Then, Sara S.

Introduction: Non-invasive assays are needed to better discriminate patients with prostate cancer (PCa) to avoid over-treatment of indolent disease. We analyzed 14 methylated DNA markers (MDMs) from urine samples of patients with biopsy-proven PCa relative to healthy controls and further studied discrimination of clinically significant PCa (csPCa) from healthy controls and Gleason 6 cancers. Methods: To evaluate the panel, urine from 24 healthy male volunteers with no clinical suspicion for PCa and 24 men with biopsy-confirmed disease across all Gleason scores was collected. Blinded to clinical status, DNA from the supernatant was analyzed for methylation signal within specific DNA sequences across 14 genes (HES5, ZNF655, ITPRIPL1, MAX.chr3.6187, SLCO3A1, CHST11, SERPINB9, WNT3A, KCNB2, GAS6, AKR1B1, MAX.chr3.8028, GRASP, ST6GALNAC2) by target enrichment long-probe quantitative-amplified signal assays. Results: Utilizing an overall specificity cut-off of 100% for discriminating normal controls from PCa cases across the MDM panel resulted in 71% sensitivity (95% CI: 49–87%) for PCa detection (4/7 Gleason 6, 8/12 Gleason 7, 5/5 Gleason 8+) and 76% (50–92%) for csPCa (Gleason ≥ 7). At 100% specificity for controls and Gleason 6 patients combined, MDM panel sensitivity was 59% (33–81%) for csPCa (5/12 Gleason 7, 5/5 Gleason 8+). Conclusions: MDMs assayed in urine offer high sensitivity and specificity for detection of clinically significant prostate cancer. Prospective evaluation is necessary to estimate discrimination of patients as first-line screening and as an adjunct to prostate-specific antigen (PSA) testing.

Background: Radiographic surveillance of colorectal cancer (CRC) after curative-intent therapy is costly and unreliable. Methylated DNA markers (MDMs) detected primary CRC and metastatic recurrence with high sensitivity and specificity in cross-sectional studies. This study evaluated using serial MDMs to detect recurrence and monitor the treatment response to anti-cancer therapies. Methods: A nested case-control study was drawn from a prospective cohort of patients with CRC who completed curative-intent therapy for CRC of all stages. Plasma MDMs were assayed vis target enrichment long-probe quantitative-amplified signal assays, normalized to B3GALT6, and analyzed in combination with serum carcinoembryonic antigen to yield an MDM score. Clinical information, including treatment and radiographic measurements of the tumor burden, were longitudinally collected. Results: Of the 35 patients, 18 had recurrence and 17 had no evidence of disease during the study period. The MDM score was positive in 16 out of 18 patients who recurred and only 2 of the 17 patients without recurrence. The MDM score detected recurrence in 12 patients preceding clinical or radiographic detection of recurrent CRC by a median of 106 days (range 90–232 days). Conclusions: Plasma MDMs can detect recurrent CRC prior to radiographic detection; this tumor-agnostic liquid biopsy approach may assist cancer surveillance and monitoring.

