Abstract PR-007: Single-nucleus and spatial transcriptomics of archival pancreatic ductal adenocarcinoma reveals multi-compartment reprogramming after neoadjuvant treatment

Molecular subtyping of pancreatic ductal adenocarcinoma (PDAC) remains in its nascent stages and does not currently inform clinical management or therapeutic development. Previously identified bulk expression subtypes in the untreated setting were influenced by contaminating stroma whereas single cell RNA-seq (scRNA-seq) of fresh tumors under-represented key cell types. Two consensus subtypes have arisen from these prior efforts: (1) classical-pancreatic, encompassing a spectrum of pancreatic lineage precursors, and (2) basal-like/squamous/quasi-mesenchymal, characterized by loss of endodermal identity and aberrations in chromatin modifiers. Basal-like tumors were associated with poorer responses to chemotherapy and worse survival in the metastatic setting but attempts to refine this binary classification have failed to further stratify patient survival. Recent clinical trials have supported the increasing adoption of neoadjuvant therapy to aggressively address the risk of micro-metastatic spread and to circumvent concerns of treatment tolerance in the postoperative setting. There is an urgent need to understand how preoperative treatment reprograms residual tumor cells to identify additional therapeutic vulnerabilities that can be exploited in combination with neoadjuvant CRT. Here, we developed a robust single-nucleus RNA-seq (snRNA-seq) technique for frozen archival PDAC specimens and used it to study both untreated tumors (n = 15) and those that received neoadjuvant CRT (n = 11). Gene expression programs learned across malignant cell and fibroblast profiles uncovered a clinically relevant molecular taxonomy with improved prognostic stratification (median survival: 11.2 months in highest risk group to 44.7 months in lowest risk group) compared to prior classifications. Moreover, in the neoadjuvant treatment context, there was lower expression of classical-like phenotypes in malignant cells in favor of basal-like phenotypes associated with TNF-NFkB and interferon signaling as well as the presence of novel acinar and neuroendocrine classical-like states, which may be more resilient to cytotoxic treatment. These results suggest that differentiated endodermal phenotypes are only prevalent enough to be detected under treatment selection pressure and when observed in treatment-naive bulk studies, may reflect normal cell contamination. Spatially-resolved transcriptomics revealed an association between malignant cells expressing basal-like programs and higher immune infiltration with increased lymphocytic content, whereas those exhibiting classical-like programs were linked to sparser macrophage-predominant microniches, perhaps pointing to distinct therapeutic susceptibilities. Our refined molecular taxonomy and spatial resolution may help advance precision oncology in PDAC through informative stratification in clinical trials and insights into differential therapeutic targeting leveraging the immune system. Citation Format: William L. Hwang, Karthik A. Jagadeesh, Jimmy A. Guo, Hannah I. Hoffman, Eugene Drokhlyansky, Nicholas Van Wittenberghe, Samouil Farhi, Denis Schapiro, Jason Reeves, Daniel R. Zollinger, George Eng, Jason M. Schenkel, William A. Freed-Pastor, Clifton Rodrigues, Domenic Abbondanza, Debora Ciprani, Jennifer Y. Wo, Theodore S. Hong, Andrew J. Aguirre, Orit Rozenblatt-Rosen, Mari Mino-Kenudson, Carlos Fernandez-del Castillo, Andrew S. Liss, Tyler E. Jacks, Aviv Regev. Single-nucleus and spatial transcriptomics of archival pancreatic ductal adenocarcinoma reveals multi-compartment reprogramming after neoadjuvant treatment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PR-007.