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Background: Pancreatic ductal adenocarcinoma (PDAC) is a disease with an abysmal prognosis. Unfortunately, no reliable prognostic or predictive blood-based biomarkers are currently available. An increasingly promising prognostic and predictive tool in cancer is the analysis of genetic and epigenetic alterations in cell-free DNA. One such alteration, promoter hypermethylation (ph) of SFRP1, has recently been linked to poor prognosis in gemcitabine-treated patients with stage IV PDAC. This study aimed to further examine phSFRP1 as a clinically relevant prognostic and predictive biomarker in FOLFIRINOX-treated patients with stage IV PDAC. Methods: Serum samples from patients with stage IV PDAC, treated with first-line FOLFIRINOX, were obtained from two danish biobanks. All blood samples were drawn before the initiation of palliative treatment. Based on a bisulfite treatment, we conducted a methylation-specific polymerase chain reaction analysis of the promoter region of the gene SFRP1. Methylation status was dichotomized based on the measured Cycle threshold. Survival according to SFRP1 promoter methylation status was assessed with Kaplan-Meier curves, log-rank tests, and Cox proportional hazard regression models. The adjusted Cox regression model included SFRP1 promoter methylation status, WHO performance status, age > 65 years, and sex. Results: Fifty-two FOLFIRINOX-treated patients with stage IV PDAC were included in the study. Patients with phSFRP1 (n = 23) had a median overall survival (mOS) of 6.8 months, significantly shorter than patients with unmethylated (um) SFRP1 (n = 29) who had a mOS of 15.7 months (log-rank test, p < 0.01). Likewise, in both crude and adjusted Cox-regression analysis, patients with phSFRP1 had an hazard ratio for death of 2.78 (95% CI 1.55-4.99) and 2.82 (95% CI 1.55-5.14) respectively, compared to patients with umSFRP1. Conclusions: Results indicate that FOLFIRINOX-treated stage IV PDAC patients with phSFRP1 have significantly shorter survival than patients with umSFRP1. Combined with previous literature, this indicates the value of phSFRP1 as a blood-based prognostic biomarker in patients with stage IV PDAC, regardless of the given first-line palliative chemotherapy. In addition, it indicates that phSFRP1 may also have predictive value for chemotherapy sensitivity in patients with stage IV PDAC. This knowledge may facilitate individualized treatment of patients with stage IV PDAC. Additionally, SFRP1 could be a valuable target for treatment with hypomethylating drugs to improve treatment response. Citation Format: Benjamin E. Stubbe, Stine D. Henriksen, Poul H. Madsen, Anders C. Larsen, Henrik B. Krarup, Inge S. Pedersen, Julia S. Johansen, Carsten P. Hansen, Ole Thorlacius-Ussing. Promoter hypermethylation of SFRP1 as a prognostic and predictive blood-based biomarker in patients with stage IV pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B047.

Background: While detection of circulating tumor DNA (ctDNA) is associated with poor cancer prognosis, the clinical utility for guiding treatment decisions is unresolved. Patients with minimal residual disease (MRD) often have less than one genome equivalent of ctDNA per 10 mL blood. Consequently, it is stochastic whether a 10 mL sample contains ctDNA from a particular genomic locus. Consequently, the sensitivity of ctDNA detection methods targeting a limited number of tumor loci is heavily affected by sampling bias. To overcome this challenge, we developed MRDetect; a whole genome sequencing (WGS) approach, which detects ctDNA using the patient-specific cumulative signal from tens of thousands of mutations throughout the genome. Recently, we showed how MRDetect found ctDNA fractions down to 10-4. Here, we performed a validation study to confirm the prognostic impact of MRDetect. Aim: Validation of MRDetect for sensitive ctDNA detection to monitor residual disease in stage III colorectal cancer (CRC) patients treated with curative intent. Methods: From a large, uniform cohort of stage III CRC patients n = 146), we had plasma samples collected every third month (n = 938, median = 9 per patient) and a median follow-up of 34 months. For each patient, a genome-wide mutational signature was established by WGS of tumor and matched