FGFR1 amplifications have recently been identified in 22% of squamous non-small cell lung cancers (sqNSCLC) (Weiss et al., Sci Transl Med., 2(62), 2010) and in pre-clinical models this amplification confers sensitivity to FGFR inhibitor induced cell growth arrest and death (Zhang et al., Clin. Can. Res., In press). Ongoing clinical trials of FGFR inhibitors are selecting patients based on FGFR1 amplifications, as determined by FISH. The development of additional biomarker assays to evaluate the expression of FGFRs, FGF ligands and other potential modulators of response to support analysis of clinical tissues is warranted.

We validated the Nanostring platform to allow gene expression (mRNA) analysis of 194 genes, including FGFRs and FGF ligands, from one 5μm section of formalin fixed paraffin embedded clinical tumor tissue. Intra- and inter-assay variability was low, as was variability between adjacent sections from the same tumor sample. Further validation of a subset of these genes by RT-PCR revealed a good correlation between platforms. A panel of 50 sqNSCLC tumors were analysed for FGFR1 gene amplification by FISH and for gene expression by Nanostring. FGFR1 amplfied tumours were enriched for high FGFR1 mRNA expression (p<0.02) compared to non-amplified samples, although a subset of non-amplified tumours also demonstrated increased FGFR1 mRNA expression. Immunohistochemical analysis, employing an FGFR1 specific antibody, revealed a correlation between FGFR1 mRNA levels and protein expression in these tumors. Nanostring analysis of additional genes on the 8p12 region, revealed that several of these, for example PPAPDC1B, ASH2L, TACC1 and in particular the FGFR1 neighbouring gene WHSC1L1 are highly expressed in FGFR1 amplified tumors (p<0.0001 for WHSC1L1). In contrast, expression of LETM2, located between WHSCL1 and FGFR1, was not significantly increased in amplified tumors.

An FGF ligand expression profile was generated for each tumor, showing that a-FGF, b-FGF and FGF7 were the most abundantly expressed ligands in the sqNSCLC samples, with increased b-FGF expression detected in high FGFR1 expressing tumours. The expression of a panel of potential mediators of resistance to FGFR inhibitor therapy such as c-MET, EGFR and IGF1R were also profiled across the samples, revealing differential expression of these factors across each of the tumors and an inverse correlation between FGFR1 expression and ERBB3 expression.

Taken together, these data validate the utility of the Nanostring platform for expression analysis of FGFR pathway genes using mRNA isolated from FFPE tumor samples and this platform can be used to assess the level of expression of potential modulators of clinical outcome.

Citation Format: Claire Rooney, Dawn Baker, Neil R. Smith, Victoria Williams, Elizabeth A. Harrington, Carl Barrett, Paul D. Smith, Elaine Kilgour. Expression profiling of FGF-receptor pathway genes in squamous NSCLC tissue by Nanostring. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2407. doi:10.1158/1538-7445.AM2013-2407