Abstract Background: Reliable non-invasive biomarkers for human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPX) are lacking. We performed a discovery and validation study of methylated DNA markers (MDMs) for detection of OPX. In addition, we sought to evaluate the MDM profiles in saliva gargle samples from normal controls (nSAL). Methods: Formalin fixed paraffin embedded OPX and gender-balanced normal oropharyngeal tissue (NOP) were pathologically reviewed and punched for DNA extraction and sequenced using reduced representation bisulfite sequencing (RRBS). A tiling algorithm was used to define candidate MDMs. Highest ranked (e.g., area under the receiver operating characteristic curve (AUC), fold-change, p-value) candidate MDMs were validated by qMSP in independent OPX and NOP samples. nSAL DNA was used to assess MDM background signal. Discrimination between OPX tissue, NOP, and nSAL was assessed using AUC with corresponding 95% confidence intervals. HPV was confirmed with p16 staining or DNA in situ hybridization. Results: RRBS discovery assessed 18 OPX, 18 NOP, and 18 normal WBC samples. 234 MDM candidates from approximately 3 × 106 CpGs at >10X coverage were identified. 36 MDMs were selected for validation in independent patient samples which included 32 OPX, 36 NOP, and 35 nSAL specimens. OPX patients were older (median 51; IQR 45-54 years) than NOP patients (median 29; IQR 24-42). 69 and 61% of OPX and NOP patients were female, respectively. The majority of OPX patients were Stage I (n=27, 84%). We observed excellent discrimination between OPX and NOP in the validation set; the median AUC across 36 MDMs was 0.90 (IQR 0.87-0.95). The 10 highest performing markers (PARP15, EPDR1, SHROOM1, EMBP1.rs, IFFO1, MAX.chr19.118, ZNF763, ITGB4, FAM19A2, and MT1IP) exhibited AUCs ranging from 0.95-0.99. Strong discrimination was also observed between OPX and nSAL (Median AUC=0.97; IQR=0.93-0.99). All 10 of the highest-performing markers in tissue exhibited AUCs of at least 0.97 for discriminating OPX versus nSAL. Median fold change between NOP and nSAL across all MDMs was 1.9 (IQR 1.4-3.8), demonstrating low background signal in nSAL. Conclusions: We found strong discrimination in a panel of 36 MDMs between OPX case and control tissue as well as between OPX case tissue and normal saliva. The low background signal observed in normal saliva makes this media a strong candidate for non-invasive detection of OPX. Further studies are in development to assess the utility of measuring these MDMs in both case and control saliva and blood for detecting OPX non-invasively. Citation Format: Benjamin R. Gochanour, David M. Routman, Kathleen R. Bartemes, William R. Taylor, Douglas W. Mahoney, Rondell P. Graham, Xiaoming Cao, Calise K. Berger, Sara S. Then, Karen A. Doering, Kelli N. Burger, Patrick H. Foote, John B. Kisiel, Kathryn M. Van Abel. Discovery and validation of methylated DNA markers of human papillomavirus associated oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-039.

Abstract Objective: Upper tract urothelial cancers (UTUC) can be difficult to characterize, and non-invasive surveillance tools are lacking. UTUC are often treated with nephroureterectomy (NU). To address these gaps, we sought to discover and validate methylated DNA markers (MDMs) for detection of UTUC. Methods: Archival formalin fixed paraffin embedded (FFPE) tissue samples from NU were pathologically reviewed and punched prior to DNA extraction. Reduced representation bisulfite sequencing (RRBS) was used to identify candidate MDMs that discriminated between UTUC and age-balanced control urothelial tissues (uninvolved ureter/renal pelvic of renal cell carcinoma at radical nephrectomy). Highest ranked (fold-change and p-value) candidate MDMs were biologically validated by quantitative methylation specific PCR in a 2nd independent set of UTUC and age-balanced controls. DNA from healthy donor urine and buffy coat samples was used to assess MDM background signal. Discrimination between UTUC and controls was assessed as the area under the receiver operator characteristic curve (AUC) with corresponding 95% confidence intervals. Results: RRBS assessed 33 UTUC and 26 ureter/renal pelvis controls. 3.01 x 106 CpGs mapped to the reference genome with at least 10X read depth. From 358 candidates, 20 MDMs were selected for biological validation in independent patient samples which included 20 renal pelvis & 16 ureter UTUC and 17 renal pelvis & 15 ureter control specimens. Of UTUC and control patients, 36% and 65% of were men, respectively. Median AUC for comparison of UTUC vs control tissue was 0.87 (IQR 0.81-0.88); AUCs with corresponding 95% CIs are shown for the 10 most accurate MDMs (Table) across all control types. Conclusion: We report discovery and validation of highly sensitive and specific MDMs with potential for accurate non-invasive screening supported by low background in urine and blood. AUCs (95% CI) for selected methylated DNA markers for UTUC vs control tissue, urine, and blood UTUC vs Marker Control Tissue Control Urine Control Buffy C2orf55 0.92 (0.84-1) 0.93 (0.87-1) 0.96 (0.91-1) ANKRD35 0.91 (0.84-0.99) 0.93 (0.86-1) 0.98 (0.94-1) DLX6 0.91 (0.84-0.98) 0.78 (0.66-0.9) 0.97 (0.93-1) Chr8.9998 0.90 (0.82-0.97) 0.91 (0.84-0.97) 0.98 (0.96-1) SP9 0.88 (0.8-0.97) 0.95 (0.9-1) 0.99 (0.98-1) Chr13.1127 0.88 (0.8-0.97) 0.94 (0.89-1) 0.92 (0.85-1) Septin9.r2 0.88 (0.79-0.97) 0.95 (0.88-1) 0.98 (0.94-1) LBX2 0.88 (0.79-0.97) 0.94 (0.88-1) 0.96 (0.91-1) SIM2.r1 0.88 (0.79-0.97) 0.91 (0.82-1) 0.94 (0.86-1) Chr14.1037 0.88 (0.78-0.97) 0.93 (0.85-1) 0.96 (0.9-1) Citation Format: Ojas Bhagra, William R. Taylor, Benjamin R. Gochanour, Patrick H. Foote, Calise K. Berger, Xiaoming Cao, Douglas W. Mahoney, Sara S. Then, Anna M. Gonser, Karen A. Doering, Kelli N. Burger, John C. Cheville, Matthew T. Gettman, John B. Kisiel. Discovery and validation of methylated DNA markers of upper tract urothelial tract cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3349.

