The College of American Pathologists/American Society of Clinical Oncology recommends HER2testing prior to initiation of targeted therapy for patients with advanced Gastroesophageal adenocarcinoma (GEA), using immunohistochemistry (IHC) followed by fluorescence in situ hybridization (FISH) in cases with an equivocal (score 2 + ) result on IHC. The FISH results are considered indeterminate if the HER2/CEP17 ratio is <2.0 with an average CEP17 copy number of ≥3.0 and a HER2copy number ≥4.0 and ≤6.0 after counting additional tumor cells. Indeterminate results may be resolved by using an alternative chromosome 17 probe such as RAI1. The purpose of this study is to review our experience with RAI1alternate probe in HER2FISH testing of GEA in a large reference laboratory setting. Esophageal, gastroesophageal, and gastric adenocarcinomas received for HER2FISH testing in our lab between 9/2018 and 1/2020 were included. HER2/CEP17 and HER2/ RAI1ratios, and the average HER2, CEP17, RAI1signals per cell were recorded. 328 GEA had HER2testing performed in our lab during the study period. 101 (30.8%) were amplified, 169 (51.5%) were non-amplified and 58 (17.7%) were indeterminate. Following RAI1testing, 42 (72.4%) of 58 indeterminate cases were reclassified as non-amplified and 16 (27.6%) were reclassified as amplified, increasing the total amplified cases to 117 (35.7%). The correlation between the average CEP17 and RAI1copy number for all cases was weak (R2= 0.095). In summary, using the alternate probe RAI1reclassifies 27.6% of original HER2FISH indeterminate gastroesophageal carcinomas as amplified, which makes them eligible for targeted therapies.