our study might have also contributed to the low marker sensitivity.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    A molecular-based FOBT has potential to simplify patient experience by avoiding separate stool sampling and reduce material costs of the multi-target stool test.

    “A molecular-based FOBT has potential to simplify the patient experience by avoiding a separate stool sampling and also reduce material costs of the multi-target stool test.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The FIT component of the multi-target stool DNA test requires separate stool sampling by the patient because the EDTA stabilizing buffer for DNA interferes with the protein-based FIT assay and an assay platform different from that used for the DNA markers.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Samples had gone through no more than one previous freeze-thaw cycle to minimize variation.

    “To minimize variation in sample quality due to potential freeze–thaw artifact, samples had gone through no more than one previous freeze–thaw cycle.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Mature hsa-miR-144-3p and hsa-miR-451a regulate erythropoiesis.

    “Mature hsa-miR-144-3p (major product of precursor hsa-miR-144) and hsa-miR-451a regulate erythropoiesis.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [24] Dore LC, Amigo JD, Dos Santos CO, et al. A GATA-1-regulated microRNA locus essential for erythropoiesis. Proc Natl Acad Sci USA. 2008;105:3333–3338. source
    • cites [25] Rasmussen KD, Simmini S, Abreu-Goodger C, et al. The miR-144/451 locus is required for erythroid homeostasis. J Exp Med. 2010;207:1351–1358. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    All pathologies were reviewed by expert gastrointestinal pathologists.

    “All pathologies were reviewed by expert gastrointestinal pathologists.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    CRC cases in both cohorts had been recruited from symptomatic patients in referral setting, and this early assessment may not be representative of performance in screening population.

    “It is important to note that CRC cases in both cohorts had been recruited from symptomatic patients in the referral setting, and this early assessment of miRNA marker discrimination in stool may not be representative of performance in a screening population.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Transcription of this locus is regulated by hematopoietic transcription factor GATA-1.

    “Transcription of this locus is regulated by hematopoietic transcription factor GATA-1 [24, 25].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [24] Dore LC, Amigo JD, Dos Santos CO, et al. A GATA-1-regulated microRNA locus essential for erythropoiesis. Proc Natl Acad Sci USA. 2008;105:3333–3338. source
    • cites [25] Rasmussen KD, Simmini S, Abreu-Goodger C, et al. The miR-144/451 locus is required for erythroid homeostasis. J Exp Med. 2010;207:1351–1358. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisereasoning

    hsa-miR-144-5p, being the minor product, is not known to play important roles in erythrocytes.

    “hsa-miR-144-5p, being the minor product, is not known to play important roles in erythrocytes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    In both sample sets, all stool samples met criteria of >1 μg extracted total RNA and >20% external control recovery.

    “In both sample sets, all stool samples had met the criteria of above 1 µg in extracted total RNA amount and above 20% external control recovery efficiency.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    A multi-target stool test comprising DNA markers and FIT has been approved by FDA for CRC screening, incorporated into guidelines, and become available.

    “A multi-target stool test, comprising a panel of DNA markers and FIT [9–11], has been approved by the Food and Drug Administration (FDA) for CRC screening, been incorporated into CRC screening guidelines by the American Cancer Society and United States Preventive Services Task Force [14, 15], and become available to patients.”
    Quote in the paperSubstantial claim0 of 5 sources checked
    • cites [9] Imperiale TF, Ransohoff DF, Itzkowitz SH, et al. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med. 2014;370:1287–1297. source
    • cites [10] Lidgard GP, Domanico MJ, Bruinsma JJ, et al. Clinical performance of an automated stool DNA assay for detection of colorectal neoplasia. Clin Gastroenterol Hepatol. 2013;11:1313–1318. source
    • cites [11] Redwood DG, Asay ED, Blake ID, et al. Stool DNA testing for screening detection of colorectal neoplasia in Alaska Native people. Mayo Clin Proc. 2016;91:61–70. source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [14] Bibbins-Domingo K, Grossman DC, Curry SJ, et al. Screening for colorectal cancer: US preventive services task force recommendation statement. JAMA. 2016;315:2564–2575. source
    • cites [15] El-Shami K, Oeffinger KC, Erb NL, et al. American cancer society colorectal cancer survivorship care guidelines. CA Cancer J Clin. 2015;65:428–455. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Samples with less than 1 μg extracted total RNA were excluded from further analysis.

