Abstract P6-07-16: Quantitative proteomic analysis of FGFR by mass spectrometry may improve identification of FGFR amplified tumors sensitive to inhibitor therapy
Authors: Schwartz S , Tian Y , Fasani R , Diaz Delgado M , Hierro C , Rodon J , Sellappan S , Cecchi F , Hembrough T , Nuciforo P 
Published: March 2017

Background: The fibroblast growth factor receptors 1 and 2 (FGFR1 and FGFR2) have been reported as amplified in multiple solid tumors, including lung, breast and gastric cancers. The FGFR family of proteins is an attractive target for therapy due to the importance of FGFR signaling in the pathogenesis of diverse tumor types. However, pan-FGFR inhibitor have shown only modest efficacy in patients with FGFR1 or FGFR2 gene amplification as determined by fluorescence in situ hybridization (FISH). Gene copy number alterations may not be the optimal predictive biomarker since the targets of these drugs are FGFR proteins; recent findings suggest that direct measurement of the protein targets of FGFR inhibitor therapies may be necessary to assess their treatment efficacy. We therefore developed quantitative FGFR protein assays using selected reaction monitoring mass spectrometry (SRM-MS). We sought to correlate levels of FGFR1, FGFR2 and pan-FGFR (FGFR1-4) proteins as measured by SRM-MS with FGFR gene amplification status as determined by FISH.

Methods: Formalin-fixed, paraffin-embedded (FFPE) tissue sections from breast (n=18), esophageal (n=1), gastric (n=1), lung (n=2), and endometrial (n=1) tumors were obtained. A board-certified pathologist marked the tumor area for laser microdissection. Tumor cell proteins were extracted using the Liquid Tissue® process and subjected to SRM-MS for analysis of protein expression levels of FGFR1, FGFR2 and FGFR1-4, as well as other targetable proteins including MET, EGFR, and PD-L1. We compared FGFR protein levels with FGFR amplification defined as FGFR to CEP FISH ratio &gt;2.2.

Results: Of 23 tumor samples analyzed, the pan-FGFR1-4 proteomic assay detected FGFR protein in 11 cases (protein expression range: 217.8-3199.5 amol/ug). Ten of these 11 samples (91%) showed FGFR gene amplification. Only a single non-amplified case showed protein expression (235.6 amol/ug). FGFR protein (of 1, 2, and 1-4) was undetectable in 12 samples, of which 5 (42%) harbored FGFR1 amplification. Two of 2 (100%) of FGFR2-amplified cases (average copy number=38) showed high FGFR2 protein expression (3063.0 and 3199.5 amol/ug). Sensitivity of the pan-FGFR1-4 assay was superior to single FGFR assays.

Conclusion: A subset of FGFR-amplified tumors does not express FGFR protein when assessed by highly-sensitive SRM-MS. Patients whose tumors do not express FGFR protein are not likely to respond to FGFR inhibitor therapy, as supported by previous findings in squamous cell lung tumors, in which FGFR1 mRNA and/or protein expression levels greatly outperformed FGFR1 gene copy number in predicting sensitivity to a pan-FGFR inhibitors. An approach combining quantitative proteomics and FISH analyses may accurately identify patients most likely to respond to anti-FGFR protein agents.

Citation Format: Schwartz S, Tian Y, Fasani R, Diaz Delgado M, Hierro C, Rodon J, Sellappan S, Cecchi F, Hembrough T, Nuciforo P. Quantitative proteomic analysis of FGFR by mass spectrometry may improve identification of FGFR amplified tumors sensitive to inhibitor therapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-16.