Citations

Abstract Purpose: Lung cancer is the leading cause of cancer deaths worldwide. Most present symptomatically at late stage with high lethality. Early detection reduces mortality but accurate and readily accessible tools for population screening are limited. By whole methylome sequencing, we have identified novel methylated DNA markers (MDMs) for lung cancer in tissue (Giakoumopoulos et al. ASCO 2016). Using top candidate MDMs in the present study, we now explore their clinical accuracy for lung cancer detection when assayed from plasma. Experimental Procedures: Archival plasmas from two independent study groups were tested in blinded fashion. Lung cancer cases and controls (apparently healthy smokers) for each group were balanced on age and sex (Group 1: 64 cases, 231 controls; Group 2: 23 cases, 80 controls). Using multiplex PCR followed by QuARTS (Quantitative Allele-Specific Real-time Target and Signal amplification) assay, a post-bisulfite quantification of MDMs on DNA extracted from plasma was performed. We selected 31 MDM candidates for initial evaluation in Group 1 (1 ml plasma/patient); top individual MDMs were subsequently tested in Group 2 to identify optimal MDM panels for lung cancer detection (2 ml/patient). Results: From Group 1 analyses, 13 high performance MDMs were selected for further testing (CYP26C1, SOBP, SUCLG2, SHOX2, ZDHHC1, NFIX, FLJ45983, HOXA9, B3GALT6, ZNF781, SP9, BARX1, EMX1) with individual areas under the receiver operator curve (AUCs) ranging from 0.593 to 0.939. Discrimination by individual MDMs was corroborated in Group 2 in which data was analyzed using two methods: a logistic regression fit and a regression partition tree approach. The logistic fit model identified a 4-marker panel (ZNF781, BARX1, EMX1, and SOBP) with an AUC of 0.96 and an overall sensitivity of 91% and 90% specificity. Analysis of the data using a regression partition tree approach identified 4 markers (ZNF781, BARX1, EMX1, and HOXA9) with AUC of 0.93 and an overall sensitivity of 96% and specificity of 94%. For both approaches, B3GALT6 was used as a standardizing marker of total DNA input. Conclusion: A panel of MDMs assayed in plasma achieved high sensitivity and specificity for all types of lung cancer. Further clinical evaluation and validation of this promising panel in larger patient groups are clearly indicated. Citation Format: Hatim T. Allawi, Maria Giakoumopoulos, Evan Flietner, Austin Oliphant, Carla Volkmann, Brian Aizenstein, Tamara Sander, Drew Eckmayer, Ashley Poenitzsch Strong, Melissa Gray, Barry Berger, Tracy Yab, William Taylor, Douglas Mahoney, John B. Kisiel, David E. Midthun, David A. Ahlquist, Graham P. Lidgard. Detection of lung cancer by assay of novel methylated DNA markers in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 712. doi:10.1158/1538-7445.AM2017-712

341 Background: Precursors to 1/3 of colorectal cancer (CRC), serrated polyps have historically been underdetected by screening due to their inconspicuous, nonhemorrhagic, and proximal nature. New generation sDNA-MT show high sensitivity for both CRC and advanced adenomas (Gastroenterology 2012;142:248), but screen detection of serrated polyps by this approach requires further validation. Aim: To assess and compare noninvasive detection of sessile serrated polyps >1cm (SSP) by sDNA-MT and a commercial quantitative 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 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-MT in batches using optimized analytical methods (Gastroenterology 2012;142:S770). The sDNA-MT 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: Age/sex distributions were similar in cases and controls: 40% vs 45% > 65 years and 51% vs 59% women, p=NS respectively. SSP median size was 1.2cm (range 1-3cm), 93% were proximal, and 64% had synchronous diminutive polyps. Among sDNA-MT markers, mBMP3 proved highly discriminant for SSP detection (AUC=0.87, p<0.00001); other DNA markers were less informative and provided no incremental sensitivity. Hemoglobin alone showed no discrimination (AUC=0.50, p=NS). At matched specificities, SSP detection by stool mBMP3 was significantly greater than by either FIT-50 or FIT-100 (Table). Conclusions: From a screening and surveillance setting, SSP can be detected noninvasively by stool assay of exfoliated DNA markers, especially mBMP3. FIT appears to have no value in SSP detection. [Table: see text]

