Automated immunohistochemical stainer

To confirm the BC subtype listed in the original diagnostic pathology reports, we utilized formalin fixed, paraffin-embedded tumor blocks from a subset of women (n=100) identified with a subsequent cancer to examine the following markers: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Briefly, the slides were deparaffinized and rehydrated. Antigen retrieval was performed, followed by immunohistochemical staining using a Ventana automated immunohistochemical stainer, counterstained (when necessary), dehydrated, and mounted. The pathologist was blinded to the clinical characteristics and prior pathology report associated with the tissue. All antibodies were sourced from DAKO, and the specific conditions and positivity assessment are as follows: ER, clone 1D5, 1:100 dilution, positive if greater than 1% of nuclei stained; PR, clone PgR636, 1:100 dilution, positive if greater than 1% of nuclei stained; HER2, Hercept Test (pre-diluted), greater than 30% of cells showed circumferential intense and uniform staining. There was approximately a 90% concordance between the prior reports and the repeated analysis (data not shown), thus the receptor data abstracted from the pathology reports was used.

IHC was performed on 4 μm‐thick tissue sections with the automated immunohistochemical stainer (Ventana, Tucson, AZ). The primary antibodies used in the study included anti‐PD‐L1 (Clone 28‐8, Abcam, Cambridge, MA), anti‐MLH1 (Clone M1, Ventana), anti‐PMS2 (Clone EPR3947, Ventana), anti‐MLH2 (Clone G219‐1129, Ventana), anti‐MLH6 (Clone 44, Ventana), and anti‐ erbB‐2 (HER2) (Clone 4B5, Ventana). Omission of primary antibody and substitution by non‐specific immunoglobins were used as negative controls. The appropriate specificity and sensitivity of the antibody against PD‐L1 staining were determined using human placenta as the positive control. Appropriate positive controls were run concurrently for all antibodies tested. PD‐L1 expression showed membranous staining and/or cytoplasmic staining. The proportion of immunostained cells was evaluated among tumor cell and tumor‐infiltrating immune cells. The patients with at least 1% PD‐L1 staining of tumor cells or immune cells were considered positive. The cases showed preserved nuclear expression of 4 MMR proteins were considered MMR‐proficient (pMMR). HER2 expression was graded using a score scale of 0 to 3 15.