Kisiel, John B.

10545 Background: Approximately 20–30% of colorectal cancers (CRC) arise from the serrated polyp pathway. The multitarget stool DNA (mt-sDNA) test has greater sensitivity to detect sessile serrated polyps (SSPs) than a leading fecal immunochemical test (FIT). However, most modeling analyses do not account for the contribution of the SSP pathway to risk of CRC. We used the CRC-AIM model to assess the impact of the SSP pathway on predicted CRC outcomes with mt-sDNA or FIT screening. Methods: A simulated cohort of average-risk US patients underwent triennial mt-sDNA or annual FIT screening from ages 50–75. The percentage of CRCs arising from the SSP pathway were modeled at 0% (base case), 20%, and 30%, with stool screening adherence based on theoretical (100%) or previously reported (mt-sDNA 71%; FIT 43%) rates. Published SSP sensitivities for mt-sDNA and FIT were used. All other model inputs were identical to CISNET models. Sensitivity analyses used screening adherence rates of 40–70%. Key outcomes were life-years gained (LYG), CRC incidence and CRC mortality per 1000 patients. Results: Including SSPs in the model demonstrated a greater loss of LYG with FIT than mt-sDNA (Table). At 100% adherence, compared with base case, modeling 20% or 30% SSP pathway CRCs resulted in a decrease of 9–15 LYG with FIT and 2–4 LYG with mt-sDNA, a decrease in CRC incidence reduction of 3.9–6.1% with FIT and 0.7–1.1% with mt-sDNA, and a decrease in CRC mortality reduction of 2.6–4.0% with FIT and 0.4–0.8% with mt-sDNA. Using previously reported adherence, compared with base case, modeling 20% or 30% SSP pathway CRCs resulted in a decrease of 13–20 LYG with FIT and 2–5 LYG with mt-sDNA, a decrease in CRC incidence reduction of 4.4–6.9% with FIT and 0.6–1.1% with mt-sDNA, and a decrease in CRC mortality reduction of 3.5–5.4% with FIT and 0.4–0.9% with mt-sDNA. Assuming reported adherence and 30% SSP pathway CRCs, mt-sDNA had 48 more LYG, 14.6% greater CRC incidence reduction, and 12.4% greater CRC mortality reduction than FIT. Assuming 30% SSP pathway CRCs, outcomes favored mt-sDNA vs FIT even after modeling equivalent adherence rates ranging from 40–70%. Conclusions: After incorporating the SSP pathway into the model, outcomes with mt-sDNA neared those of FIT at 100% screening adherence rates and surpassed FIT at more realistic reported screening adherence rates.[Table: see text]

5526 Background: HR-HPV DNA testing, with or without cervical cytology, provides excellent sensitivity for detection of cervical cancer (CC) and its precursors; negative test results indicate that risk of disease is extremely low and enable women to undergo reduced screening with safety. However, management of women who screen positive remains challenging as many will prove to have self-limited HR-HPV infections. DNA methylation is an early event in carcinogenesis that could enhance CC screening specificity. Methods: For discovery, DNA from 70 FFPE CC (36 squamous, 34 adenocarcinoma) tissues that were reviewed microscopically, 18 fresh frozen benign cervicovaginal (BCV) tissues collected at the time of benign hysterectomy, and 18 buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify MDMs associated with CC. Candidate MDM selection was based on area under the receiver operating characteristic curve (AUC) discrimination, methylation fold change, and low background methylation among benign controls. Candidate MDMs were re-tested using methylation-specific PCR (MSP) to confirm performance. Blinded biological validation was performed using MSP on DNA extracted from independent FFPE CC (38 squamous, 43 adenocarcinoma) and BCV (40) tissues. The performance of CC MDMs was also tested in DNA extracted from cervical dysplasia (36 adenocarcinoma in situ (AIS), 32 cervical intraepithelial neoplasia (CIN) 2/3, 11 CIN 1) FFPE tissues. Results: From RRBS discovery and technical validation via MSP, 30 candidate MDMs showed marked methylation fold changes (10 to >1000) across both CC histologies compared to BCV tissue from cancer-free women. Each of the 30 MDMs highly discriminated CC from BCV tissue with 9 MDMs having an AUC >0.90 (Table). CC MDMs also highly discriminated AIS from BCV but did not perform well in CIN 2/3 and CIN 1 (Table). Conclusions: Whole methylome sequencing, stringent filtering criteria, and biological validation have yielded outstanding candidate MDMs for CC that highly discriminate CC from BCV, notably with high specificity. Performance in cervical dysplasias varied with higher positivity rates in AIS than in CIN 2/3 and CIN 1. Translation to testing these novel MDMs in lower genital tract biospecimens and the addition of HR-HPV to the CC panel are warranted.[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]

