Publication protocol
Tissue microarrays (TMA) were constructed using previously described methods.14,15 These included 297 cases from New York, Boston, Mexico, Japan and Romania to perform p16, p53, Progesterone receptor (PR), Androgen receptor (AR), Vimentin, HER2, HIK1083, MUC6, CAIX, SATB2, HNF1beta, PAX8, CK7, CK20, CDX2, GATA3, p63, p40 (Table 2). Each of the tumors from the New York, Mexico, and Romania centers was represented by three 0.6 mm cores, while cases from Japan were represented by single 3 mm cores. Except for ER, CK20, napsin A and GATA-3, which were scored by 1 pathologist (RAS or TK), stains were scored by 2 study pathologists (RAS and SS) reaching a consensus. Disagreements were extremely rare (approximately 2-3%) and were adjudicated by re-reviewing stated criteria for positivity, as described below. In some cases, only 1 or 2 cores remained on the stained slide; and were still considered eligible for scoring. p16 was interpreted as positive if diffuse, if block-like staining was found in all cores; no staining, or patchy staining, was interpreted as negative. p53 was scored as positive if ≥75% of tumor cell nuclei were strongly positive or if no staining was present in the background of an intact internal control. ER, PR, AR, PAX8, CK7, CK20, HNF-1beta and napsin A were interpreted as positive if >25% (score 3 or 4) of tumor cell nuclei or cytoplasm (CK7, CK20 and napsin A) were stained as follows: Score 0: <5%; score 1+: 5–10%; score 2+: 11–25%; score 3+: 26–75%; score 4+: more than 75%. Vimentin was scored as positive if ≥50% of tumor cells showed membranous/cytoplasmic staining. HER2 was scored using the CAP guidelines for gastric carcinoma: 3+ membranous positive.16 HIK 1083, MUC 6 and CAIX, SATB2, GATA3, p63, p40 and CDX2 were considered positive if any nuclear staining was noted in >5% of tumor cells. HIK 1083 is currently not available in the US.
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