Immunohistochemistry

TP53 IHC was performed on formalin-fixed and paraffin-embedded (FFPE) tissue sections of 4 um thickness. The majority of staining was performed at the Department of Pathology and Laboratory Medicine, University of Calgary, Canada, including 80/113 (70.8%) cases from the initial GAMuT cohort, all quality control full sections, and all TMAs from the OTTA cohort. Cases were stained using a previously published protocol (9). After 30 minutes of heat-induced pre-treatment using the high pH retrieval buffer, the DAKO Omnis protocol H30-10M-30 with the ready-to-use clone DO-7 (catalog # GA61661-2; DAKO) was utilized. From the remaining initial cohort, 24/113 cases were stained using full sections at the Department of Pathology, Peter MacCallum Cancer Centre, Australia, and 5 cases in TMAs were stained at the originating center, also using clone DO-7. For 4 cases, tissue was not available for analysis and therefore, the TP53 IHC status was retrieved from pathology reports issued for these cases.

Interpretation was initially performed according to established criteria for ovarian and endometrial carcinomas (15). IHC and sequencing results were independently interpreted with evaluators of either component blinded to the other result. Abnormal (also variably referred to as mutation-type or aberrant) TP53 staining showed one of 3 patterns: overexpression (OE) with virtually all viable tumor cell nuclei showing strong nuclear staining; complete absence (CA; also referred to as “null pattern”) with no nuclear staining of tumor cells but with normal control staining in non-tumor stromal or immune cells providing an internal control; and cytoplasmic (CY) with unequivocal cytoplasmic staining and variable nuclear staining. By contrast, tumors with normal (also referred to as wild-type pattern) staining showed nuclear expression of variable intensity and cellular distribution. If no staining was seen in any cells (CA in tumor cells and no internal control), the sample was excluded from the study.

Based on the observation of intratumoral heterogeneity (subclonal abnormal patterns) and terminal differentiation in ovarian mucinous tumors, the criteria were refined. In cases demonstrating OE, the percentage area of tumor cells exhibiting contiguous strong nuclear staining was estimated to the nearest 5%, and the average percentage of OE across all scorable TMA cores from each case was calculated. Following review of discrepant cases regarding the TP53 status by established IHC criteria, we modified the threshold for OE: strong nuclear staining in contiguous areas of at least 5% of the tumor qualified as abnormal OE. Criteria for scoring other staining patterns (WT, CA, CY) remained unchanged. Two observers (MK, EK) independently scored TP53 IHC on 144 cases from the OTTA cohort using the refined criteria and agreement was assessed to evaluate interobserver reproducibility. A consensus was reached for discordant cases. During this we noted that the minimal threshold of 5% translated into ≥12 consecutive cells with strong nuclear staining. In the fallopian tube and the endometrium, a TP53 signature requiring at least 12 consecutive cells with abnormal-pattern TP53 staining has been shown to accurately predict TP53 mutations (16). Hence, ≥12 consecutive cells with strong nuclear staining was applied as an alternative threshold for minimal abnormal OE.