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Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intratumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in > 90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell–cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intratumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC. Significance: A high-throughput pipeline using multiplex immunofluorescence in pancreatic cancer reveals striking expression subtype intratumoral heterogeneity with implications for therapy selection and identifies co-expressor cells that may serve as intermediates during subtype switching.

We conducted next-generation DNA sequencing on 335 biliary tract cancers and characterized the genomic landscape by anatomic site within the biliary tree. In addition to frequent FGFR2 fusions among patients with intrahepatic cholangiocarcinoma (IHCC), we identified FGFR2 extracellular domain in-frame deletions (EID) in 5 of 178 (2.8%) patients with IHCC, including two patients with FGFR2 p.H167_N173del. Expression of this FGFR2 EID in NIH3T3 cells resulted in constitutive FGFR2 activation, oncogenic transformation, and sensitivity to FGFR inhibitors. Three patients with FGFR2 EIDs were treated with Debio 1347, an oral FGFR1/2/3 inhibitor, and all showed partial responses. One patient developed an acquired L618F FGFR2 kinase domain mutation at disease progression and experienced a further partial response for 17 months to an irreversible FGFR2 inhibitor, futibatinib. Together, these findings reveal FGFR2 EIDs as an alternative mechanism of FGFR2 activation in IHCC that predicts sensitivity to FGFR inhibitors in the clinic.Significance:FGFR2 EIDs are transforming genomic alterations that occur predominantly in patients with IHCC. These FGFR2 EIDs are sensitive to FGFR inhibition in vitro, and patients with these alterations benefited from treatment with FGFR inhibitors in the clinic.This article is highlighted in the In This Issue feature, p. 2355