Spatially resolved protein profiling in pancreatic adenosquamous carcinoma.

760 Background: Pancreatic adenosquamous carcinoma (PASC) is a rare histopathological type of pancreatic cancer that is more aggressive than pancreatic ductal adenocarcinoma (PDAC). Although PASC has a unique tumor biology and poorer prognosis, there are no guidelines for differentially treating PASC patients, and its rarity makes it significantly understudied. We used Nanostring GeoMX digital spatial profiling (DSP) to provide a targeted proteomic characterization of key tumor-promoting proteins in PASC and PDAC. Methods: FFPE tissue from PASC (n=10 primary, n= 4 met) and PDAC (n=21 primary, n=5 met) cases were analyzed using the DSP platform. Sections were stained with an 82-protein cocktail of antibodies conjugated to UV-photocleavable DNA barcodes. A pan-cytokeratin morphology marker was used to select tumor regions of interest (ROI) and inferred surrounding stromal (i.e., pan-CK negative) ROIs. UV excitation of the defined ROIs was used to release the DNA barcodes and were quantified on the NanoString nCounter system. For PASC, squamous enriched ROI were inferred from corresponding p40 IHC. Corresponding clinical and mutational status for all cases were abstracted from medical records. Results: TP53 (PDAC, 80%; PASC, 83%) and KRAS (PDAC, 76%; PASC, 100%) were the most prevalent mutations . In comparing the tumor ROIs of PASC and PDAC (all cases), DSP-assessed global expression and activation of EGFR-MAPK signaling is significantly increased in PASC, whereas PI3K-AKT signaling is significantly decreased. Compared to PDAC, EGFR expression and phosphorylation (p-EGFR), as well as p-MEK1, are significantly increased in PASC. PASC showed significantly lower levels of PLCG1 and INPP4B, while the majority of other AKT signaling effectors were unchanged. For DNA damage response signaling, PASC showed significantly higher levels of p-ATM, p-CHK2, and p21, but not p-ATR. Likewise, expression of oncoproteins c-MYC and BCL6 were significantly increased in PASC (vs. PDAC). Conclusions: Given that the normal pancreas lacks squamous cells, their histological presence is a uniquely aberrant feature that can be used to guide deep spatially-resolved interrogation of complex cellular and molecular programs. Our preliminary findings show that regions of squamous cell differentiation within PASC have intrinsically higher levels of active EGFR signaling. With emerging RAS-targeted agents poised to alter the treatment landscape, high EGFR signaling in PASC suggest a possible compensatory resistance mechanism that may require additional targeting. Likewise, reliance on ATM signaling highlights this as preferential target for DDR-directed therapies. Building on these results with additional multiomic assays will inform strategies for improving the management of patients with PASC.