A nCounter CNV Assay to Detect HER2 Amplification: A Correlation Study with Immunohistochemistry and In Situ Hybridization in Advanced Gastric Cancer.

Aim: Screening amplified genes for targeted therapy with high-throughput technology is very important. The NanoString nCounter system allows multiplexed digital quantification of target molecules through the use of color-coded barcodes with the great advantage that formalin-fixed, paraffin-embedded (FFPE) tissue can be utilized.

Methods: We tested nCounter custom copy number variation (CNV) panels in 220 gastric cancer samples and evaluated the utility of this method as a screening tool for the detection of CNV using HER2. For the validation of results, we compared the nCounter results with immunohistochemistry (IHC), and we further performed in situ hybridization (ISH) in discrepant cases.

Results: The average HER2 gene copy numbers (CNs) by nCounter were 17.25, 2.0 and 2.61 for the HER2 IHC positive (3+), equivocal (2+), and negative cases, respectively. Out of the 16 IHC 3+ cases, 13 (81.3 %) were reported as HER2 CN gain (≥4). Gastric cancers with homogeneous HER2 overexpression or high tumor purity showed HER2 CN ≥10. Among the 192 cases with HER2 IHC negative and without HER2 gene amplification, 29 showed a HER2 CN ≥4 with the nCounter assay. The nCounter assay had a concordance rate of 83.4 % (kappa value, 0.35), a sensitivity of 66.7 %, a specificity of 85.2 %, a negative predictive value of 96 %, and a positive predictive value of 32.6 % compared with HER2 IHC/ISH results. Fresh frozen (FF) samples revealed a higher concordance rate (91.5 %, kappa value, 0.59) than FFPE samples (78.5 %, kappa value 0.27) and showed a high specificity (97.2 %).

Conclusion: The nCounter CNV assay is a reliable and practical method to detect high CN variations. Given the intra-tumoral HER2 heterogeneity and normal cell contamination, additional IHC and/or FISH is necessary and needs caution in interpretation, especially in FFPE tissue samples.