Kisiel, John B.

Abstract Introduction: Laparoscopy with peritoneal lavage enhances staging accuracy in patients with pancreatic ductal adenocarcinoma (PDAC). Occult intra-abdominal metastases are identified by staging laparoscopy in approximately 20% of patients with radiologically localized PDAC. Peritoneal fluid cytology has limited sensitivity in detecting occult intra-abdominal metastasis. Methylated DNA markers (MDMs) can accurately detect PDAC from tumor tissue, blood, and pancreatic secretions. This study aims to assess the feasibility of assaying MDMs in peritoneal lavage fluid obtained at the time of staging laparoscopy for patients with PDAC. Methods: Peritoneal lavage fluid (25-60 mL) obtained from patients with PDAC at the time of staging laparoscopy was centrifuged for 10 minutes at 1200 × g. The pellet was resuspended and aliquoted into 200µL tubes and frozen at -80°C. DNA extracted from one aliquot was assayed for mutant KRAS (G12A and G13D) and then bisulfite converted to measure 13 MDMs (GRIN2D, CD1D, ZNF781, FER1L4, RYR2, CLEC11A, AK055957, LRRC4, MAX.CHR5.4295, HOXA1, PRKCB, SHISA9, NTRK3) and a normalizing reference gene (B3GALT6) using quantitative methylation specific PCR. The association of KRAS and the average signal from the 13 MDMs (MDMscore) with intra-abdominal metastasis (Y/N) was assessed with the area under the receiver operator characteristic curve (AUC). Results: Among 48 PDAC patients, staging laparoscopy revealed intra-abdominal metastasis in 15 (31%). Of the 15 intra-abdominal metastases, 6 were found to have biopsy-confirmed intra-abdominal metastasis and positive peritoneal cytology, 4 were positive on peritoneal cytology alone, and 5 had biopsy-confirmed intra-abdominal metastasis alone. The sensitivity of the MDMscore for intra-abdominal metastasis was 87% (CI: 60-98%) at 90% (CI: 76-98%) specificity whereas the sensitivity for KRAS was 40% (CI: 16-68%) at 85% (CI: 68-95) specificity. The AUC for the MDMscore was 0.98 (CI: 0.94-1.00) compared to 0.62 (CI: 0.48-0.77) for that of KRAS (p<0.0001). Conclusion: MDMs assayed from peritoneal lavage fluid collected during staging laparoscopy is a novel tool for accurate molecular staging of PDAC. These early results warrant further study in a larger cohort to assess impact on peritoneal staging and its clinical utility in the neoadjuvant treatment paradigm of PDAC. Citation Format: Kalika Maheshwari, Calise K. Berger, William R. Taylor, Douglas W. Mahoney, Kelli N. Burger, Adriana M. Delgado, Karen A. Doering, Heather M. Streich, Patrick H. Foote, Sean P. Cleary, Travis E. Grotz, Micheal L. Kendrick, Rory L. Smoot, Patrick P. Starlinger, Cornelius A. Thiels, Mark J. Truty, Susanne G. Warner, John B. Kisiel, Shounak Majumder. Methylated DNA markers for detection of peritoneal metastasis in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3784.

Abstract Introduction: Screening for prostate cancer (PCa) with molecular markers is hindered by low tumor allele fraction (TAF) in liquid biopsy samples such as blood or urine. Others have suggested that tumor allele enrichment can be achieved by evaluating fragment-level methylation patterns generated by next generation sequencing (NGS). Here, we test this concept using target capture NGS on cfDNA from prediagnostic collected urine samples to discriminate PCa from otherwise healthy men. Methods: Buffered urines were collected from 23 patients with biopsy confirmed PCa (Gleason score 6-9) and 24 otherwise healthy age matched cancer-free controls. cfDNA was isolated and enzymatic methyl-seq libraries were prepared and enriched by hybrid capture using probes mapping to 197 differentially methylated regions (DMRs) from a previous PCa/normal tissue marker discovery study in our lab. These DMRs were subsequently vetted independently using a methylome-wide dataset from a cohort of 36 clean and disease control urines to ensure low non-cancer background signal (<1%). Indexed libraries were sequenced at 560X deduplicated coverage and fragment-level CpG methylation information was extracted from individual sample .bam files. Mapped sequencing reads exhibiting concordant methylation were used to derive a quantitative α-score. Reads had to contain at minimum 5 CpGs and have an α-value (% 5mC per read) > 60%. Positivity for the 197 markers required an α-score greater than 6 sigma (σ) above the mean of the control arm. Values were variance-adjusted to ensure equal weighting. Beta (β) analysis (mean % 5mC across all reads) of the data was also performed for comparison. Results: Of the 197 markers, 187 were positive for one or more PCa patients. Tumor-derived fragments were detected in 22 of 23 cases, which included 8 G-6, 9 G-7, 2 G-8, and 3 G-9 patients. 1 G-7 was missed, however the sequencing coverage for this sample was significantly lower than the others due to poor library enrichment. 2 of the 24 healthy controls were positive, one with extremely high fragment counts. Upon re-review of the full control cohort, this patient had been diagnosed with bladder cancer 2 years after urine collection. The 2nd patient had was diagnosed with lung cancer in a similar timeframe. The other 22 confirmed cancer-free controls had α-scores below the 6σ threshold. Using a co-captured reference gene (B3GALT6) to mimic genomic equivalents, the TAFs ranged from 0.015% to 8%. β analysis of the data detected only 13 PCa patients (>6σ) and marker positivity was lower than the fragment-level approach. Conclusion: Target capture NGS coupled with fragment-level analysis is a rational approach for sensitive and specific detection of low tumor-derived DNA in liquid biopsy samples. For PCa assay in urine, we were able to uncover positivity in cancer patients approaching 1 in 10,000 ctDNA dilution levels. These results warrant further validation studies with this and other cancers. Citation Format: Tibor Bedekovics, William R. Taylor, Zhifu Sun, Saurabh Baheti, Xiaoming Cao, Kelli N. Burger, Karen A. Doering, Patrick H. Foote, Benjamin Gochanour, Douglas, W. Mahoney, Matthew T. Gettman, John B. Kisiel. Detection of prostate cancer in urine by fragment-level DNA methylation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2424.

