Objective: Patients with pancreatic cancer (PDAC) who undergo surgical resection and receive effective chemotherapy have the best chance of long-term survival. Unfortunately, we lack predictive biomarkers to guide optimal systemic treatment. Ex-vivo generation of patient-derived organoids (PDO) for pharmacotyping may serve as predictive biomarkers in PDAC. The goal of the current study was to demonstrate the clinical feasibility of a PDOguided precision medicine framework of care.Methods: PDO cultures were established from surgical specimens and endoscopic biopsies, expanded in Matrigel, and used for high-throughput drug testing (pharmacotyping).Efficacy of standard-of-care chemotherapeutics was assessed by measuring cell viability after drug exposure.Results: A framework for rapid pharmacotyping of PDOs was established across a multiinstitutional consortium of academic medical centers. Specimens obtained remotely and shipped to a central biorepository maintain viability and allowed generation of PDOs with 77% success. Early cultures maintain the clonal heterogeneity seen in PDAC with similar phenotypes (cystic-solid). Late cultures exhibit a dominant clone with a pharmacotyping profile similar to early passages. The biomass required for accurate pharmacotyping can be minimized by leveraging a high-throughput technology. Twenty-nine cultures were pharmacotyped to derive a population distribution of chemotherapeutic sensitivity at our center. Pharmacotyping rapidly-expanded PDOs was completed in a median of 48 (range 18-102) days.Conclusions: Rapid development of PDOs from patients undergoing surgery for PDAC is eminently feasible within the perioperative recovery period, enabling the potential for pharmacotyping to guide post-operative adjuvant chemotherapeutic selection. Studies validating PDOs as a promising predictive biomarker are ongoing.

Rationale: Patient-derived organoids (PDOs) are a promising technology to support precision medicine initiatives for patients with pancreatic ductal adenocarcinoma (PDAC). PDOs may improve clinical next-generation sequencing (NGS) and enable rapid ex vivo chemotherapeutic screening (pharmacotyping). Methods: PDOs were derived from tissues obtained during surgical resection and endoscopic biopsies and studied with NGS and pharmacotyping. PDO-specific pharmacotype is assessed prospectively as a predictive biomarker of clinical therapeutic response by leveraging data from a randomized-controlled clinical trial. Results: Clinical sequencing pipelines often fail to detect PDAC-associated somatic mutations in surgical specimens that demonstrate a good pathological response to previously administered chemotherapy. Sequencing the PDOs derived from these surgical specimens, after biomass expansion, improves the detection of somatic mutations and enables quantification of copy number variants. The detection of clinically relevant mutations and structural variants is improved following PDO biomass expansion. On clinical trial, PDOs were derived from biopsies of treatment naïve patients prior to treatment with FOLFIRINOX (FFX). Ex vivo PDO pharmacotyping with FFX components predicted clinical therapeutic response in these patients with borderline resectable or locally advanced PDAC treated in a neoadjuvant or induction paradigm. PDO pharmacotypes suggesting sensitivity to FFX components were associated with longitudinal declines of tumor marker, CA-19-9 and favorable RECIST imaging response. Conclusion: PDOs establishment from tissues obtained from patients previously receiving cytotoxic chemotherapies can be accomplished in a clinically-certified laboratory. Sequencing PDOs following biomass expansion improves clinical sequencing quality. High in-vitro sensitivity to standard-of-care chemotherapeutics predicts good clinical response to systemic chemotherapy in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy driven by a heterogeneous tumor microenvironment enriched with cancer associated fibroblasts (CAFs) that influence its overall immunosuppressive composition. We examined inflammation in PDAC by leveraging our existing patient-derived organoid (PDO) model and a novel PDO-CAF co-culture. We first identified induction of major histocompatibility complex class II expression following treatment with interferon gamma. In parallel, we collated an atlas of 6 published single-cell RNA-sequencing datasets (174,394 cells) combining 61 PDAC (142,807 cells) and 16 non-malignant samples. By combining in silico modeling and in vitro PDO co-culture, we define a gene expression pattern of inflammatory processes in epithelial tumor cells. Following computational inferences, we examined interactions between epithelial tumor cells and CAFs, focusing on VEGF-A and ITGB1 pathways. This work, integrating computational and biological approaches, highlights the value of convergence to accelerate our understanding of key drivers of PDAC.