Hpa 019 051

A case series of 251 primary ovarian cancers was analysed on TMA. 6 TMAs were prepared with all the analysed samples [33 (link)]. Each tissue was examined for representability, and subsequently immunostained with anti-CCDC6 antibody (HPA 019,051, from Sigma-Aldrich), CCDC6 immunoreactivity of tumour cells was annotated. Visual annotation included staining intensity (negative, weak, moderate or strong), fraction of stained cells (% of total counted tumour cells), and subcellular localization. Following acquisition of anti-CCDC6 immunostained glass slides with a digital scanner, we also calculated the H-score on digital images by QuPath image analysis software [34 (link), 35 (link)]. Immunohistochemistry was performed as already described [30 (link)]. Median H-score was 64.2 (IQR 28.8; 97.2); Mean H-score was 67.5 (sd 47.1). The frequency distribution of digitally evaluated CCDC6 H-Score is summarised in Additional File 3: Table S2. A summary of study population is shown in Additional File 4: Table S3.
PDX FFPE sample slides were immunostained with anti-CCDC6 antibody (HPA 019051, from Sigma-Aldrich) and IHC signal was quantified on digital slides with Positive Cell Count function of QuPath software. CCDC6 protein expression was reported as optical density arbitrary units. All the glass slides were digited with Aperio AT2 digital scanner (Leica).

For light microscopy, tissues (25 whole sections and TMA) were fixed by immersion in 10% formalin and embedded in paraffin by standard procedures; 4 μm sections were stained with haematoxylin and eosin (H&E) or processed for immunohistochemistry with anti-CCDC6 antibody, HPA-019051 (Sigma-Aldrich, Co. LLC), and anti-USP7 antibody, HPA-015641 (Sigma-Aldrich, Co. LLC). The immunohistochemical staining of USP7 and CCDC6 was evaluated semiquantitatively as the percentage of positive cells (with either nuclear or cytoplasmic localization). Cells were classified as low staining, [e.g. 0 (<5%) and + (5-25%)] and high staining [++ (26-50%) and +++ (>50%)].