Mary E. Devens

Fecal calprotectin levels predict colorectal inflammation among patients with chronic diarrhea referred for colonoscopy

Fecal calprotectin levels predict colorectal inflammation among patients with chronic diarrhea referred for colonoscopy

Prospective evaluation of fecal calprotectin as a screening biomarker for colorectal neoplasia.

OBJECTIVES:Stool testing is a well established method of screening for colorectal neoplasia. Emerging data suggest that novel biomarkers may offer performance advantages over fecal occult blood. In this large, prospective study, we assessed fecal calprotectin (a leukocyte-derived protein) as a screening biomarker for colorectal neoplasia. Fecal calprotectin was directly compared to fecal hemoglobin (Hb) and colonoscopy as the existing criterion standards for stool screening and structural evaluation, respectively.METHODS:Subjects included colonoscopy patients with a personal history of colorectal neoplasia, family history of colorectal cancer, or iron deficiency anemia. Stool specimens were collected before purgation, processed appropriately, and quantitatively analyzed for calprotectin (Nycomed Pharma, Oslo, Norway) and for Hb (Mayo Medical Laboratories, Rochester, MN) by masked technicians. Colonoscopies were performed by experienced endoscopists without prior knowledge of the fecal assay results.RESULTS:Among 412 subjects, 97 (24%) subjects had one or more colorectal neoplasms (including three with adenocarcinomas). Fecal calprotectin levels did not differ significantly between subjects with versus subjects without colorectal neoplasms (p= 0.33). Neither tumor number (p= 0.85) nor tumor size (p= 0.86) significantly influenced the observed fecal calprotectin concentrations. Estimates of the sensitivity, specificity, and positive and negative predictive values of fecal calprotectin for any colorectal neoplasms were 37%, 63%, 23%, and 76%, respectively. Comparable performance estimates for fecal Hb were 3%, 97%, 27%, and 77%, respectively.CONCLUSIONS:In this cohort of colonoscopy patients at above average risk, fecal calprotectin was a poor screening biomarker for colorectal neoplasia. Further investigation of tumor-derived, rather than blood-based, biomarkers may be a more rewarding approach to stool screening for colorectal neoplasia.

Endoscopic overestimation of colorectal polyp size

BACKGROUND AND AIMS Polyp size ≥ 1 cm triggers more frequent colonoscopic surveillance, yet size is typically based on subjective endoscopic estimates. We sought to compare contemporary assessments of polyp size by endoscopic estimation and pathology measurement. METHODS Colonoscopy and pathology reports were reviewed from the 2012 medical records at a large institution. Only polyps resected in toto with both endoscopic estimates and pathology measurements were included. Pathology measurements were considered the criterion standard. Factors affecting endoscopic miscall rates were assessed by multivariate analyses. RESULTS From 6067 polyps resected, both endoscopic and pathology sizes were available on 1528. Distribution of polyp size appraised by endoscopy but not by pathology revealed modal clustering, particularly around 1 cm. Among 99 polyps endoscopically called 1 cm, 72% were <1 cm on pathology. Of all 222 polyps estimated as ≥ 1 cm on endoscopy, 46% were <1 cm on pathology; of 1306 polyps estimated as <1 cm, 3.9% were ≥ 1 cm on pathology. By histology, 39% of adenomatous, 59% of sessile serrated, and 73% of hyperplastic polyps were overcalled; P = .008. By configuration, 34% of pedunculated, 49% of sessile, and 61% of flat polyps were overcalled; P = .014. Endoscopic overestimation was more common in women (54%) than in men (40%) (P = .03) and with proximal (56%) than distal (40%) sites; P = .02. Miscall rates were unaffected by endoscopist covariates. CONCLUSIONS Substantial discordance exists between endoscopic and pathology-based assessments of polyp size. Almost half of polyps called advanced on endoscopic estimates of size ≥ 1 cm fell below this threshold on actual pathology measurements.

T2036 Pan-Detection of Gastrointestinal Neoplasms By Stool DNA Testing: Establishment of Feasibility