Cholangiocarcinoma (CCA) has poor prognosis due to late‐stage, symptomatic presentation. Altered DNA methylation markers may improve diagnosis of CCA. Reduced‐representation bisulfite sequencing was performed on DNA extracted from frozen CCA tissues and matched to adjacent benign biliary epithelia or liver parenchyma. Methylated DNA markers (MDMs) identified from sequenced differentially methylated regions were selected for biological validation on DNA from independent formalin‐fixed, paraffin‐embedded CCA tumors and adjacent hepatobiliary control tissues using methylation‐specific polymerase chain reaction. Selected MDMs were then blindly assayed on DNA extracted from independent archival biliary brushing specimens, including 12 perihilar cholangiocarcinoma, 4 distal cholangiocarcinoma cases, and 18 controls. Next, MDMs were blindly assayed on plasma DNA from patients with extrahepatic CCA (eCCA), including 54 perihilar CCA and 5 distal CCA cases and 95 healthy and 22 primary sclerosing cholangitis controls, balanced for age and sex. From more than 3,600 MDMs discovered in frozen tissues, 39 were tested in independent samples. In the clinical pilot of 16 MDMs on cytology brushings, methylated EMX1 (empty spiracles homeobox 1) had an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.95‐1.0). In the clinical pilot on plasma, a cross‐validated recursive partitioning tree prediction model from nine MDMs was accurate for de novo eCCA (AUC, 0.88 [0.81‐0.95]) but not for primary sclerosing cholangitis–associated eCCA (AUC, 0.54 [0.35‐0.73]). Conclusion : Next‐generation DNA sequencing yielded highly discriminant methylation markers for CCA. Confirmation of these findings in independent tissues, cytology brushings, and plasma supports further development of DNA methylation to augment diagnosis of CCA.

Abstract Background Certolizumab pegol (CZP) has been successfully used for the treatment of Crohn disease (CD); however, real-world data regarding the utility of CZP trough levels (CTLs) are lacking. We aimed to correlate CTL with CD outcomes and to determine frequency of CZP antibodies. Methods Retrospective evaluation of all CD patients on maintenance CZP with CTL obtained between 2016 and 2019. Outcomes included: median CTL, presence of anti-CZP antibodies, biochemical response (BR), clinical response (CR), radiologic response (RR), radiologic healing (RH), and mucosal healing (MH). Results Seventy-seven CD patients were included. Median CTL was 18.9 µg/mL (interquartile range, 7.6–35.4). Twenty-three patients (27.3%) had positive antibody levels, with lower median CTL compared to patients with no antibodies (0.0 vs 29.8; P < 0.0001). Median CTL levels were higher in patients with vs without CR (30.4 vs 10.3 µg/mL; P = 0.0015) and RR (29.6 vs 5.8 µg/mL; P = 0.006). CZP dosing at least every 2 weeks was associated with higher odds of achieving MH (odds ratio, 3.2; 95% confidence interval, 1.03–9.97). CTL resulted in change in clinical management in 62.7% of cases and presence of CMZ antibodies was associated with an odds ratio of 5.83 (95% confidence interval, 1.57–21.73) of change in management. Receiver operating characteristic curve and quartile analysis suggested that CTL >19 µg/mL is associated with increased rates of CR and RR. Conclusions Higher CTL was significantly associated with CR and RR. The rate of CZP antibodies was 27.3%. Our data suggest maintenance CTL of ≥19 µg/mL should be achieved in order to optimize outcomes in clinical practice.

Abstract Polyphenon E (Poly E) is a green tea polyphenol preparation whose most active component is epigallocatechin gallate (EGCG). We studied the cancer preventive efficacy and safety of Poly E in subjects with rectal aberrant crypt foci (ACF), which represent putative precursors of colorectal cancers. Eligible subjects had prior colorectal advanced adenomas or cancers, and had ≥5 rectal ACF at a preregistration chromoendoscopy. Subjects (N = 39) were randomized to 6 months of oral Poly E (780 mg EGCG) daily or placebo. Baseline characteristics were similar by treatment arm (all P >0.41); 32 of 39 (82%) subjects completed 6 months of treatment. The primary endpoint was percent reduction in rectal ACF at chromoendoscopy comparing before and after treatment. Among 32 subjects (15 Poly E, 17 placebo), percent change in rectal ACF number (baseline vs. 6 months) did not differ significantly between study arms (3.7% difference of means; P = 0.28); total ACF burden was also similar (−2.3% difference of means; P = 0.83). Adenoma recurrence rates at 6 months were similar by arm (P > 0.35). Total drug received did not differ significantly by study arm; 31 (79%) subjects received ≥70% of prescribed Poly E. Poly E was well tolerated and adverse events (AE) did not differ significantly by arm. One subject on placebo had two grade 3 AEs; one subject had grade 2 hepatic transaminase elevations attributed to treatment. In conclusion, Poly E for 6 months did not significantly reduce rectal ACF number relative to placebo. Poly E was well tolerated and without significant toxicity at the dose studied. Prevention Relevance: We report a chemoprevention trial of polyphenon E in subjects at high risk of colorectal cancer. The results show that polyphenon E was well tolerated, but did not significantly reduce the number of rectal aberrant crypt foci, a surrogate endpoint biomarker of colorectal cancer.