Abstract Objectives: We hypothesize that a validated panel of methylated DNA markers (MDMs) for HPV(+)CSCC will be similar to but distinct from a cohort of HPV(+)OPSCC. Methods: Patients were excluded for recurrent tumor, prior pelvic or head or neck cancer/neoplasia, chemotherapy < 1 year, prior therapeutic radiation to the target region, prior transplant, insufficient target tissue (<5mm), or if <18 yrs. MDMs identified from sequenced differentially methylated regions were selected from a panel validated for HPV(+)CSCC and evaluated on DNA from independent FFPE HPV(+)OPSCC, HPV(+)CSCC, normal oropharynx tonsil, and normal cervix tissue using methylation-specific PCR. White blood cells (WBCs) were used as a background control. Results: 34 HPV(+)OPSCC, 36 HPV(+)CSCC, 26 normal tonsil tissue, and 24 normal cervical tissue patients met criteria. The HPV(+)OPSCC cohort was older (57 vs 44 yrs, p=0.027), had more frequent alcohol exposure (85% vs 64%, p=0.02), but similar ACE-27 comorbidity scores (p=0.078) and tobacco use (p=0.066). Tumor stages were stage I (47% and 83%), stage II (44% and 11%) and stage III (9% and 6%) for HPV(+)OPSCC and HPV(+)CSCC, respectively. Twenty-one MDMs were evaluated and areas under the receiver operator characteristic curve (AUC) are reported (Table 1). 95% confidence intervals excluded a non-diagnostic null hypothesis AUC of 0.5. 5/21 (24%) achieved an AUC ≥ 0.90, 15/21 (71%) achieved an AUC ≥ 0.8, and 19/21 (90%) exhibited better than chance classifications relative to control tonsil tissue (all p<0.001). Conclusion: MDMs have the potential to discriminate both HPV(+)OPSCC and HPV(+)CSCC from their normal tissue controls. 90% of the MDMs discovered for HPV(+)CSCC had better than chance discrimination in HPV(+)OPSCC suggesting a distinct signature between the two subtypes. Future discovery efforts specific to HPV(+)OPSCC is warranted. Methylated DNA Markers in HPV(+)OPSCC and HPV(+)CSCC CSCC AUC (95% CI) OPSCC AUC (95% CI) AUC CSCC vs AUC OPSCC Pvalue GRIN2D 0.72 (0.59-0.85) 0.91 (0.83-0.99) 0.019 EMX1 0.93 (0.86-0.99) 0.77 (0.65-0.89) 0.0191 VWC2 0.88 (0.79-0.97) 0.57 (0.42-0.72) 0.0001 ZNF610 0.99 (0.98-1) 0.89 (0.8-0.97) 0.0162 ZNF781.F 0.92 (0.86-0.99) 0.79 (0.67-0.91) 0.0585 TBX15 0.99 (0.96-1) 0.82 (0.72-0.93) 0.0025 TSPYL5 0.94 (0.87-1) 0.82 (0.72-0.93) 0.058 LOC645323 0.95 (0.89-1) 0.88 (0.8-0.97) 0.1952 ASCL1 0.98 (0.93-1) 0.86 (0.76-0.95) 0.0187 ABCB1 0.97 (0.93-1) 0.74 (0.61-0.86) 0.0001 ZNF69 0.89 (0.8-0.97) 0.95 (0.9-1) 0.2017 MAX.chr9 0.97 (0.92-1) 0.82 (0.72-0.93) 0.0113 ARHGAP12 0.91 (0.83-0.99) 0.9 (0.81-1) 0.9223 C1orf114 0.94 (0.86-1) 0.88 (0.8-0.97) 0.3366 MAX.chr6 0.99 (0.97-1) 0.86 (0.76-0.96) 0.0099 NEUROG3 0.95 (0.89-1) 0.7 (0.57-0.83) 0.0001 NID2 0.98 (0.95-1) 0.86 (0.77-0.95) 0.0207 TMEM200C 0.95 (0.89-1) 0.54 (0.39-0.69) 0.0001 TTYH1 0.99 (0.98-1) 0.82 (0.71-0.93) 0.0023 ZNF773 0.93 (0.86-1) 0.9 (0.82-0.98) 0.5696 ZNF781.R 1 (1-1) 0.92 (0.86-0.99) 0.0198 Citation Format: Kathryn M. Van Abel, David M. Routman, Daniel J. Ma, Karen A. Doering, Kelli N. Burger, Patrick H. Foote, William R. Taylor, Douglas W. Mahoney, Calise K. Berger, Xiaoming Cao, Sara S. Then, Alyssa M. Larish, Eric J. Moore, Joaquin G. Garcia, Rondell P. Graham, Jamie N. Bakkum-Gamez, John B. Kisiel. Comparison of methylated DNA markers between human papillomavirus mediated carcinoma in oropharynx and cervical cancer: A pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3742.

