Kisiel, John B

Abstract Lynch syndrome (LS) markedly increases risks of colorectal and endometrial cancers. Early detection biomarkers for LS cancers could reduce the needs for invasive screening and surgical prophylaxis. To validate a panel of methylated DNA markers (MDM) previously identified in sporadic colorectal cancer and endometrial cancer for discrimination of these cancers in LS. In a case–control design, previously identified MDMs for the detection of colorectal cancer and endometrial cancer were assayed by qMSP on tissue-extracted DNA. Results were normalized to ACTB values within each sample. Least absolute shrinkage and selection operator models to classify colorectal cancer and endometrial cancer were trained on sporadic cases and controls and then applied to classify colorectal cancer and endometrial cancer, in those with LS, and cross-validated. We identified colorectal cancer cases (23 with LS, 48 sporadic), colorectal controls (32 LS, 48 sporadic), endometrial cancer cases (30 LS, 48 sporadic), and endometrial controls (29 LS, 37 sporadic). A 3-MDM panel (LASS4, LRRC4, and PPP2R5C) classified LS-CRC from LS controls with an AUC of 0.92 (0.84–0.99); results were similar for sporadic colorectal cancer. A 6-MDM panel (SFMBT2, MPZ, CYTH2, DIDO1, chr10.4479, and EMX2OS) discriminated LS-EC from LS controls with an AUC of 0.92 (0.83–1.0); the AUC for sporadic endometrial cancer versus sporadic controls was nominally higher, 0.99 (0.96–1.0). MDMs previously identified in sporadic endometrial cancer and colorectal cancer discriminate between endometrial cancer and benign endometrium and colorectal cancer and benign colorectum in LS. This supports the inclusion of patients with LS within future prospective clinical trials evaluating endometrial cancer and colorectal cancer MDMs and may provide a new avenue for cancer screening or surveillance in this high-risk population. Prevention Relevance: Lynch syndrome (LS) markedly increases risks of colorectal and endometrial cancers. Early detection biomarkers for LS cancers could reduce the needs for invasive screening and surgery. Methylated DNA markers previously identified in sporadic endometrial cancer and colorectal cancer discriminate between benign and cancer tissue in LS.

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.

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.

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.

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 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.