Barry M. Berger

Multitarget Stool DNA Testing for Colorectal-Cancer Screening

BACKGROUND An accurate, noninvasive test could improve the effectiveness of colorectal-cancer screening. METHODS We compared a noninvasive, multitarget stool DNA test with a fecal immunochemical test (FIT) in persons at average risk for colorectal cancer. The DNA test includes quantitative molecular assays for KRAS mutations, aberrant NDRG4 and BMP3 methylation, and β-actin, plus a hemoglobin immunoassay. Results were generated with the use of a logistic-regression algorithm, with values of 183 or more considered to be positive. FIT values of more than 100 ng of hemoglobin per milliliter of buffer were considered to be positive. Tests were processed independently of colonoscopic findings. RESULTS Of the 9989 participants who could be evaluated, 65 (0.7%) had colorectal cancer and 757 (7.6%) had advanced precancerous lesions (advanced adenomas or sessile serrated polyps measuring ≥1 cm in the greatest dimension) on colonoscopy. The sensitivity for detecting colorectal cancer was 92.3% with DNA testing and 73.8% with FIT (P=0.002). The sensitivity for detecting advanced precancerous lesions was 42.4% with DNA testing and 23.8% with FIT (P<0.001). The rate of detection of polyps with high-grade dysplasia was 69.2% with DNA testing and 46.2% with FIT (P=0.004); the rates of detection of serrated sessile polyps measuring 1 cm or more were 42.4% and 5.1%, respectively (P<0.001). Specificities with DNA testing and FIT were 86.6% and 94.9%, respectively, among participants with nonadvanced or negative findings (P<0.001) and 89.8% and 96.4%, respectively, among those with negative results on colonoscopy (P<0.001). The numbers of persons who would need to be screened to detect one cancer were 154 with colonoscopy, 166 with DNA testing, and 208 with FIT. CONCLUSIONS In asymptomatic persons at average risk for colorectal cancer, multitarget stool DNA testing detected significantly more cancers than did FIT but had more false positive results. (Funded by Exact Sciences; ClinicalTrials.gov number, NCT01397747.).

Next-Generation Stool DNA Test Accurately Detects Colorectal Cancer and Large Adenomas

BACKGROUND & AIMS Technical advances have led to stool DNA (sDNA) tests that might accurately detect neoplasms on both sides of the colorectum. We assessed colorectal neoplasm detection by a next-generation sDNA test and effects of covariates on test performance. METHODS We performed a blinded, multicenter, case-control study using archived stool samples collected in preservative buffer from 252 patients with colorectal cancer (CRC), 133 with adenomas ≥ 1 cm, and 293 individuals with normal colonoscopy results (controls); two-thirds were randomly assigned to a training set and one-third to a test set. The sDNA test detects 4 methylated genes, a mutant form of KRAS, and the α-actin gene (as a reference value) using quantitative, allele-specific, real-time target and signal amplification; it also quantifies hemoglobin. We used a logistical model to analyze data. RESULTS The sDNA test identified 85% of patients with CRC and 54% of patients with adenomas ≥1 cm with 90% specificity. The test had a high rate of detection for all nonmetastatic stages of CRC (aggregate 87% detection rate for CRC stages I-III). Detection rates increased with adenoma size: 54% ≥ 1 cm, 63% >1 cm, 77% >2 cm, 86% >3 cm, and 92% >4 cm (P < .0001). Based on receiver operating characteristic analysis, the rate of CRC detection was slightly greater for the training than the test set (P = .04), whereas the rate of adenoma detection was comparable between sets. Sensitivities for detection of CRC and adenoma did not differ with lesion site. CONCLUSIONS Early-stage CRC and large adenomas can be detected throughout the colorectum and with high levels of accuracy by the sDNA test. Neoplasm size, but not anatomical site, affected detection rates. Further studies are needed to validate the findings in a larger population and optimize the sDNA test.