Non-apoptotic human DNA (long DNA) in stool is a discriminant marker for colorectal cancer (Gastroenterology 2000; 119:1219). Fecal long DNA may originate from either the exfoliation of dysplastic cells or from the luminal hemorrhage or exudation of leukocytes. The contribution of these potential sources long DNA in stool has not been examined. If leukocytes are the major source, then fecal human DNA and occult blood levels should correlate. If exfoliation is the major source, then fecal human DNA and occult blood should be complementary markers for colorectal cancer. AIM: To quantify and correlate human DNA and hemoglobin in stools from patients with colorectal neoplasia, and to consider a simple stool assay for cancer screening. METHODS: Subjects comprised 74 patients with colorectal cancer, 27 with adenomas >1cm, and 100 with normal colonoscopy. One stool per subject was collected in stabilization buffer before or > 1 week after colonoscopy. Fecal long DNA and occult blood were quantified in blinded fashion by real-time Alu PCR (CEBP 2006 15:1115) and the HemoQuant assay (Mayo Medical Laboratories), respectively. HemoQuant assay results are not affected by fecal storage (Ann Intern Med 1984;101:297) and could be reliably applied to archival specimens for this study. RESULTS: Fecal long DNA and occult blood showed no correlation (R2=0). At 90% specificities, long DNA testing detected 70% of colorectal cancers and 46% of adenomas while occult blood testing detected 50% and 12%, respectively. Combining these tests detected 80% colorectal cancers and 46% of adenomas at 90% specificity. For detection of colorectal cancer, AUC values were 0.82, 0.78, and 0.90 for fecal long DNA, occult blood, and combination testing, respectively (p<0.05, long DNA or occult blood vs combination). The median (range) fecal long DNA level was 181 ng/g stool (0-7120 ng/g) with stage 1-2 cancers and 910 ng/g (0-19140 ng/ g) with stage 3-4 cancers, p<0.05. The median fecal hemoglobin level was 7.0 mg Hb/g (0.2-26.4 mg/g) with proximal cancers and 2.5 mg/g (0.1-36.1 mg/g) with distal ones, p<0.05. Median size was 4.0 cm (1.0-15.0) for neoplasms detected by the combined tests and 3.7 cm (1.0-7.0) for neoplasms missed, p=0.02. Neoplasm detection rates by combined tests were affected by neither tumor site nor stage. CONCLUSIONS: Long DNA and occult blood in stools are complementary stool markers for detection of colorectal cancer, and combined assay of these markers provides higher sensitivity for cancer than assay of either alone. The combined assay offers a simple and inexpensive screening approach that warrants further evaluation.

Aberrantly Methylated Gene Marker Levels in Stool: Effects of Demographic, Exposure, Body Mass, and Other Patient Characteristics

Background: Selected aberrantly methylated genes represent sensitive candidate stool markers for colorectal cancer (CRC) screening. We assessed the impact of demographic, exposure, body mass, and other patient variables on stool levels of highly informative methylated gene markers — BMP3, NDRG4, vimentin, and TFPI2. Methods: We studied freezer-archived stools from 500 patients with normal colonoscopy (median age 64 (range 44-85); 53% women). On supernatants from thawed aliquots, target gene sequences were purified by hybrid capture; bisulfite treated, and assayed using the analytically-sensitive QuARTS method (quantitative allele-specific real-time target and signal amplification). The reference human gene β-actin was assayed along with the 4 methylated genes. Results: Only age significantly influenced all methylated marker levels in stool (p<0.0001 for each). The relative increase per standard deviation of age was greatest with TFPI2 at 49.4% and least with BMP3 at only 0.21%; levels of β-actin did not change across age. Other demographic variables (sex, race, and residence), exposures (smoking, alcohol, or analgesic use), family or personal history of colorectal neoplasia, body mass, and diabetes mellitus had no effect on methylated marker levels. Conclusions: Although stool levels of candidate methylated markers increase with age to variable extents, most common clinical covariates have no effect. Impact: These findings have important implications on CRC screening compliance, as patients using a stool test that incorporates these markers would not have to make life-style or medication adjustments. Furthermore, age effect can be mitigated by adjustment of cut-off levels based on age or by selection of markers least influenced by age.

T2037 Quantitative Stool DNA Testing for Detection of Both Colorectal Cancer and Advanced Adenoma