Abstract The multi-target stool DNA (mt-sDNA) test screens for colorectal cancer by analyzing DNA methylation/mutation and hemoglobin markers to algorithmically derive a qualitative result. A new panel of highly discriminant candidate methylated DNA markers (MDM) was recently developed. Performance of the novel MDM panel, with hemoglobin, was evaluated in a simulated screening population using archived stool samples weighted to early-stage colorectal cancer and prospectively collected advanced precancerous lesions (APL). Marker selection study (MSS) and separate preliminary independent verification studies (VS) were conducted utilizing samples from multi-center, case–control studies. Sample processing included targeted MDM capture, bisulfite conversion, and MDM quantitation. Fecal hemoglobin was quantified using ELISA. Samples were stratified into 75%/25% training-testing sets; model outcomes were cross-validated 1,000 times. All laboratory operators were blinded. The MSS included 232 cases (120 colorectal cancer/112 APLs) and 490 controls. The VS featured 210 cases (112 colorectal cancer/98 APLs) and 567 controls; APLs were 86.7% adenomas and 13.3% sessile serrated lesions (SSL). Average age was 65.5 (cases) and 63.2 (controls) years. Mean sensitivity in the VS from cross-validation was 95.2% for colorectal cancer and 57.2% for APLs, with specificities of 89.8% (no CRC/APLs) and 92.4% (no neoplasia). Subgroup analyses showed colorectal cancer sensitivities of 93.4% (stage I) and 94.2% (stage II). APL sensitivity was 82.9% for high-grade dysplasia, 73.4% for villous lesions, 49.8% for tubular lesions, and 30.2% for SSLs. These data support high sensitivity and specificity for a next-generation mt-sDNA test panel. Further evaluation of assay performance will be characterized in a prospective, multi-center clinical validation study (NCT04144738). Prevention Relevance: This study highlights performance of the next-generation mt-sDNA test, which exhibits high sensitivity and specificity for detecting colorectal cancer and APLs. This noninvasive option has potential to increase screening participation and clinical outcomes. A multi-center, clinical validation trial is underway. See related commentary by Bresalier, p. 93

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.

The Alaska Tribal Health System is working to increase colorectal cancer (CRC) screening among Alaska Native people, who experience the highest CRC rates in the world. This study examined CRC screening provider- and system-level barriers and facilitators from the perspective of healthcare providers serving Alaska Native people in rural/remote communities. A total of 28 provider (physicians, advanced practice, and Community Health Aides/Practitioners) interviews were held from 1 February to 30 November 2021. Colonoscopy provider-level barrier themes included time, competing priorities, and staffing, while system-level barriers included travel costs, weather, and the COVID-19 pandemic. Multi-target stool DNA (mt-sDNA) barrier themes included test viability and unfamiliarity, and previous stool tests experiences. For both tests, limited medical record reminders was a major barrier. Facilitator themes for both tests included community outreach, cultural competency and patient navigation, and clinic/system improvements. In-depth interviews with tribal health providers showed that adding mt-sDNA testing may help address system-level colonoscopy barriers such as waitlists and travel costs, but other barriers remain. Further research is needed into patient barriers and facilitators, as well as the effectiveness of integrating mt-sDNA into a geographically dispersed tribal health system to reduce cancer disparities and build equity in CRC prevention among Alaska Native people.

Plasma-based MDMs may offer a cost-effective alternative to imaging for melanoma detection. Early results show 80% sensitivity and 100% specificity against cancer-free controls. Further study needed. #MelanomaResearch #EarlyDetection