Kisiel, John B.

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]

AbstractExtracellular vesicles (EVs) are of growing interest due to their potential diagnostic, disease surveillance, and therapeutic applications. While several studies have evaluated EV isolation methods in various biofluids, there are few if any data on these techniques when applied to stool. The latter is an ideal biospecimen for studying EVs and colorectal cancer (CRC) because the release of tumour markers by luminal exfoliation into stool occurs earlier than vascular invasion. Since EV release is a conserved mechanism, bacteria in stool contribute to the overall EV population. In this study, we assessed five EV separation methods (ultracentrifugation [UC], precipitation [EQ‐O, EQ‐TC], size exclusion chromatography [SEC], and ultrafiltration [UF]) for total recovery, reproducibility, purity, RNA composition, and protein expression in stool supernatant. CD63, TSG101, and ompA proteins were present in EV fractions from all methods except UC. Human (18s) and bacterial (16s) rRNA was detected in stool EV preparations. Enzymatic treatment prior to extraction is necessary to avoid non‐vesicular RNA contamination. Ultrafiltration had the highest recovery, RNA, and protein yield. After assessing purity further, SEC was the isolation method of choice. These findings serve as the groundwork for future studies that use high throughput omics technologies to investigate the potential of stool‐derived EVs as a source for novel biomarkers for early CRC detection.

To expedite the development and application of potentially transformative multicancer early detection assays, an international summit was hosted by the Mayo Clinic Cancer Center, the American Cancer Society, and the Union for International Cancer Control on World Cancer Day 2020 at the historic Mayowood Mansion in Rochester, Minnesota. Insights shared during this unique summit have been formulated into a series of expert‐authored commentaries, which accompany this introductory article.

Minimally invasive molecular biomarkers have been applied to the early detection of multiple cancers in large scale case‐control and cohort studies. These demonstrations of feasibility herald the potential for permanent transformation of current cancer screening paradigms. This commentary discusses the major opportunities and challenges facing the preclinical development and clinical validation of multicancer early detection test strategies. From a diverse set of early detection research perspectives, the authors recommend specific approaches and highlight important questions for future investigation.

63 Background: Population-based colorectal cancer (CRC) screening can reduce mortality by detecting and removing advanced precancerous lesions (APL) and early-stage invasive disease. One guideline-included strategy is the multi-target stool DNA test (mt-sDNA), which combines detection of methylation DNA markers (MDMs), KRAS mutations, and fecal hemoglobin. Since the mt-sDNA pivotal study was conducted, novel biomarkers have been discovered. A panel of highly discriminant MDMs ( LASS4, LRRC4, PPP2R5C, and reference marker ZDHHC1) was identified through a blinded, case-control study of archival specimens. Here, we evaluated the performance of this novel mt-sDNA panel, combined with fecal hemoglobin, in archival stool samples weighted to early-stage CRC and prospectively collected APL, simulating a screening population. Methods: The verification study featured 777 samples—210 cases (112 CRC [49 stage I, 38 stage II, 17 stage III, and 8 stage IV] and 98 APL) and 567 controls (176 non-APL and 391 colonoscopy-negative)—from three trials (NCT01397747, NCT01260168, and NCT02503631). Median APL size was 12 mm (interquartile range: 10 mm to 15 mm), with 86.7% adenomas (n = 85) and 13.3% sessile serrated polyps (SSPs; n = 13). The average age was 65.5 years for cases (57% men) and 63.2 for controls (47% men). Samples were processed through homogenization, targeted MDM capture, bisulfite conversion, and MDM quantitation using Long‐probe Quantitative Amplified Signal (LQAS). Fecal hemoglobin was quantified using enzyme-linked immunosorbent assay (ELISA). Samples were split into stratified 75%/25% training-testing sets and model outcomes were cross-validated 1,000 times. Results: Mean performance from the cross-validation analysis is summarized in the table below. Overall sensitivity was 95.2% for CRC and 57.2% for APL, with specificities of 89.8% (no CRC/APL) and 92.4% (no neoplasia). Subgroup analyses showed high sensitivity for early-stage CRC, with 93.4% at stage I and 94.2% at stage II. By APL subtype, sensitivity was 82.9% for high-grade dysplasia, 73.4% for villous lesions, 49.8% for tubular lesions, and 30.2% for SSPs. Conclusions: These data support high sensitivity and specificity for a second-generation mt-sDNA panel. A multicenter pivotal trial evaluating the panel is underway (NCT04144738). [Table: see text]