Abstract Purpose: We have previously identified tissue methylated DNA markers (MDMs) associated with pancreatic ductal adenocarcinoma (PDAC). In this case–control study, we aimed to assess the diagnostic performance of plasma MDMs for PDAC. Experimental Design: Thirteen MDMs (GRIN2D, CD1D, ZNF781, FER1L4, RYR2, CLEC11A, AK055957, LRRC4, GH05J042948, HOXA1, PRKCB, SHISA9, and NTRK3) were identified on the basis of selection criteria applied to results of prior tissue experiments and assays were optimized in plasma. Next, 340 plasma samples (170 PDAC cases and 170 controls) were assayed using target enrichment long-probe quantitative amplified signal method. Initially, 120 advanced-stage PDAC cases and 120 healthy controls were used to train a prediction algorithm at 97.5% specificity using random forest modeling. Subsequently, the locked algorithm derived from the training set was applied to an independent blinded test set of 50 early-stage PDAC cases and 50 controls. Finally, data from all 340 patients were combined, and cross-validated. Results: The cross-validated area under the receiver operating characteristic curve (AUC) for the training set was 0.93 (0.89–0.96) for the MDM panel alone, 0.91 (95% confidence interval, 0.87–0.96) for carbohydrate antigen 19-9 (CA19-9) alone, and 0.99 (0.98–1) for the combined MDM-CA19-9 panel. In the test set of early-stage PDAC, the AUC for MDMs alone was 0.84 (0.76–0.92), CA19-9 alone was 0.87 (0.79–0.94), and combined MDM-CA19-9 panel was 0.90 (0.84–0.97) significantly better compared with either MDMs alone or CA19-9 alone (P = 0.0382 and 0.0490, respectively). At a preset specificity of 97.5%, the sensitivity for the combined panel in the test set was 80% (28%–99%) for stage I disease and 82% (68%–92%) for stage II disease. Using the combined datasets, the cross-validated AUC was 0.9 (0.86–0.94) for the MDM panel alone and 0.89 for CA19-9 alone (0.84–0.93) versus 0.97 (0.94–0.99) for the combined MDM-CA19-9 panel (P ≤ 0.0001). Overall, cross-validated sensitivity of MDM-CA19-9 panel was 92% (83%–98%), with an observed specificity of 92% at the preset specificity of 97.5%. Conclusions: Plasma MDMs in combination with CA19-9 detect PDAC with significantly higher accuracy compared with either biomarker individually.

Abstract High-specificity colorectal cancer screening is desirable to triage patients <50 years for colonoscopy; however, most endorsed colorectal cancer screening tests have not been rigorously evaluated in younger populations. This prospective cross-sectional study determined the specificity of the multitarget stool DNA (mt-sDNA) test in an average-risk screening population of 45 to 49 year-olds. Specificity was the primary outcome and was measured in participants without colorectal cancer or advanced precancerous lesions [APL– advanced adenomas (AA), and sessile serrated lesions ≥10 mm], and in the subgroup of participants with negative colonoscopic findings. APL sensitivity was a secondary outcome. The evaluable cohort included those who completed the study without protocol deviations and had a usable mt-sDNA test. Of 983 enrolled participants, 816 formed the evaluable cohort, with a mean age of 47.8 (SD, 1.5) years; 47.7% were women. No participants had colorectal cancer, 49 had APL, 253 had nonadvanced adenomas (NAA), and 514 had negative colonoscopic findings. mt-sDNA test specificity was 95.2% (95% CI, 93.4–96.6) in participants with NAA or negative findings [96.3% (confidence interval (CI), 94.3%–97.8%)] in those with negative findings, and did not differ by sex (P = 0.75) or race (P = 0.36) in participants with NAA or negative findings. Sensitivity for APL was 32.7% (CI, 19.9–47.5%), with most APL (83.7%) measuring 10–19 mm and none having high-grade dysplasia. The area under the ROC curve for discriminating between APL and lesser findings was 0.72 (CI, 0.64–0.81). mt-sDNA's high specificity would help minimize risk from unnecessary diagnostic procedures in this age group. This study shows that mt-sDNA has high specificity among average-risk 45 to 49-year olds, supporting its use as a noninvasive option for colorectal cancer screening. Prevention Relevance: This study shows that mt-sDNA has high specificity among average-risk 45–49 year olds, supporting its use as a non-invasive option for colorectal cancer screening.