    “Samples with less than 1 µg extracted total RNA could have insufficient analyte and were not further analyzed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Age and gender were balanced between blood and colorectal epithelia groups in small RNA sequencing.

    “Age and gender were balanced between groups.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    High abundance of miRNAs in erythrocytes and ability to quantify them by RT-qPCR render miRNAs as rational and appealing candidate class of markers for occult blood.

    “Although depleted of DNA and large-sized RNAs, the high abundance of miRNAs in erythrocytes and the ability to quantify them by RT-qPCR approaches render miRNAs as rational and appealing candidate class of markers for occult blood.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    FIT is human-specific, not affected by dietary artifact, and detects primarily lower intestinal bleeding.

    “FIT has been increasingly the FOBT of choice for CRC screening, as it is human-specific, not affected by dietary artifact, and detects primarily lower intestinal bleeding [3, 6, 7].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [3] Ahlquist DA, Young GP. Approach to the patient with occult gastrointestinal bleeding. In: Yamada T, Alpers DH, eds. Principles of clinical gastroenterology. New York: Wiley; 2008:152–169. source
    • cites [6] Harewood GC, McConnell JP, Harrington JJ, Mahoney DW, Ahlquist DA. Detection of occult upper gastrointestinal tract bleeding: performance differences in fecal occult blood tests. Mayo Clin Proc. 2002;77:23–28. source
    • cites [7] Rockey DC, Auslander A, Greenberg PD. Detection of upper gastrointestinal blood with fecal occult blood tests. Am J Gastroenterol. 1999;94:344–350. source

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    miRNA that indicates presence of blood could be assayed from stool by a PCR-based platform without modification of collection device or FDA-approved nucleic acid buffer.

    “Conceptually, miRNA that indicates the presence of blood could be assayed from stool by a PCR-based platform, without modification of collection device or FDA-approved nucleic acid buffer.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    For any combination of two group comparisons, minimum detectable AUC from null 0.5 is 0.69 assuming group sizes of 30 or more and 80% power with one-sided .05.

    “For any combination of two group comparisons, the minimum detectable AUC from the null value of 0.5 is 0.69 assuming group sizes of 30 or more and 80% power with a one-sided test of significance of .05.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    Observed detection rates for advanced precancers were lower than those reported for FIT with advanced conventional adenomas and similar to those reported with SSAs.

    “observed detection rates for these advanced precancers were lower than those reported for FIT with advanced conventional adenomas [9, 11, 26] and similar to those reported with SSAs [9, 11, 13].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [9] Imperiale TF, Ransohoff DF, Itzkowitz SH, et al. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med. 2014;370:1287–1297. source
    • cites [11] Redwood DG, Asay ED, Blake ID, et al. Stool DNA testing for screening detection of colorectal neoplasia in Alaska Native people. Mayo Clin Proc. 2016;91:61–70. source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [26] Park DI, Ryu S, Kim YH, et al. Comparison of guaiac-based and quantitative immunochemical fecal occult blood testing in a population at average risk undergoing colorectal cancer screening. Am J Gastroenterol. 2010;105:2017–2025. source
    • cites [13] Heigh RI, Yab TC, Taylor WR, et al. Detection of colorectal serrated polyps by stool DNA testing: comparison with fecal immunochemical testing for occult blood (FIT). PLoS ONE. 2014;9:e85659. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim.

    Premisecitation

    The FDA-approved multi-target stool DNA test combines FIT and a DNA marker panel.