Association of Large Serrated Polyps With Synchronous Advanced Colorectal Neoplasia

OBJECTIVES:Serrated polyps of the colorectum are a histologically and genetically heterogeneous group of lesions, which include classic hyperplasic polyps, sessile serrated adenomas (SSAs), and traditional serrated adenomas. Accumulating evidence suggests that they may have different malignancy potentials. This study sought to determine the association between the presence of large serrated colorectal polyps and synchronous advanced colorectal neoplasia.METHODS:Among 4,714 asymptomatic subjects who underwent screening colonoscopy, cases of advanced colorectal neoplasia (tubular adenoma ≥1 cm, adenoma with any villous histology, adenoma with carcinoma in situ / high-grade dysplasia, or invasive adenocarcinoma) were compared with controls without advanced neoplasia with respect to candidate predictors, including age, sex, family history of colorectal cancer, body mass index, the presence and number of small tubular adenomas (<1 cm), the presence of multiple small serrated polyps (<1 cm), and the presence of large serrated polyps (≥1 cm). Independent predictors of advanced neoplasia were determined by multivariate logistic regression analysis.RESULTS:Among 467 cases and 4,247 controls, independent predictors of advanced colorectal neoplasia were increasing age (odds ratio (OR)=4.51; 95% confidence interval (CI), 1.43–14.3; P=0.01 for subjects ≥80 years vs. 50–54 years of age); non-advanced tubular adenomas (OR=2.33; 95% CI 1.37–3.96, P=0.0017 for 3 or more); and large serrated polyps (OR=3.24; 95% CI 2.05–5.13, P<0.0001). In total, 109 subjects (2.3% of the study population) had large serrated polyps. Right- and left-sided large serrated polyps had a similar association with advanced colorectal neoplasia (OR=3.38 vs. 2.66, P=0.62).CONCLUSIONS:Large serrated polyps are strongly and independently associated with synchronous advanced colorectal neoplasia. Our results suggest that large serrated polyps may be a marker for advanced colorectal neoplasia. Further studies are needed to determine whether the association with advanced neoplasia differs among subsets of serrated polyps, particularly SSAs and classic hyperplastic polyps.

A simplified, noninvasive stool DNA test for colorectal cancer detection.

BACKGROUND:As a noninvasive colorectal cancer (CRC) screening test, a multi-marker first generation stool DNA (sDNA V 1.0) test is superior to guaiac-based fecal occult blood tests. An improved sDNA assay (version 2), utilizing only two markers, hypermethylated vimentin gene (hV) and a two site DNA integrity assay (DY), demonstrated in a training set (phase 1a) an even higher sensitivity (88%) for CRC with a specificity of 82%.AIM:To validate in an independent set of patients (phase 1b) the sensitivity and specificity of sDNA version 2 for CRC.METHODS:Forty-two patients with CRC and 241 subjects with normal colonoscopy (NC) provided stool samples, to which they immediately added DNA stabilizing buffer, and mailed their specimen to the laboratory. DNA was purified using gel-based capture, and analyzed for hV and DY using methods identical to those previously published.RESULTS:Using the same cutpoints as the 1a training set (N = 162; 40 CRCs, 122 normals), hV demonstrated a higher and DY a slightly lower sensitivity, for a combined sensitivity of hV + DY of 86%. Optimal cutpoints based on the combined phase 1a + 1b dataset (N = 445; 82 CRCs, 363 normals) yielded a CRC sensitivity of 83%. The vast majority of cancers were detected regardless of tumor stage, tumor location, or patient age. Assay specificity in the phase 1b dataset for hV, DY, and hV + DY was 82%, 85%, and 73%, respectively, using the phase 1a cutpoints. Optimal cutpoints based on the combined phase 1a + 1b dataset yield a specificity of 82%.CONCLUSIONS:This study provides validation of a simplified, improved sDNA test that incorporates only two markers and that demonstrates high sensitivity (83%) and specificity (82%) for CRC. Test performance is highly reproducible in a large set of patients. The use of only two markers will make the test easier to perform, reduce the cost, and facilitate distribution to local laboratories.

