Oldham-Haltom, Rebecca

Abstract We have demonstrated that colorectal cancer (CRC) and advanced pre-cancers can be detected non-invasively by a manual multi-target stool DNA-based test (sDNA-MT) comprising exfoliated DNA markers (methylated BMP3 and NDRG4, mutant KRAS (7 mutations, codons 12, 13), plus β-actin) and fecal hemoglobin (Hb) (Lidgard, Gastroenterology 2012;142(5);S-770). We now report the clinical performance of this sDNA-MT test using an optimized automated analytic platform and logistic algorithm. This platform could facilitate the routine performance of sDNA-MT for CRC screening by molecular diagnostics capable clinical laboratories. Method: Stool samples were collected from 1003 subjects at 36 study sites after informed consent and prior to colonoscopy bowel preparation from those presenting for average risk CRC screening (283) or surveillance (176) at 2 sites. From referred subjects with CRC, Advanced Adenoma (AA) or Sessile Serrate Adenoma ≥ 1 cm, (SSA) stool was collected at least 7 days post-colonoscopy and prior to surgery or chemo-radiation (135; 21 sites) and similarly for subjects with no neoplastic findings on colonoscopy (Neg) (409; 13 sites). The study population included: cases (207), 58% male, median age 65 yrs. (38-87), CRC (93), AA (84), SSA ≥ 1 cm (30) and controls (796), 42% male, median age 65 yrs. (50-84), Neg (641) and non-advanced adenomas (NA) (155). Stool sample collection and DNA isolation were previously described. Automated methylation, mutation and actin assays were performed with a Hamilton STARlet fluid handler (Hamilton Robotics, Reno NV), and QuARTS (Quantitative Allele-specific Real-time Target and Signal amplification) run on an ABI 7500 FastDx real time thermal cycler (Applied Biosystems, Foster City, CA). Fecal Hb (ng/ml buffer) analysis was performed by automated sandwich ELISA. A “Positive” or “Negative” result was determined with an algorithm that included the methylation and mutation results and a logistic regression score, which combines DNA marker results with Hb and actin results. Algorithm results exceeding a threshold were called “Positive”. The algorithm provided good discriminative ability, stability, sensitivity and specificity. Robustness was tested with computer simulations and statistical techniques (leave-one-out and 10-fold cross validation). Results: At a 90% nominal specificity, sDNA-MT sensitivity was 98% for CRC (91/93) [Stage: I, 95% (20/21), II, 100% (23/23), III 96% (26/27), IV 100% (7/7) and I-III combined 97% (69/71)], 57% (65/114) for precursors ≥1 cm (AA, SSA), and 86% (12/14) for precursors with high grade dysplasia. CRC patients were typically referred to colonoscopy for symptoms and test sensitivity may be elevated relative to that seen with screening. Conclusion: With this study using a novel automated sDNA-MT analytic platform with logistic algorithm, we corroborate our earlier findings using a manual process and demonstrate a platform that allows testing to be performed routinely by molecular diagnostic capable laboratories. The high sensitivity of sDNA-MT for CRC across all stages and for advanced precursors with high-grade dysplasia could lead to improved non-invasive CRC screening performance with wide accessibility to patients. A large multi-site pivotal CRC screening study (DeeP-C study clinicaltrials.gov, NCT01397747) to support such use is underway. Citation Format: Graham P. Lidgard, Michael J. Domanico, Janelle J. Bruinsma, James Light, Zubin D. Gagrat, Rebecca L. Oldham-Haltom, Keith D. Fourrier, Hatim Allawi, Tracy C. Yab, Julie A. Simonson, Mary Devens, Russell I. Heigh, David A. Ahlquist, Barry M. Berger. An optimized molecular stool test for colorectal cancer screening: Evaluation of an automated analytic platform and logistic algorithm. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B12.

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States, yet it is potentially the most treatable and preventable cancer with effective screening. Exact Sciences has developed a multi-biomarker CRC screening test that targets DNA methylation, DNA mutations, and hemoglobin in stool. The aim of this study was to evaluate the analytical accuracy of a novel assay for the detection of KRAS mutation sequences at Codons 12 and 13 in well-characterized colorectal tissues. Methodology: A multiplexed KRAS assay was designed utilizing QuARTS (Quantitative Allele-specific Real-time Target and Signal amplification), a highly sensitive technology that combines allele-specific DNA amplification with invasive cleavage chemistry to generate signal during each amplification cycle similar to real-time PCR. The assay, which detects seven KRAS mutations and the reference gene β-actin, was used to assess 87 colorectal tissue samples (52 CRCs, 16 adenomas ≥ 1cm, and 19 normal epithelia) as determined by Mayo Clinic Pathology. Samples were obtained by microdissection of fresh frozen tissue biopsies. DNA was extracted by Mayo Clinic using a standardized chloroform/phenol methodology. The genotypes of each sample were established using dye terminator dideoxy sequencing in both the forward and reverse orientations. Copy numbers of KRAS mutations and β-actin were determined by conventional comparison against standard curves. KRAS data are reported as percent mutation and calculated by dividing mutant copies by β-actin copies and multiplying by 100. Results: Based on sequencing data: the 52 CRC samples contained 22 KRAS mutations and 30 wild-type genotypes, the 16 adenomas ≥ 1cm contained 8 mutations and 8 wild-type genotypes, and the 19 normal tissues contained all wild-type genotypes. The QuARTS assay detected 100% of the KRAS mutations in the CRC and adenomas and provided excellent differentiation between wild-type and mutation, with the highest percent KRAS mutation of normal wild-type samples at 0.55% and the lowest percent mutation of KRAS positive samples at 8.34%. Based on this data, this assay has the potential to be more sensitive analytically than standard sequencing. Conclusion: The QuARTS assay detects KRAS mutations in tissue with high sensitivity and specificity. This assay method could provide an accurate and high throughput approach to detection of KRAS mutations in biospecimens for multiple clinical applications. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A22.