Background: Patient inclusion to the primary efficacy population of TRIO-013/LOGiC trial of lapatinib in combination with chemotherapy in advanced HER2-positive UGI adenocarcinomas required evidence of HER2 gene amplification confirmed by central lab. As no HER2 tests were approved for use in UGI cancers at trial start, the HER2 FISH assay used in the 2 trial central labs was PathVysion. A study was conducted in a cohort of UGI adenocarcinomas to determine concordance of PathVysion HER2 FISH assay between the 2 central labs and concordance between PathVysion HER2 FISH and HER2 IQFISH pharmDx, an assay recently approved in UGI adenocarcinomas. Methods: 488 UGI adenocarcinoma cases, procured as formalin-fixed, paraffin-embedded blocks, were screened by HercepTest to ensure representation of 4 immunochemistry (IHC) staining intensities. All IHC3+ and 2+cases were included; IHC1+ and 0 cases were binned by primary tumor (gastric, gastro-esophageal junction [GEJ] and esophageal) then randomly selected. Selected cases were sent to 2 trial central labs for HER2 FISH testing, blinded to the IHC results. Overall acceptance criteria were predefined as positive percent agreement (PPA) and N (negative) PA of at least 90%. Results: 159 cases were selected: 31 IHC3+; 20 IHC2+; 55 IHC1+; 53 IHC0. Of the 159 cases, 105 were gastric, 35 GEJ and 19 esophageal adenocarcinomas. All 159 cases were tested by 1 central lab using both assays; the PPA (95% Confidence Interval [CI]) and NPA (95% CI) were 97.9% (89.1, 99.6) and 99.1% (94.8, 99.8), respectively. Preliminary analysis in 126 cases tested by both central labs resulted in PPA of 95.6% (85.2, 98.8) and NPA of 94.5% (86.7, 97.8). Majority of IHC1+/0 cases were HER2 non-amplified by both assays (89%) and in both central labs (89% versus 93%). Of the 31 IHC3+ cases, 2 were considered HER2 non-amplified; both exhibited heterogeneity in HER2 IHC staining: 1 case was HER2 non-amplified by 1 central lab for both assays, yet amplified by the other central lab; other case was HER2 non-amplified by PharmDx assay only. Conclusions: There was reasonably high level of concordance within laboratory for different HER2 FISH assays as well as between labs for the same HER2 FISH assay. Clinical trial information: NCT00680901.