Cytotoxic T cell depletion with increasing epithelial abnormality in women with benign breast disease

We obtained Institutional Review Board approval prior to conducting this research. We utilized samples from an existing study of breast tissues from normal donor women and women with benign breast disease [8]. Briefly, normal breast tissues were obtained from the Susan G. Komen^® for the Cure Tissue Bank at IU Simon Cancer Center (i.e. Komen Tissue Bank = KTB), a unique resource of tissue from donor women with no known clinical breast abnormalities [11]. Breast tissues with benign disease were obtained from the Mayo benign breast disease (BBD) Cohort [9], a well-annotated cohort of 13,000 women with follow-up information on later breast cancer events. Within the cohort, a nested set of 100 cases (BBD women with later BC) and 100 controls (BBD cancer-free women) were randomly selected from the latter years of the cohort (1992–2001) and were matched on age, year at biopsy, and length of follow-up. Then an age-matched normal breast tissue donor was randomly selected from the KTB samples to create an age-matched triplet: KTB normal tissue donor, BBD woman with later BC, and BBD cancer-free woman. Groups were also frequency matched for first-degree family history of breast cancer. From these age-matched triplets, ten were randomly selected for evaluation of CD8/CD103 staining.

For each study sample, an H&E section was digitally scanned with the Aperio ScanScope^® XT slide scanner (Leica Biosystems^®, Buffalo Grove, IL) using the 20 × objective lens. The H&E slide was assessed by the study breast pathologist (DWV) for an overall histologic impression of the greatest severity of abnormality according to established categories of benign breast lesions: no histologic abnormality, nonproliferative changes, proliferative changes without atypia, or atypical hyperplasia. From each digital H&E image, 10 representative lobules (or all lobules if < 10 present) were selected, circled digitally, and annotated using Imagescope. Each circled lobule was individually classified as normal, nonproliferative, or proliferative (with or without atypia), according to established criteria that are based upon the degree of epithelial proliferation and cytologic and architectural atypia, as these categories have stratified later BC risk in multiple studies [9, 12]. Examples of nonproliferative lesions include columnar cell alteration, cystic change, fibroadenoma, apocrine metaplasia; proliferative lesions include columnar cell hyperplasia, usual or florid ductal hyperplasia, radial scar, sclerosing adenosis, flat epithelial atypia, and atypical hyperplasia. A serial formalin-fixed paraffin-embedded tissue section was stained for CD8 and CD103 and also digitally scanned scanned with the Aperio Scanscope^® FL slide scanner (Leica Biosystems^®) using the 20 × objective lens. Matched samples (KTB donor, BBD woman with later BC, BBD cancer-free woman) underwent immunostaining within the same batch to minimize batch effects. The following immunostains were performed with the following antibodies: CD8 (DAKO clone C4/188B, 1:100) and secondary antibody conjugated with AlexaFluor 488 (Life technology, 1:1000); CD103 (AbCam clone EPR4166, 1:2500) and secondary antibody conjugated with AlexaFluor 547 (Life technology, 1:1000).

Samples were deparaffinized with 3 changes of xylene and rehydrated in a series of ethanol baths (100%, 90%, and then 70% Ethanol) and rinsed well in running distilled water. Slides were then placed in a preheated Antigen Retrieval Solution (pH 8.0 EDTA buffer, Vector Lab) for 25 min and then cooled in the buffer for 30 min followed by a 5 min rinse in running distilled water. After the heat-inactivated epitope retrieval step, slides were placed on the DAKO Autostainer for the following procedure (at room temperature). Sections were incubated with phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA), 2% fetal bovine serum (FBS) to block non-specific staining for 30 min. Sections were incubated in primary antibody at dilutions listed above for 60 min at room temperature. Sections were rinsed with PBS three times, followed by another 60-min incubation with secondary antibody as listed above. The slides were rinsed with PBS three times. Sections were then mounted with 1 drop of Prolong Diamond Antifade Mountant and coverslipped. Digital images were analyzed using Aperio ImageScope Software, version 12.1.0.5029 (Leica Biosystems^®, Buffalo Grove, IL). The annotated lobules from the H&E slide were identified; then each lobule was circled digitally and the area was calculated. The RGB image was unmixed into separate channels for absorbance at 488 and 594. Within each lobule, positive cells were annotated individually for each color (with all other colors masked). After all positive cells were identified, colors and annotations were unmasked, and both singly and double positive cells were counted.

Cell densities were calculated as the number of positively stained cells per mm² within each outlined lobule area. Number of CD8+/CD103+ cells was analyzed as a count variable using a negative binomial regression model with the logarithm of the lobule area as offset. A generalized linear mixed model approach was used to account for the correlation among multiple lobules measured for each subject. Analysis was performed using PROC GLIMMIX in SAS^® (SAS^® Institute Inc., Version 9.4). p values < 0.05 were considered statistically significant.