INTRODUCTION: Vulvar involvement is a rare complication of Crohn's disease (CD). The optimal treatment of vulvar CD is unknown. METHODS: We conducted a 25-year retrospective cohort study of vulvar CD from 3 referral centers. Clinical features and outcomes were studied. RESULTS: Fifty patients were identified. The most common vulvar symptoms were pain (74%), edema (60%), ulcerations (46%), nodules (36%), and abscess (34%). Medical management leading to symptomatic improvement varied, and 5 patients ultimately required surgery. DISCUSSION: Vulvar CD manifests with a broad spectrum of symptoms. Aggressive medical management was frequently effective, although surgery was required in 10% of cases.

AbstractBackground:The role of methylation in pancreatic cancer risk remains unclear. We integrated genome and methylome data to identify CpG sites (CpG) with the genetically predicted methylation to be associated with pancreatic cancer risk. We also studied gene expression to understand the identified associations.Methods:Using genetic data and white blood cell methylation data from 1,595 subjects of European descent, we built genetic models to predict DNA methylation levels. After internal and external validation, we applied prediction models with satisfactory performance to the genetic data of 8,280 pancreatic cancer cases and 6,728 controls of European ancestry to investigate the associations of predicted methylation with pancreatic cancer risk. For associated CpGs, we compared their measured levels in pancreatic tumor versus benign tissue.Results:We identified 45 CpGs at nine loci showing an association with pancreatic cancer risk, including 15 CpGs showing an association independent from identified risk variants. We observed significant correlations between predicted methylation of 16 of the 45 CpGs and predicted expression of eight adjacent genes, of which six genes showed associations with pancreatic cancer risk. Of the 45 CpGs, we were able to compare measured methylation of 16 in pancreatic tumor versus benign pancreatic tissue. Of them, six showed differentiated methylation.Conclusions:We identified methylation biomarker candidates associated with pancreatic cancer using genetic instruments and added additional insights into the role of methylation in regulating gene expression in pancreatic cancer development.Impact:A comprehensive study using genetic instruments identifies 45 CpG sites at nine genomic loci for pancreatic cancer risk.

Abstract Purpose: Screening for high prevalence cancers (breast, colorectum and uterine cervix) reduces mortality; however, most cancer deaths arise in unscreened organs. An ideal solution would accurately detect multiple currently unscreened cancers in a single round of testing. We aimed to assess the feasibility of a targeted assay panel of methylated DNA markers (MDMs) and proteins for detection of highly lethal cancers in a multi-cancer case-control study. Experimental Procedures: Prospectively collected plasmas from 180 cases of 6 cancer types (stage A-D liver (n=36), and TNM stage II-IV esophageal (n=18), lung (n=36), ovarian (n=30), pancreatic (n=30), and stomach (n=30)) and 257 smoking status, age-, and gender-matched asymptomatic controls were tested in blinded fashion. To quantify 26 MDMs previously found to be common among many cancers, multiplex PCR followed by LQAS (Long probe Quantitative Amplified Signal) assay was performed on bisulfite converted DNA extracted from 3 mL of plasma. Additionally, 5 proteins were tested from paired serum aliquots and combined with MDMs for a multi-analyte analysis. Two-thirds of the cases and controls were used to develop prediction algorithms, both logistic regression and random forest models, and the remaining 1/3 were used to validate the models. Results: Using stepwise logistic regression, a combination of 3 proteins (CEA, CA-125, CA19-9) and 5 MDMs (ZNF671, GRIN2D, NDGR4, SHOX2, B3GALT6) resulted in an AUC of 0.95 and an overall sensitivity of 87% for all cancers at 95% specificity. The logistic model on the validation set of the samples resulted in an AUC of 0.96 and a sensitivity of 83% with an observed specificity of 94%. The cancer-specific sensitivities ranged from 78% for lung cancer to 90% for ovarian and pancreatic cancer (Table). Random forest and logistic analyses were in agreement. Conclusion: Targeted assay of select MDMs and proteins show high discrimination for multiple cancers in a case control study, which included early stage patients. Performance with 95% confidence intervals of multi-target assay for multi-cancer detectionSensitivitySpecificityModelOverallBy cancer typeLungEsophagealGastricPancreaticLiverOvarianLogisticTraining set87%(79-92%)79%83%90%95%88%85%95%(90-97%)Test set83%(72-91%)75%100%80%80%75%100%94%(87%-98%)Training + Test186%(80-90%)78%89%87%90%83%90%95%(91-97%)Training + Test275%(68-81%)50%89%83%76%81%80%98%(96-99%)Random forest377%(70-83%)58%78%83%77%81%87%95%(92-97%)195% specificity goal, 5 MDMs and 3 proteins.298% specificity goal, 5 MDMs and 3 proteins.3With 500-fold in silico cross-validation on all samples and all markers (26 MDMs + 5 proteins) Citation Format: Slava Katerov, Abram Vaccaro, Jacquelyn Hennek, Jennifer Carlson, William R. Taylor, Douglas Mahoney, John B. Kisiel, Hatim T. Allawi. Accurate multi-cancer detection using methylated DNA markers and proteins in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 111.

