Phosphorylated FRS2 (pFRS2) as a biomarker for FGFR mutation or fusion activity assessment in formalin-fixed paraffin-embedded tumor sections

Genes encoding the Fibroblast Growth Factor Receptor family (FGFR1, FGFR2, FGFR3 and FGFR4) are a recurrent target of genomic alterations in several solid tumor types. Alteration types include gene amplification, gene fusion and point mutations which can affect both extracellular and intracellular domains. The FGFR inhibitors, pemigatinib, erdafitinib and futibatinib, have FDA approvals to treat selected solid tumors with FGFR pathway alterations. Because FRS2 is a common substrate for each of these kinases, we investigated whether the level and subcellular localization of phosphorylated FRS2 (pFRS2) would be an effective biomarker to read out pathway activity in clinical FFPE tumor specimens. The Gundersen Precision Oncology Cohort includes 1,046 patients who received next-generation sequencing at Gundersen Health System during their cancer care. A total of 29 patients had FGFR alterations (13 x FGFR1, 9 x FGFR2 and 7 x FGFR3) with fusions, point mutations and gene amplifications represented. The most common tumor types were urothelial (7), lung (7) and biliary tract (6). Here we report a pFRS2 immunofluorescence assay that permits high-resolution readout of both the level and subcellular localization of the activity of genetically altered FGFRs. This enables quantitative distinction between specimens with high pathway activity (including fusions and some point mutations) from specimens with lower pathway activity (some amplifications), suggesting utility as a companion diagnostic for identification of likely responders to currently approved and newly emerging FGFR therapies. In particular, the ability to determine subcellular localization of pathway activity may prove useful for trials of both antibody-drug conjugates and bispecific antibodies which might be expected to be less effective in tumors where the oncogenic FGFR activity is primarily internal and not cell-surface associated.