Abstract Background: Adenomatous polyps are the most common precursor to colorectal cancer, the second leading cause of cancer-related death in the United States. We sought to learn more about early events of carcinogenesis by investigating shifts in the gut microbiota of patients with adenomas. Methods: We analyzed 16S rRNA gene sequences from the fecal microbiota of patients with adenomas (n = 233) and without (n = 547). Results: Multiple taxa were significantly more abundant in patients with adenomas, including Bilophila, Desulfovibrio, proinflammatory bacteria in the genus Mogibacterium, and multiple Bacteroidetes species. Patients without adenomas had greater abundances of Veillonella, Firmicutes (Order Clostridia), and Actinobacteria (family Bifidobacteriales). Our findings were consistent with previously reported shifts in the gut microbiota of colorectal cancer patients. Importantly, the altered adenoma profile is predicted to increase primary and secondary bile acid production, as well as starch, sucrose, lipid, and phenylpropanoid metabolism. Conclusions: These data hint that increased sugar, protein, and lipid metabolism along with increased bile acid production could promote a colonic environment that supports the growth of bile-tolerant microbes such as Bilophilia and Desulfovibrio. In turn, these microbes may produce genotoxic or inflammatory metabolites such as H2S and secondary bile acids, which could play a role in catalyzing adenoma development and eventually colorectal cancer. Impact: This study suggests a plausible biological mechanism to explain the links between shifts in the microbiota and colorectal cancer. This represents a first step toward resolving the complex interactions that shape the adenoma–carcinoma sequence of colorectal cancer and may facilitate personalized therapeutics focused on the microbiota. Cancer Epidemiol Biomarkers Prev; 26(1); 85–94. ©2016 AACR.

Abstract Purpose: Methylated DNA markers can discriminate upper from lower GI neoplasms (Gastroenterology 2013;144(5):S84). It is unknown if organ-specific molecular prediction of GI tumor site can be achieved. Experimental design: An unbiased search for neoplasm markers at each major GI organ site was conducted by reduced representation bisulfite sequencing. Top marker candidates were then blindly validated by methylation specific PCR on independent tissue DNA samples from normal mucosa [17 esophagus (E), 13 stomach (S), 33 pancreas (P), 35 bile duct/liver (BD), 21 colorectal (CR)] and neoplasia [42 E, 43 S, 36 P, 48 BD, 97 CR]. Recursive partitioning (rPart) trees modeled all stepwise combinations of single markers to classify controls vs neoplasms and then neoplasms by site (CR, gastroesophageal or pancreaticobiliary). A 2nd model was designed from tissue data for blinded clinical piloting on 42 independent plasma DNA samples (14 normal and 28 cancers (14 P, 14 CR). Results: From the top 100 markers, 95 were validated and used for rPart modeling. A 3- marker panel (chr12.133484978-5047, BMP3, chr11.123301058-255) was selected for the universal detection of GI neoplasms, as it classified neoplasms and control tissues with 95% accuracy. Two markers (chr7.25896389-501, QKI) then assigned lower vs upper GI neoplasms with 94% accuracy. Finally, 3 markers (PDGFD, ELOVL2, PCBP3) called pancreaticobiliary vs gastroesophageal neoplasms with 94% accuracy. All 8 markers applied in a single model (Table) accurately predicted control vs tumor by site [88% (p &lt;0.0001)]. In plasma, a 2-marker panel (BMP3, QKI) was 78% accurate in assigning patients to cancer or control groups (p&lt;0.0009) and 83% accurate in assigning cancer origin to CR or P sites (p&lt;0.0001). Predicted by ModelTissue SourceControlsColorectal neoplasiaPancreatico-biliary cancerGastro-esophageal neoplasiaControls1169102Colorectal neoplasia18617Pancreatico-biliary cancer10654Gastro-esophageal neoplasia12872 Conclusion: Panels of methylated DNA markers were identified for both universal detection of GI neoplasia and accurate prediction of tumor site. Pilot testing of these markers in plasma supports potential clinical feasibility. Citation Format: John B. Kisiel, William R. Taylor, Tracy C. Yab, Hassan M. Ghoz, Patrick H. Foote, Mary E. Devens, Douglas W. Mahoney, Thomas C. Smyrk, Lisa A. Boardman, Gloria M. Petersen, Navtej S. Buttar, Lewis R. Roberts, Graham P. Lidgard, David A. Ahlquist. Accurate site prediction of gastrointestinal cancer by novel methylated DNA markers: Discovery & validation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4252. doi:10.1158/1538-7445.AM2015-4252