5091 Background: Controversy surrounding prostate-specific antigen (PSA) based screening for prostate cancer (PCa) highlights the need for non-invasive assays that better discriminate patients with and without clinically significant prostate cancer (csPCa). Such distinction avoids overtreatment in patients with absent or indolent disease while capturing pathology that would derive benefit from intervention. We analyzed the presence of specific methylated DNA markers (MDMs) within urine samples of patients with biopsy proven PCa and assessed the ability to discriminate these patients from healthy controls with no clinical suspicion for PCa. Methods: 24 healthy volunteers with no clinical suspicion for PCa were age-matched to 24 patients with biopsy-confirmed disease across all Gleason scores. Urine collected from subjects was centrifuged with the cell-free supernatant analyzed in a blinded fashion for methylation signal within specific DNA sequences across 14 genes ( HES5, ZNF655, ITPRIPL1, MAX.chr3.193, SLCO3A1, CHST11, SERPINB9, WNT3A, KCNB2, GAS6, AKR1B1, MAX.chr3.727, GRASP, ST6GALNAC2) by target enrichment long-probe quantitative-amplified signal assays. A patient was considered to have a positive MDM panel if any individual marker exceeded the corresponding100% specificity cut-off value (overall MDM panel specificity of 100%). MDM panel positivity was used to evaluate the sensitivity for distinguishing patients with PCa (any Gleason score) from controls as well as discriminate csPCa cancers from PCa Gleason 6. Results: Median age of healthy controls and PCa patients was 70 years (IQR 67-72) and 65 years (IQR 61-71), respectively. Median PSA was 6.4 (IQR 4.9-9.0) among PCa patients, including median PSA of 5 (IQR 4.0-6.1) for Gleason 6, and 7.8 (IQR 5.1-9.2) for Gleason ≥7 disease. Gleason 6, Gleason 7, and Gleason 8+ cancer was noted in 8, 10, and 6 patients, respectively. Utilizing an overall specificity cut-off of 100% for discriminating normal controls from PCa cases across the MDM panel revealed an overall sensitivity of 83% (95% CI: 63-95%) for detection of PCa (6 of 8 Gleason 6, 9 of 10 Gleason 7, 5 of 6 Gleason 8+) and 88% (95% CI: 62-98%) for csPCa (Gleason ≥ 7). When considering a 100% specificity threshold for controls and Gleason 6 patients, the sensitivity the MDM panel was 69% (95% CI: 41-89%) for csPCa (6 of 10 Gleason 7, 5 of 6 Gleason 8+). Conclusions: We describe a panel of 14 MDMs within urine that offer high specificity and sensitivity for detection of prostate cancers as well as selective identification of clinically significant disease states. Prospective comparison between urine MDMs and PSA blood testing is necessary to discern the differential clinical impact of each screening methodology.

