The Immune Microenvironment of Breast Ductal Carcinoma in Situ

Elizabeth Thompson; Janis M Taube; Hillary Elwood; Rajni Sharma; Alan Meeker; Hind Nassar Warzecha; Pedram Argani; Ashley Cimino-Mathews; Leisha A Emens


Mod Pathol. 2016;29(3):249-258. 

In This Article

Materials and Methods

Case Selection and Tissue Microarray Construction

This study was approved by the Institutional Review Board of the Johns Hopkins Medical Institutions. We evaluated tissue microarrays previously constructed from archived, paraffin-embedded blocks of primary DCIS,[25,26] with 27 cases evaluable. Each DCIS case was sampled with 2–5 cores/tumor, with each core measuring 1.4 mm in diameter; 1 core/case sampled benign breast lobules. Clinicopathologic characteristics were recorded, including patient age, gender, race, tumor size, presence or absence of associated invasive carcinoma, DCIS nuclear grade, presence or absence of necrosis, ER, PR and HER-2 status, and patient outcome (local recurrence, metastasis, and survival). Carcinoma recurrence was defined as any in situ or invasive carcinoma recurrence in the ipsilateral breast, axilla, or chest wall. Younger women were defined as ≤40 years old, and older women as >40 years old.

Immunohistochemistry and Tumor Infiltrating Lymphocyte Quantification

To characterize the DCIS subtype, tissue microarrays were stained by immunohistochemistry for ER (clone 6F11; Leica Microsystems, Bannockburn, IL, USA), PR (clone 16; Leica Microsystems), and HER-2 (Ventana 4B5; Ventana Medical Systems, Tucson, AZ, USA) using standard automated methods. Tumors were classified as Luminal A (ER+/PR+/HER-2), Luminal B (ER+/PR+/HER-2+), HER-2+ (ER/PR/HER-2+), and triple negative (ER/PR/HER-2). HER-2 positivity was defined as ≥10% complete strong membranous DCIS cell labeling as per the 2012 ASCO/CAP guidelines for HER-2 immunohistochemistry.

Each case was assigned a qualitative stromal tumor infiltrating lymphocyte density score on the hematoxylin and eosin (H&E) stained sections: 0 (no tumor infiltrating lymphocytes), 1 (mild, <5% tumor area with tumor infiltrating lymphocytes), 2 (moderate, 5–50% tumor area with tumor infiltrating lymphocytes), and 3 (diffuse/marked, >50% infiltration).[19,24,27] Tumor infiltrating lymphocytes were scored by two pathologists (ET and ACM) blinded to clinicopathologic characteristics. Tissue microarrays were stained for CD20 (monoclonal, clone MS/L26, catalog no. 760-2531, Ventana Medical Systems), CD3 (mouse monoclonal, clone PS1, catalog no. ORG-8982, Leica Microsystems), CD4 (rabbit monoclonal, clone SP35, catalog no. q790-4423, Ventana Medical Systems), CD8 (mouse monoclonal, clone C8/C8144B, catalog no. 760-4250, Cell Marque, Rockin, CA, USA), and FoxP3 (mouse monoclonal, clone 236A/E7, catalog no. 14-4777-80, dilution 1:50; eBioscience, San Diego, CA, USA) to characterize tumor infiltrating lymphocytes as previously described.[23] CD20, CD3, CD4, and CD8 expression was defined by membranous lymphocyte labeling, and FoxP3 expression was defined by nuclear labeling in lymphocytes. The total number of tumor infiltrating lymphocytes/high power field was manually counted in one high power field/tissue microarray tumor core, and averaged across the case to give the mean number of tumor infiltrating lymphocytes/high power field. Each high power field was chosen as representative of the overall tumor lymphocytic infiltration in each tumor core.

Tissue microarrays were stained for PD-L1 (B7-H1) using the murine anti-human PD-L1 antibody, clone 5H1 (2 μg/ml) with a paired isotype murine IgG1 control as previously described.[19] PD-L1 staining was scored by two pathologists (ET and ACM) blinded to patient clinicopathologic characteristics; discrepancies were adjudicated by a third pathologist (JT). PD-L1 staining on carcinoma cells and tumor infiltrating lymphocytes was scored by percent membranous staining. PD-L1 positivity by DCIS carcinoma cells was defined as >5% membranous staining. PD-L1 positivity in tumor infiltrating lymphocytes was scored as none (0), focal (1+; <5%), moderate (2+; 5–50%), or marked (3+; 51–100%) percentage of tumor infiltrating lymphocytes expressing PD-L1. PD-L1 expression was also scored as low (≤50% tumor infiltrating lymphocytes) or high (>50% of tumor infiltrating lymphocytes).

Statistical Analysis

Statistical analysis was performed using Fisher's exact test and paired, two-tailed Student's T-test.