The Stool DNA Test Is More Accurate Than the Plasma Septin 9 Test in Detecting Colorectal Neoplasia

BACKGROUND & AIMS Several noninvasive tests have been developed for colorectal cancer (CRC) screening. We compared the sensitivities of a multimarker test for stool DNA (sDNA) and a plasma test for methylated septin 9 (SEPT9) in identifying patients with large adenomas or CRC. METHODS We analyzed paired stool and plasma samples from 30 patients with CRC and 22 with large adenomas from Mayo Clinic archives. Stool (n = 46) and plasma (n = 49) samples from age- and sex-matched patients with normal colonoscopy results were used as controls. The sDNA test is an assay for methylated BMP3, NDRG4, vimentin, and TFPI2; mutant KRAS; the β-actin gene, and quantity of hemoglobin (by the porphyrin method). It was performed blindly at Exact Sciences (Madison, Wisconsin); the test for SEPT9 was performed at ARUP Laboratories (Salt Lake City, Utah). Results were considered positive based on the manufacturers specificity cutoff values of 90% and 89%, respectively. RESULTS The sDNA test detected adenomas (median, 2 cm; range, 1-5 cm) with 82% sensitivity (95% confidence interval [CI], 60%-95%); SEPT9 had 14% sensitivity (95% CI, 3%-35%; P = .0001). The sDNA test identified patients with CRC with 87% sensitivity (95% CI, 69%-96%); SEPT9 had 60% sensitivity (95% CI, 41%-77%; P = .046). The sDNA test identified patients with stage I-III CRC with 91% sensitivity (95% CI, 71%-99%); SEPT9 had 50% sensitivity (95% CI, 28%-72%; P = .013); for stage IV CRC, sensitivity values were 75% (95% CI, 35%-97%) and 88% (95% CI, 47%-100%), respectively (P = .56). False positive rates were 7% for the sDNA test and 27% for SEPT9. CONCLUSIONS Based on analyses of paired samples, the sDNA test detects nonmetastatic CRC and large adenomas with significantly greater levels of sensitivity than the SEPT9 test. These findings might be used to modify approaches for CRC prevention and early detection.

Stool DNA screening for colorectal neoplasia: biological and technical basis for high detection rates

Summary Colorectal cancer (CRC), the second most common cause of cancer-related mortality worldwide, is preventable with effective screening and removal of precursor lesions. Yet, screening efforts have been hampered by low participation rates and by performance limitations of the screening tools themselves. Stool DNA testing has emerged as a biologically rational and user-friendly strategy for the non-invasive detection of both CRC and critical precursor lesions. Unlike most conventional screening tools, stool DNA testing detects proximal and distal colorectal neoplasms equally well. Several key technical advances have led to increasingly accurate approaches for stool DNA testing including use of a DNA preservative buffer with stool collection, efficient target capture and amplification methods, broadly informative marker panels, and automated assay components. Based on recent studies, advanced multi-marker stool DNA tests including methylated markers, mutation markers and an assessment of faecal haemoglobin have been shown to detect CRC at sensitivities of 85% and higher and adenomas >1 cm at 60% and higher in a case-control environment. If the high accuracy of multi-marker stool tests is corroborated in multicentre screening studies on average-risk persons currently underway, then these stool tests could influence our CRC screening paradigm. This review discusses the biological basis, key technical advances, and recent clinical performance validation of stool DNA testing.

Detection of Colorectal Serrated Polyps by Stool DNA Testing: Comparison with Fecal Immunochemical Testing for Occult Blood (FIT)