e15567 Background: Radiographic surveillance of colorectal cancer (CRC) after therapy is costly and insensitive. We have reported high cross-sectional performance of methylated DNA markers (MDMs) for detection of primary CRC and metastatic recurrence. This study evaluated MDMs to detect recurrence and measure response to anti-cancer therapy in a longitudinal cohort. Methods: This was a nested case-control study drawn from a prospective cohort of adult patients with CRC who completed definitive treatment and followed forward from the start of surveillance by cross-sectional imaging (CT or MRI) and carcinoembryonic antigen (CEA), collected every 3-6 months under routine care. Blood was collected in LBgard® tubes (Biomatrica, San Diego CA). Cases had recurrent CRC (defined by RECIST v1.1 or radiographic findings that justify re-initiation of anti-cancer therapy as determined by treating clinicians). Controls remained NED (no radiographic recurrence and off therapy for ≥4 surveillance visits). From 6mL of plasma, MDMs ( CNNM1, ANKRD13B, FER1L4, ZNF568, CHST2, ZNF671, VAV3, QKI, GRIN2D, DTX1, PDGFD, SFMBT2, JAM3) were measured by target enrichment long-probe quantitative-amplified signal assays, normalized to B3GALT6, in blinded fashion. Previously published random forest models of MDMs +/- CEA, developed on independent patients with intact primary tumors, were used to develop a summary score. Results: With a median follow-up of 276 days (IQR 187-343) and 3 visits per patient (54 visits total), 18 patients had recurrence after start of surveillance. Another 18 patients remained NED after median 555 days (IRQ 469-662) and 4 surveillance visits per patient (76 visits total). Using a 90th %-ile cut-off summary score from 60 NED patients in a prior cross-sectional measurement, the MDM panel was positive in 15 of 18 patients who recurred and only 2 of the 18 NED patients (Table). Of the 76 patient visits for NED patients, 67 (88%) were below the threshold. The MDM panel score crossed the positivity threshold by a median of 127 days (IRQ 0-233) preceding clinical or radiographic documentation of recurrence. A random forest model with CEA was more sensitive but less specific (Table). In patients who resumed therapy, the MDM panel scores correlated closely with subsequent RECIST scores (not shown). Conclusions: Plasma MDMs rise in anticipation of recurrent CRC prior to radiographic detection and remain low in patients without radiographic disease over serial measurements, warranting further development of this tumor-agnostic liquid biopsy approach as a cost-effective tool to assist cancer surveillance.[Table: see text]

e15511 Background: Aberrantly methylated DNA marker (MDM) candidates are strongly associated with primary colorectal cancer (CRC) before treatment and detect CRC recurrence with high sensitivity when assayed from plasma. The relationship of these MDMs in association to chemotherapy treatment response is unknown. Methods: In a prospective cohort of patients receiving systemic therapy for advanced CRC, peripheral blood was collected serially during restaging visits. 15 patients were retrospectively identified to have partial response (PR), stable disease (SD) and progressive disease (PD) to treatment (n=5 for each group) based on the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Using paired samples from each patient before and after response assessment, we analyzed 11 MDMs ( GRIN2D, ZNF671, ANKRD13B, QKI, VAV3, JAM3, SFMBT2, CHST2, ZNF568, FER1L4 and CNNM1) to assess correlation with treatment response. Cell-free DNA was extracted and bisulfite treated before MDMs were quantified by target enrichment long-probe quantitative-amplified signal assay and normalized to a methylated sequence of B3GALT6. Continuous variables are summarized as a median with corresponding interquartile ranges (IQR) and comparisons between subgroups were based on the Wilcox Rank Sums test. Results: The median interval between pre- and post-response assessment visits was 69 days (IQR: 63-83 days) and the level of tumor burden at pre-assessment was similar across all response types (Table 1). Patients with PD had higher levels of methylated GRIN2D, ZNF671 and ANKRD13B than those with PR or SD at baseline and may offer additional prognostic value over CEA which was similar in the PR and PD groups before treatment (Table 1). Elevation of pre-assessment MDMs preceded radiographic evidence of disease progression by 82 days (IQR 69-83 days). Conclusions: Three MDMs, GRIN2D, ZNF671 and ANKRD13B, were found to reflect treatment response (PD vs. PR + SD) as shown in the table. Although this pilot study was limited by a small sample size, it demonstrated the feasibility of using plasma-based MDMs in monitoring treatment response to systemic therapy for advanced CRC and should be compared to CEA in a larger study.[Table: see text]