Non-apoptotic human DNA (long DNA) in stool is a discriminant marker for colorectal cancer (Gastroenterology 2000; 119:1219). Fecal long DNA may originate from either the exfoliation of dysplastic cells or from the luminal hemorrhage or exudation of leukocytes. The contribution of these potential sources long DNA in stool has not been examined. If leukocytes are the major source, then fecal human DNA and occult blood levels should correlate. If exfoliation is the major source, then fecal human DNA and occult blood should be complementary markers for colorectal cancer. AIM: To quantify and correlate human DNA and hemoglobin in stools from patients with colorectal neoplasia, and to consider a simple stool assay for cancer screening. METHODS: Subjects comprised 74 patients with colorectal cancer, 27 with adenomas >1cm, and 100 with normal colonoscopy. One stool per subject was collected in stabilization buffer before or > 1 week after colonoscopy. Fecal long DNA and occult blood were quantified in blinded fashion by real-time Alu PCR (CEBP 2006 15:1115) and the HemoQuant assay (Mayo Medical Laboratories), respectively. HemoQuant assay results are not affected by fecal storage (Ann Intern Med 1984;101:297) and could be reliably applied to archival specimens for this study. RESULTS: Fecal long DNA and occult blood showed no correlation (R2=0). At 90% specificities, long DNA testing detected 70% of colorectal cancers and 46% of adenomas while occult blood testing detected 50% and 12%, respectively. Combining these tests detected 80% colorectal cancers and 46% of adenomas at 90% specificity. For detection of colorectal cancer, AUC values were 0.82, 0.78, and 0.90 for fecal long DNA, occult blood, and combination testing, respectively (p<0.05, long DNA or occult blood vs combination). The median (range) fecal long DNA level was 181 ng/g stool (0-7120 ng/g) with stage 1-2 cancers and 910 ng/g (0-19140 ng/ g) with stage 3-4 cancers, p<0.05. The median fecal hemoglobin level was 7.0 mg Hb/g (0.2-26.4 mg/g) with proximal cancers and 2.5 mg/g (0.1-36.1 mg/g) with distal ones, p<0.05. Median size was 4.0 cm (1.0-15.0) for neoplasms detected by the combined tests and 3.7 cm (1.0-7.0) for neoplasms missed, p=0.02. Neoplasm detection rates by combined tests were affected by neither tumor site nor stage. CONCLUSIONS: Long DNA and occult blood in stools are complementary stool markers for detection of colorectal cancer, and combined assay of these markers provides higher sensitivity for cancer than assay of either alone. The combined assay offers a simple and inexpensive screening approach that warrants further evaluation.

Long-term Follow-up of Patients Having False-Positive Multitarget Stool DNA Tests after Negative Screening Colonoscopy: The LONG-HAUL Cohort Study

Background: Studies of colorectal cancer screening by multitarget stool DNA (MT-sDNA) show false-positive (FP) rates of 7% to 13%. It is unclear whether FP patients are at increased long-term risk of adverse outcomes. Methods: We compared subsequent clinical events among patients with apparent FP MT-sDNA with those in patients reported as true negative (TN). This was a retrospective cohort study of participants in pre-FDA approval MT-sDNA studies having nonadvanced or negative baseline colonoscopy findings from a single referral center. Per-protocol and calibrated cutoffs defined FP and TN groups. From the time of stool collection, we measured differences between FP and TN groups in time to death, subsequent cancer diagnosis, and onset of alarm symptoms. Results: Of 1,050 eligible patients, only 6 were lost to follow-up. Median age was 65.6 years [interquartile range (IQR), 56.8–72.3]; 54% were female. Median follow-up time was 4 years (IQR, 3.5–5.3). Eight aerodigestive (lung and gastrointestinal tract) cancers occurred. FP status by calibrated, but not per-protocol, cutoffs was associated with subsequent aerodigestive cancer; however, cumulative incidence did not exceed SEER expectations from the general population. By any cutoff method, FP status was not associated with mortality or alarm symptoms. Conclusions: Although FP status was associated with long-term aerodigestive cancers, new cases were not temporally related and did not exceed incidence estimates from general population. Impact: These observations do not justify aggressive follow-up evaluation for patients with FP MT-sDNA at this time. Larger studies are needed to confirm these early findings. Cancer Epidemiol Biomarkers Prev; 26(4); 614–21. ©2016 AACR.

T2034 Stool DNA and Occult Blood for Detection of Colorectal Cancer: Complementary Markers

Non-apoptotic human DNA (long DNA) in stool is a discriminant marker for colorectal cancer (Gastroenterology 2000; 119:1219). Fecal long DNA may originate from either the exfoliation of dysplastic cells or from the luminal hemorrhage or exudation of leukocytes. The contribution of these potential sources long DNA in stool has not been examined. If leukocytes are the major source, then fecal human DNA and occult blood levels should correlate. If exfoliation is the major source, then fecal human DNA and occult blood should be complementary markers for colorectal cancer. AIM: To quantify and correlate human DNA and hemoglobin in stools from patients with colorectal neoplasia, and to consider a simple stool assay for cancer screening. METHODS: Subjects comprised 74 patients with colorectal cancer, 27 with adenomas >1cm, and 100 with normal colonoscopy. One stool per subject was collected in stabilization buffer before or > 1 week after colonoscopy. Fecal long DNA and occult blood were quantified in blinded fashion by real-time Alu PCR (CEBP 2006 15:1115) and the HemoQuant assay (Mayo Medical Laboratories), respectively. HemoQuant assay results are not affected by fecal storage (Ann Intern Med 1984;101:297) and could be reliably applied to archival specimens for this study. RESULTS: Fecal long DNA and occult blood showed no correlation (R2=0). At 90% specificities, long DNA testing detected 70% of colorectal cancers and 46% of adenomas while occult blood testing detected 50% and 12%, respectively. Combining these tests detected 80% colorectal cancers and 46% of adenomas at 90% specificity. For detection of colorectal cancer, AUC values were 0.82, 0.78, and 0.90 for fecal long DNA, occult blood, and combination testing, respectively (p<0.05, long DNA or occult blood vs combination). The median (range) fecal long DNA level was 181 ng/g stool (0-7120 ng/g) with stage 1-2 cancers and 910 ng/g (0-19140 ng/ g) with stage 3-4 cancers, p<0.05. The median fecal hemoglobin level was 7.0 mg Hb/g (0.2-26.4 mg/g) with proximal cancers and 2.5 mg/g (0.1-36.1 mg/g) with distal ones, p<0.05. Median size was 4.0 cm (1.0-15.0) for neoplasms detected by the combined tests and 3.7 cm (1.0-7.0) for neoplasms missed, p=0.02. Neoplasm detection rates by combined tests were affected by neither tumor site nor stage. CONCLUSIONS: Long DNA and occult blood in stools are complementary stool markers for detection of colorectal cancer, and combined assay of these markers provides higher sensitivity for cancer than assay of either alone. The combined assay offers a simple and inexpensive screening approach that warrants further evaluation.