Abstract Purpose: Discriminant markers for pancreatic cancer detection are needed. We sought to identify and validate methylated DNA markers for pancreatic cancer using next-generation sequencing unbiased by known targets. Experimental Design: At a referral center, we conducted four sequential case–control studies: discovery, technical validation, biologic validation, and clinical piloting. Candidate markers were identified using variance-inflated logistic regression on reduced-representation bisulfite DNA sequencing results from matched pancreatic cancers, benign pancreas, and normal colon tissues. Markers were validated technically on replicate discovery study DNA and biologically on independent, matched, blinded tissues by methylation-specific PCR. Clinical testing of six methylation candidates and mutant KRAS was performed on secretin-stimulated pancreatic juice samples from 61 patients with pancreatic cancer, 22 with chronic pancreatitis, and 19 with normal pancreas on endoscopic ultrasound. Areas under receiver-operating characteristics curves (AUC) for markers were calculated. Results: Sequencing identified &gt;500 differentially hyper-methylated regions. On independent tissues, AUC on 19 selected markers ranged between 0.73 and 0.97. Pancreatic juice AUC values for CD1D, KCNK12, CLEC11A, NDRG4, IKZF1, PKRCB, and KRAS were 0.92*, 0.88, 0.85, 0.85, 0.84, 0.83, and 0.75, respectively, for pancreatic cancer compared with normal pancreas and 0.92*, 0.73, 0.76, 0.85*, 0.73, 0.77, and 0.62 for pancreatic cancer compared with chronic pancreatitis (*, P = 0.001 vs. KRAS). Conclusions: We identified and validated novel DNA methylation markers strongly associated with pancreatic cancer. On pilot testing in pancreatic juice, best markers (especially CD1D) highly discriminated pancreatic cases from controls. Clin Cancer Res; 21(19); 4473–81. ©2015 AACR.

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.

SummaryBackgroundCurrent approaches to the detection of colorectal neoplasia associated with inflammatory bowel disease (IBD‐CRN) are suboptimal.AimTo test the feasibility of using stool assay of exfoliated DNA markers to detect IBD‐CRN.MethodsThis investigation comprised tissue and stool studies. In the tissue study, gene sequencing and methylation assays were performed on candidate genes using tissue DNA from 25 IBD‐CRNs and from 25 IBD mucosae without CRN. Mutations on p53, APC, KRAS, BRAF or PIK3CA genes were insufficiently informative, but several aberrantly methylated genes were highly discriminant. In the stool study, we evaluated candidate methylated genes (vimentin, EYA4, BMP3, NDRG4) in a prospective blinded study on buffered stools from 19 cases with known IBD‐CRN and 35 age‐ and sex‐matched IBD controls without CRN. From stool‐extracted DNA, target genes were assayed using quantitative allele‐specific real‐time target and signal amplification method.ResultsIBD‐CRN cases included 17 with ulcerative colitis (UC) and two with Crohn's disease (CD); nine had cancer and 10 had dysplasia. Controls included 25 with UC and 10 with CD. Individually, BMP3, vimentin, EYA4 and NDRG4 markers showed high discrimination in stools with respective areas under the ROC curve of 0.91, 0.91, 0.85 and 0.84 for total IBD‐CRN and of 0.97, 0.97, 0.95 and 0.85 for cancer. At 89% specificity, the combination of BMP3 and mNDRG4 detected 9/9 (100%) of CRC and 80% of dysplasia, 4/4 (100%) of high grade and 4/6 (67%) of low grade.ConclusionThese findings demonstrate the feasibility of stool DNA testing for non‐invasive detection of colorectal neoplasia associated with inflammatory bowel disease.