Abstract Purpose: We aimed to assess the concordance of colorectal cancer–associated methylated DNA markers (MDM) in primary and metastatic colorectal cancer for feasibility in detection of distantly recurrent/metastatic colorectal cancer in plasma. Experimental Design: A panel of previously discovered colorectal cancer–associated MDMs was selected. MDMs from primary and paired metastatic colorectal cancer tissue were assayed with quantitative methylation-specific PCR. Plasma MDMs were measured blindly by target enrichment long-probe quantitative-amplified signal assays. Random forest modeling was used to derive a prediction algorithm of MDMs in archival plasma samples from primary colorectal cancer cases. This algorithm was validated in prospectively collected plasma samples from recurrent colorectal cancer cases. The accuracy of the algorithm was summarized as sensitivity, specificity, and area under the curve (AUC). Results: Of the 14 selected MDMs, the concordance between primary and metastatic tissue was considered moderate or higher for 12 MDMs (86%). At a preset specificity of 95% (91%–98%), a panel of 13 MDMs, in plasma from 97 colorectal cancer cases and 200 controls, detected stage IV colorectal cancer with 100% (80%–100%) sensitivity and all stages of colorectal cancer with an AUC of 0.91 (0.87–0.95), significantly higher than carcinoembryonic antigen [AUC, 0.72 (0.65–0.79)]. This panel, in plasma from 40 cases and 60 healthy controls, detected recurrent/metastatic colorectal cancer with 90% (76%–97%) sensitivity, 90% (79%–96%) specificity, and an AUC of 0.96 (0.92–1.00). The panel was positive in 0.30 (0.19–0.43) of 60 patients with no evidence of disease in post-operative patients with colorectal cancer. Conclusions: Plasma assay of novel colorectal cancer–associated MDMs can reliably detect both primary colorectal cancer and distantly recurrent colorectal cancer with promising accuracy.

OBJECTIVES: Multitarget stool DNA (MT-sDNA) testing has grown as a noninvasive screening modality for colorectal cancer (CRC), but real-world clinical data are limited in the post-FDA approval setting. The effect of previous colonoscopy on MT-sDNA performance is not known. We aimed to evaluate findings of colorectal neoplasia (CRN) at diagnostic colonoscopy in patients with positive MT-sDNA testing, stratified by patient exposure to previous colonoscopy. METHODS: We identified consecutive patients completing MT-sDNA testing over a 39-month period and reviewed the records of those with positive tests for neoplastic findings at diagnostic colonoscopy. MT-sDNA test positivity rate, adherence to diagnostic colonoscopy, and the positive predictive value (PPV) of MT-sDNA for any CRN and neoplastic subtypes were calculated. RESULTS: Of 16,469 MT-sDNA tests completed, testing returned positive in 2,326 (14.1%) patients. After exclusion of patients at increased risk for CRC, 1,801 patients remained, 1,558 (87%) of whom underwent diagnostic colonoscopy; 918 of 1,558 (59%) of these patients had undergone previous colonoscopy, whereas 640 (41%) had not. Any CRN was found in 1,046 of 1,558 patients (PPV = 67%). More neoplastic lesions were found in patients without previous colonoscopy (73%); however, the rates remained high among those who had undergone previous colonoscopy (63%, P < 0.0001). The large majority (79%) of patients had right-sided neoplasia. DISCUSSION: MT-sDNA has a high PPV for any CRN regardless of exposure to previous colonoscopy. Right-sided CRN was found at colonoscopy in most patients with positive MT-sDNA testing, representing a potential advantage over other currently available screening modalities for CRC.