Objectives Precursors to 1/3 of colorectal cancer (CRC), serrated polyps have been under-detected by screening due to their inconspicuous, non-hemorrhagic, and proximal nature. A new multi-target stool DNA test (multi-target sDNA) shows high sensitivity for both CRC and advanced adenomas. Screen detection of serrated polyps by this approach requires further validation. We sought to assess and compare noninvasive detection of sessile serrated polyps (SSP) ≥1 cm by sDNA and an occult blood fecal immunochemical test (FIT). Methods In a blinded prospective study, a single stool sample used for both tests was collected from 456 asymptomatic adults prior to screening or surveillance colonoscopy (criterion standard). All 29 patients with SSP≥1 cm were included as cases and all 232 with no neoplastic findings as controls. Buffered stool samples were processed and frozen on receipt; Exact Sciences performed sDNA in batches using optimized analytical methods. The sDNA multi-marker panel targets methylated BMP3 (mBMP3) and NDRG4, mutant KRAS, β-actin, and hemoglobin. FIT (Polymedco OC-FIT Check) was performed in separate lab ≤2 days post defecation and evaluated at cutoffs of 50 (FIT-50) and 100 ng/ml (FIT-100). Results Median ages: cases 61 (range 57–77), controls 62 (52–70), p = NS. Women comprised 59% and 51%, p = NS, respectively. SSP median size was 1.2 cm (1–3 cm), 93% were proximal, and 64% had synchronous diminutive polyps. Among multi-target sDNA markers, mBMP3 proved highly discriminant for detection of SSP≥1 cm (AUC = 0.87, p<0.00001); other DNA markers provided no incremental sensitivity. Hemoglobin alone showed no discrimination (AUC = 0.50, p = NS). At matched specificities, detection of SSP≥1 cm by stool mBMP3 was significantly greater than by FIT-50 (66% vs 10%, p = 0.0003) or FIT-100 (63% vs 0%, p<0.0001). Conclusions In a screening and surveillance setting, SSP≥1 cm can be detected noninvasively by stool assay of exfoliated DNA markers, especially mBMP3. FIT appears to have no value in SSP detection.

Colon cancer-associated DNA mutations: marker selection for the detection of proximal colon cancer.

This study evaluates the potential ability of a specific panel of DNA mutations to identify right-sided colorectal carcinomas (CRCs) that would be missed by a flexible sigmoidoscopy (FS) screening program. This panel could then be applied to stool DNA analysis for noninvasive proximal CRC detection. A series of resected right-sided CRCs from 101 patients who had no left-sided advanced colonic neoplasms distal to the splenic flexure were analyzed. Tumor DNA was isolated from microdissected tumor sections. Deletions in the BAT-26 locus, a marker of microsatellite instability, and mutations at 19 loci spread among the p53, K-ras, and Apc genes were detected following PCR amplification. Mutations were identified in 83% of successfully amplified samples and were variably present in each of the target sites:p53 (42%), Apc (37%), K-ras (28%), and BAT-26 (24%). Mutations were identified across all Dukes stages (CIS/A 6/8 [75%], B 41/51[80%], C 30/32 (94%), and D 6/9 [67%]). Our data suggest that this 20-marker mutation panel may be associated with more than 80% of cancers undetectable by FS. The adjunctive use of stool DNA mutation analysis using this marker panel in FS CRC screening programs may significantly increase the detection of proximal CRC.

Tu1189 An Optimized Multi-Marker Stool Test for Colorectal Cancer Screening: Initial Clinical Appraisal

Background: Organized colorectal cancer screening program in the Czech Republic (10.5 million inhabitants, 3.8 million aged over 50) was introduced in year 2000, based on guaiac fecal occult blood test (FOBT). In 2009, new program design was launched. To asymptomatic individuals aged 50 54, annual guaiac or immunochemical FOBT has been offered, followed by screening colonoscopy, if positive. In age of 55, there is a choice of either FOBT biannually or primary screening colonoscopy in 10 years interval. Methods: Early performance indicators (European guidelines 2010) were used to assess the efficacy of the program Results: The coverage of target population has been rising since the program introduction, reaching 23% in 2010. The overall FOBT positivity of 6.1% has been recorded. Since the beginning of on-line individual data management in year 2006, until November 2011, there were 59,320 screening colonoscopies (FOBT+) and 8,493 primary screening colonoscopies performed (basic result included in the table). A trend for earlier stage cancer detection was observed (42.7% in stage I). Average ADR and CIR were 31.2% and 94.2% respectively. In years 2006 2010, there were reported 45 cases of perforations (0.08% of all colonoscopies). Conclusion: The Czech national Colorectal Cancer Screening program is growing in performance and influential. Introduction of the new program design increases the target population coverage. Main findings on (primary) screening colonoscopies

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.