Abstract Aberrantly methylated genes represent attractive and broadly informative markers for colorectal cancer (CRC). As demographics, numerous ingestants, environmental exposures, and obesity variably influence both gene methylation rates and CRC incidence, it is important to understand the effects of such covariates on the clinical performance of CRC screening tests that incorporate assay of methylated gene markers. Based on a recent multicenter study (Ahlquist et al. Gastroenterology 2012 (in press)), stool assay of carefully selected methylated gene marker candidates (NDRG4, BMP3, vimentin, TFPI2), alone or in combination, yield high detection rates of both CRC and large adenomas. Aim: To assess the impact of demographic, exposure, body mass, and other patient variables on stool levels of the above 4 methylated gene markers. Methods: We studied buffered stools collected within 3 years of a normal colonoscopy from 500 patients undergoing average-risk screening or polyp surveillance (median age 64 (range 44-85); 53% women). Upon receipt, stools were promptly homogenized, aliquoted, and frozen at –80°C. On supernatants from thawed/spun aliquots, target genes were purified by hybrid capture, bisulfite treated, and assayed using the analytically-sensitive QuARTS method (quantitative allele-specific real-time target and signal amplification), as described (above citation). The reference gene β-actin was assayed along with methylation of NDRG4, BMP3, vimentin, TFPI2. Log-converted data were normalized to allow comparison of effects between markers, and effect size was expressed as % change relative to standard deviation for each marker (standardized relative change (SRβ)). Results: The only patient characteristic that significantly influenced all methylated marker levels in stool was age (p&lt;0.0001 for each marker); SRΔ was greatest with TFPI2 at +91.3 (p&lt;0.001 vs other markers), least with BMP3 at +29.7 (p&lt;0.001 vs others), and intermediate with vimentin at +46.0 and NDRG4 at +45.1. In contrast to methylation markers, levels of β-actin did not change across age. The other demographic variables of sex, race, and geographic residence had no effect on methylation markers. Marker levels were also not affected by exposure variables (smoking, alcohol consumption, or analgesic use), by family history of CRC or polyps, or by personal history of polyps. Finally, neither overweight status (n=176) nor obesity (n=148) had an effect on marker levels. Conclusions: Age significantly affects stool levels of tumor-associated methylated gene markers in colonoscopy-normal patients, and the degree of this age effect differs across markers. In contrast, other key patient variables had no effect on stool marker levels. Comment: Optimal use of these methylation markers for stool screening of CRC may require age-adjustment of normal cutoff levels to maximize test specificity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3572. doi:1538-7445.AM2012-3572

Abstract BACKGROUND Aberrantly methylated genes represent important markers for cancer diagnosis. We describe a multiplex detection approach to efficiently quantify these markers for clinical applications such as colorectal cancer screening. METHODS Quantitative allele-specific real-time target and signal amplification (QuARTS) combines a polymerase-based target amplification with an invasive cleavage-based signal amplification. The fluorescence signal is detected in a fashion similar to real-time PCR. We measured the dynamic range and analytical sensitivity of multiplex QuARTS reactions with titrated plasmid DNA. We used the QuARTS technology to quantify methylated BMP3, NDRG4, VIM, and TFPI2 genes on 91 DNA samples extracted from colorectal tissues, including 37 cancers, 25 adenomas, and 29 healthy epithelia. The assays were designed in triplex format that incorporated ACTB as a reference gene. Percent methylation was calculated by dividing methylated strands over ACTB strands and multiplying by 100. RESULTS The QuARTS method linearly detected methylated or unmethylated VIM gene down to 10 copies. No cross-reactivity was observed when methylated assays were used to amplify 105 copies of unmethylated gene and vice versa. The multiplex assay detected methylated genes spiked in unmethylated genes at a 0.01% ratio and vice versa. At a diagnostic specificity cutoff of 95%, methylated BMP3, NDRG4, VIM, and TFPI2 detected 84%, 92%, 86%, and 92% of colorectal cancers and 68%, 76%, 76%, and 88% of adenomas, respectively. CONCLUSIONS The QuARTS technology provides a promising approach for quantifying methylated markers. The markers assayed highly discriminated colorectal neoplasia from healthy epithelia.