Association between ERBB2 (HER2) amplification, RNA gene expression (GE), and immunohistochemistry (IHC) in GI cancers.

23 Background: ERBB2 copy number (CN) and amplification (amp) – assessed with next-generation sequencing (NGS) and in situ hybridization – and HER2 protein overexpression – assessed with IHC – are established targets in GI cancers. ERBB2 RNA GE, may serve as a surrogate for HER2 IHC and could be an informative metric to predict therapy response. Here, we explore the relationship between ERBB2 (HER2) amp and expression at the gene and protein levels in GI cancers to understand their comparative predictive value. Methods: We queried a genomic database (Foundation Medicine, Inc., Boston, MA) of comprehensive genomic profiling (CGP) results from 380 GI patients (53% CRC, 19% gastroesophageal, 18% pancreatic, 10% other) who received FoundationOne CDx +/- FoundationOne RNA and HER2 IHC testing. ERBB2 amp was defined as CN ≥ ploidy +3 and parallel analysis using a quantitative AmpRatio (gene CN/sample ploidy) was performed. GE (Research Use Only) was quantified from RNAseq data and reported as transcripts per million (TPM). HER2 IHC was performed with the Ventana 4B5 antibody and scored according to package insert. Results: For 380 GI patients (178 IHC 0, 123 IHC 1+, 63 IHC 2+, 16 IHC 3+), median ERBB2 segment AmpRatio ranged from 1.0-1.1 for IHC 0/1+/2+ cases, but was significantly elevated in IHC 3+ cases (median 12.4). Amps were never observed in IHC 0 and 1+, while 11.1% of IHC 2+ cases were amp+ vs 81.3% of IHC 3+ (p < 0.0001) with median AmpRatio 2.3 vs 15 (p = 0.002). Conversely, 65% (13/20) of amp+ cases were IHC 3+. In the 178 patients with GE, ERBB2 GE increased nonlinearly with IHC positivity, with distinct interquartile ranges between IHC 3+ and IHC < 3+ groups. Using the 95th percentile of ERBB2 GE to dichotomize, a 77.8% (7/9) positive agreement with IHC 3+ was observed. 18 patients harbored pathogenic ERBB2 mutations (mut) which were largely mutually exclusive with amps, there being only 2 ERBB2 mut amp+ cases (both IHC 3+) with AmpRatios 2.1 and 13.4, respectively. The remaining 16 mut cases were HER2- (10 IHC 0, 6 IHC 1+). The most frequently observed mut were R678Q (n = 5), S310X (n = 4), T733I and V777L (n = 2 each). Conclusions: This exploratory analysis demonstrated high concordance between IHC 3+ and ERBB2 segment AmpRatio, amp-positivity, and GE. By complementing IHC with additional detection modalities, integrated DNA and RNA CGP could identify additional patients who may benefit from anti-HER2 therapies. Clinical correlation is needed to refine ERBB2 AmpRatio and GE biomarker definitions. IHC 0 IHC 1+ IHC 2+ IHC 3+ p Total N (N w/ GE) 178 (86) 123 (59) 63 (24) 16 (9) ERBB2 amp 0 (0.0) 0 (0.0) 7 (11.1) 13 (81.3) <0.0001 ERBB2 mut 10 (5.6) 6 (4.9) 0 (0.0) 2 (12.5) 0.12 ERBB2 segment AmpRatio, Median IQR 1.000.88, 1.09 1.030.98, 1.18 1.121.05, 1.33 12.404.76, 36.39 <0.0001 ERBB2 GElog2(TPM + 1), Median IQR 11.5011.12, 11.90 11.9511.64, 12.23 12.4112.10, 12.54 14.4713.20, 15.08 <0.0001