Highly Discriminant Methylated DNA Markers for the Non-endoscopic Detection of Barrett’s Esophagus

BACKGROUND: Minimally invasive methods have been described to detect Barretts esophagus (BE), but are limited by subjectivity and suboptimal accuracy. We identified methylated DNA markers (MDMs) for BE in tissue and assessed their accuracy on whole esophagus brushings and capsule sponge samples. METHODS: Step 1: Unbiased whole methylome sequencing was performed on DNA from BE and normal squamous esophagus (SE) tissue. Discriminant MDM candidates were validated on an independent patient cohort (62 BE cases, 30 controls) by quantitative methylation specific PCR (qMSP). Step 2: Selected MDMs were further evaluated on whole esophageal brushings (49 BE cases, 36 controls). 35 previously sequenced esophageal adenocarcinoma (EAC) MDMs were also evaluated. Step 3: 20 BE cases and 20 controls were randomized to swallow capsules sponges (25 mm, 10 pores or 20 pores per inch (ppi)) followed endoscopy. DNA yield, tolerability, and mucosal injury were compared. Best MDM assays were performed on this cohort. RESULTS: Step 1: 19 MDMs with areas under the ROC curve (AUCs) >0.85 were carried forward. Step 2: On whole esophageal brushings, 80% of individual MDM candidates showed high accuracy for BE (AUCs 0.84‐0.94). Step 3: The capsule sponge was swallowed and withdrawn in 98% of subjects. Tolerability was superior with the 10 ppi sponge with minimal mucosal injury and abundant DNA yield. A 2‐marker panel (VAV3 + ZNF682) yielded excellent BE discrimination (AUC = 1). CONCLUSIONS: Identified MDMs discriminate BE with high accuracy. BE detection appears safe and feasible with a capsule sponge. Corroboration in larger studies is warranted. ClinicalTrials.gov number NCT02560623.

Patient perceptions of stool DNA testing for pan-digestive cancer screening: A survey questionnaire

AIM To explore patient interest in a potential multi-organ stool-DNA test (MUST) for pan-digestive cancer screening. METHODS A questionnaire was designed and mailed to 1200 randomly-selected patients from the Mayo Clinic registry. The 29-item survey questionnaire included items related to demographics, knowledge of digestive cancers, personal and family history of cancer, personal concern of cancer, colorectal cancer (CRC) screening behavior, interest in MUST, importance of test features in a cancer screening tool, and comparison of MUST with available CRC screening tests. All responses were summarized descriptively. χ(2) and Rank Sum Test were used for categorical and continuous variables, respectively. RESULTS Completed surveys were returned by 434 (29% aged 50-59, 37% 60-69, 34% 70-79, 52% women). Most participants (98%) responded they would use MUST. In order of importance, respondents rated multi-cancer detection, absence of bowel preparation, safety and noninvasiveness as most attractive characteristics. For CRC screening, MUST was preferred over colorectal-only stool-DNA testing (53%), occult blood testing (75%), colonoscopy (84%), sigmoidoscopy (91%), and barium enema (95%), P < 0.0001 for each. Among those not previously screened, most (96%) indicated they would use MUST if available. Respondents were confident in their ability to follow instructions to perform MUST (98%). Only 9% of respondents indicated that fear of finding cancer was a concern with MUST, and only 3% indicated unpleasantness of stool sampling as a potential barrier. CONCLUSION Patients are receptive to the concept of MUST, preferred MUST over conventional CRC screening modalities and valued its potential feature of multi-cancer detection.