normal DNA. Enhanced by an AI-based error suppression model, this signature was used to detect ctDNA in 1-2 mL plasma samples using WGS (20x coverage). We used de-novo point mutation and copy number variation analysis to investigate cancer evolution after treatment. To evaluate the reproducibility of MRDetect, aliquot samples (n = 2x190 samples) from 5 recurrence and 10 non-recurrence patients were processed and sequenced at two independent laboratories. Outcome measures: ctDNA status, tumor fraction, false positive rate, Time To ctDNA Recurrence (TTcR), and Time To radiological Recurrence (TTrR). Results: Analysis of paired samples showed great reproducibility with high agreement between both ctDNA status calls (Cohens Kappa = 0.81) and the estimated tumor fractions (r2 = 0.99). MRDetect revealed post-operative ctDNA in all recurrence patients (5/5) with detected tumor fractions down to 2 x 10-4. Median TTcR was 0.9 month (range 0.5 - 7.3 months) while median TTrR was 12.8 months (range 11.3 - 31.1 months). The false positive rate was 1% (1/100), assessed in longitudinal samples from the 10 non-relapsing patients. Tumor evolution dynamics in plasma samples revealed novel amplification and deletions, which were absent in the primary tissue but confirmed in metachronous metastases. We will present results from the full cohort at AACR 2022. Conclusion: MRDetect detects ctDNA with high sensitivity and specificity and enables effective postoperative assessment of MRD, cancer evolution dynamics and early relapse detection. Citation Format: Amanda Frydendahl, Thomas Reinert, Jesper Nors, Sunil Deochand, Dillon Maloney, Noah Friedman, Tomer Lauterman, Danielle Afterman, Imane Bourzgui, Nidhi Ramaraj, Zohar Donenhirsh, Ronel Veksler, Ravi Kandasamy, Iman Tavassoly, Jonathan Rosenfeld, Anders Husted Andersen, Uffe S. Løve, Per V. Andersen, Ole Thorlacius-Ussing, Lene Hjerrild Iversen, Kåre Andersson Gotschalck, Boris Oklander, Asaf Zviran, Claus Lindbjerg Andersen. Sensitive detection of circulating tumor DNA by whole genome sequencing: Validation of MRDetect using serial blood samples from stage III colorectal cancer patients [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 1959.

The large number of colorectal cancer survivors, coupled with the financial burden of colorectal cancer, makes it critical to identify factors associated with cancer recurrence. The human gut microbiome is one of the most densely populated bacterial communities known to exist. It plays a vital role in the host's gut physiology and potentially contributes to colorectal cancer progression. Given the prevalence and often overuse of antibiotics in the population, and their effects on the gut microbiome, the potential influence of antibiotics on the clinical course of colorectal cancer is of great public health importance. Using data from Danish medical registries, we followed 21,152 patients (5,036 with recurrences) diagnosed with stage I-III colorectal cancer from 2001 to 2011. We examined the association between pre- and post-diagnostic antibiotic use and cancer recurrence. After adjustment for potential confounders (age at diagnosis, sex, year of diagnosis, stage, tumor location, cancer treatment, comorbidities, inflammatory bowel disease, and use of statins, non-steroidal anti-inflammatory drugs and aspirin), any antibiotic prescription 90 days prior to colorectal cancer diagnosis was not associated with recurrence [adjusted hazard ratio (aHR) = 1.06, 95% confidence interval (CI): 0.98, 1.14]. However, antibiotic use after cancer diagnosis was associated with a higher risk of recurrence (aHR = 1.23, 95% CI: 1.16, 1.31). Additional ongoing analyses address cancer-specific and all-cause mortality and different time-lag periods. Their results also will be presented at the meeting. Our preliminary findings support further investigation into the possible roles of the gut microbiome and agents that alter its composition and diversity in colorectal cancer progression. Citation Format: Veronika Fedirko, Deirdre Cronin-Fenton, Anders Hammerich Riis, Henrik Toft Sørensen, Claus Lindbjerg Andersen, Ole Thorlacius-Ussing, Timothy Lash. Antibiotic prescriptions and colorectal cancer recurrence risk: A Danish nationwide prospective cohort study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-166.