    “The FDA-approved multi-target stool DNA test combines FIT and a DNA marker panel [9, 11].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [9] Imperiale TF, Ransohoff DF, Itzkowitz SH, et al. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med. 2014;370:1287–1297. source
    • cites [11] Redwood DG, Asay ED, Blake ID, et al. Stool DNA testing for screening detection of colorectal neoplasia in Alaska Native people. Mayo Clin Proc. 2016;91:61–70. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Modeling was not performed on exploratory set to avoid over-fitting.

    “It should be noted that this modeling was not performed to exploratory set to avoid over-fitting of data in a small sample size.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    miRNA expression was normalized to hsa-miR-30e-3p, which was identified in small RNA sequencing as most consistent with global mean of all miRNA reads.

    “miRNA expression was calculated by comparative Cq method normalized to hsa-miR-30e-3p, which, in small RNA sequencing, was identified to be most consistent to the global mean of all miRNA reads.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    This would obviate need for separate stool sampling, simplify patient experience, and reduce material costs.

    “This would obviate the need for separate stool sampling, simplify the patient experience, and reduce material costs of the stool collection kit and assay process.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    RT-qPCR on each clinical stool sample was performed once by an investigator blinded to clinical information.

    “RT-qPCR on each clinical stool sample was performed once, by investigator who was blinded to samples’ clinical information.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The study was approved by Mayo Clinic IRB.

    “The study was approved by the Mayo Clinic Internal Review Board.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Candidate markers are generally human-specific and are stable in stool with use of preservation buffers.

    “Candidate markers are generally human-specific and are stable in stool with use of preservation buffers.”
    Quote in the paperSubstantial claim
41 of 41

  • The paper relies on this without support.

    The FIT component of the multi-target stool DNA test requires separate stool sampling by the patient because the EDTA stabilizing buffer for DNA interferes with the protein-based FIT assay and an assay platform different from that used for the DNA markers.

    “The FIT component of this multi-target test currently requires separate stool sampling by the patient, as the EDTA stabilizing buffer for DNA interferes with the protein-based FIT assay, and an assay platform different from that used for the DNA markers.”
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Authors
Wu, C. W., Cao, X., Berger, C. K., Foote, P. H., Mahoney, D. W., Simonson, J. A., Anderson, B. W., Yab, T. C., Taylor, W. R., Boardman, L. A., Kisiel, J. B., & Ahlquist, D. A.
Affiliations
Mayo Clinic (US)
Corresponding author
Ahlquist, David A.
ORCID
Venue
Digestive Diseases and Sciences
Publisher
Springer Science and Business Media LLC
Published
2017-06-28
Volume
62
Issue
8
Pages
1985-1994
ISSN
  • 0163-2116 (ISSN-L)
  • 1573-2568
DOI
10.1007/s10620-017-4627-6
PMC
PMC6021130
OpenAlex
W2728396909
Semantic Scholar
2f5f14be961fde4fce9804db11a5ee4290d44992
Cited by
37
References
27
Citations by year
2026: 1 · 2025: 4 · 2024: 5 · 2023: 5 · 2022: 5 · 2021: 4 · 2020: 6 · 2019: 4 · 2018: 1 · 2017: 2
FWCI
1.21
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (unspecified)
Locations
  • Digestive Diseases and Sciences (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • Mayo Clinic
  • Exact Sciences
  • Clinical Core of Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Health Sciences › Medicine › Oncology › Colorectal Cancer Screening and Detection
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
Keywords
fecal occult blood test, microRNA biomarkers, colorectal cancer detection, colorectal adenoma, small RNA sequencing
MeSH terms
Erythrocytes, Feasibility Studies, Feces, Humans, Intestinal Mucosa, Occult Blood, Prospective Studies, Sensitivity and Specificity, Biomarkers, Tumor, Colorectal Neoplasms, Case-Control Studies, Sequence Analysis, RNA, MicroRNAs, Real-Time Polymerase Chain Reaction

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-06.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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