INTRODUCTION: Significant variability between colonoscopy operators contributes to postcolonoscopy colorectal cancers (CRCs). We aimed to estimate postcolonoscopy colorectal neoplasia (CRN) detection by multi-target stool DNA (mt-sDNA), which has not previously been studied for this purpose. METHODS: In a retrospective cohort of patients with +mt-sDNA and completed follow-up colonoscopy, positive predictive value (PPV) for endpoints of any CRN, advanced adenoma, right-sided neoplasia, sessile serrated polyps (SSP), and CRC were stratified by the time since previous colonoscopy (0–9, 10, and ≥11 years). mt-sDNA PPV at ≤9 years from previous average-risk screening colonoscopy was used to estimate CRN missed at previous screening colonoscopy. RESULTS: Among the 850 studied patients with +mt-sDNA after a previous negative screening colonoscopy, any CRN was found in 535 (PPV 63%). Among 107 average-risk patients having +mt-sDNA ≤9 years after last negative colonoscopy, any CRN was found in 67 (PPV 63%), advanced neoplasia in 16 (PPV 15%), right-sided CRN in 48 (PPV 46%), and SSP in 20 (PPV 19%). These rates were similar to those in 47 additional average risk persons with previous incomplete colonoscopy and in an additional 68 persons at increased CRC risk. One CRC (stage I) was found in an average risk patient who was mt-sDNA positive 6 years after negative screening colonoscopy. DISCUSSION: The high PPV of mt-sDNA 0–9 years after a negative screening colonoscopy suggests that lesions were likely missed on previous examination or may have arisen de novo . mt-sDNA as an interval test after negative screening colonoscopy warrants further study.

INTRODUCTION: Minimally invasive tests for Barrett's esophagus (BE) detection have raised the prospect of broader nonreflux-based testing. Cost-effectiveness studies have largely studied men aged 50 years with chronic gastroesophageal reflux disease (GERD) symptoms. We evaluated the comparative cost effectiveness of BE screening tests in GERD-based and GERD-independent testing scenarios. METHODS: Markov modeling was performed in 3 scenarios in 50 years old individuals: (i) White men with chronic GERD (GERD-based); (ii) GERD-independent (all races, men and women), BE prevalence 1.6%; and (iii) GERD-independent, BE prevalence 5%. The simulation compared multiple screening strategies with no screening: sedated endoscopy (sEGD), transnasal endoscopy, swallowable esophageal cell collection devices with biomarkers, and exhaled volatile organic compounds. A hypothetical cohort of 500,000 individuals followed for 40 years using a willingness to pay threshold of $100,000 per quality-adjusted life year (QALY) was simulated. Incremental cost-effectiveness ratios (ICERs) comparing each strategy with no screening and comparing screening strategies with each other were calculated. RESULTS: In both GERD-independent scenarios, most non-sEGD BE screening tests were cost effective. Swallowable esophageal cell collection devices with biomarkers were cost effective (<$35,000/QALY) and were the optimal screening tests in all scenarios. Exhaled volatile organic compounds had the highest ICERs in all scenarios. ICERs were low (<$25,000/QALY) for all tests in the GERD-based scenario, and all non-sEGD tests dominated no screening. ICERs were sensitive to BE prevalence and test costs. DISCUSSION: Minimally invasive nonendoscopic tests may make GERD-independent BE screening cost effective. Participation rates for these strategies need to be studied.