Computational protocol: Prognostic and predictive role of spatially positioned tumour infiltrating lymphocytes in metastatic HER2 positive breast cancer treated with trastuzumab

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Protocol publication

[…] Immunohistochemistry was conducted on 3.5 µm sections cut from the paraffin-embedded specimens. The sections were deparaffinized in xylene and rehydrated through graded alcohols. Antigen retrieval was conducted in a microwave oven with Tris-EDTA (pH9) buffer at 800 W for 2 min and at 150 W for 15 min. The incubation with primary antibodies (CD3, Novocastra, Leica Biosystems, mouse monoclonal, clone PS1, dilution 1:50; or CD8, Novocastra, mouse monoclonal, clone 4B11, dilution 1:200; or CD56, Cell Marque, rabbit monoclonal, clone MRQ-42, dilution 1:100; or CD68, Dako, mouse monoclonal, clone PG-M1, dilution 1:100) was conducted at room temperature for 30 min. Bound antibodies were detected using the EnVisionTM (Dako) system. Diaminobenzidine (DAB) was used as the chromogen and hematoxylin as the counterstain.For the analysis of the immune cell densities in different tumour locations (Fig. ), sections were scanned with Aperio AT2 image-capturing device (Leica Biosystems). Imagescope (Aperio Technologies) software, version 11.2 was used to view the scanned images. With the software, 3–6 (median 6) images, depending of the size of the tumour sample, were captured with ×20 magnification from the center of the tumour (CT) and the invasive margin (IM). The immune cells were counted using an earlier described and validated computerassisted counting method, that utilizes ImageJ, a freeware image analysis software. Intraepithelial (IEL) immune cells were counted manually because the automated method was not able to distinguish the intraepithelial immune cells from stromal immune cells. Tertiary lymphoid structures (TLSs) were manually counted and measured from the H-E stained sections. Final counts presented in this manuscript are the average cell densities for each tumour area (immune cells), the diameter of the largest TLS in the samples (TLS diameter) and the highest density of TLSs/2x magnification (TLS density). [...] IBM SPSS Statistics 22.0.0.0 for Windows was applied for statistical analysis. The reported p-values are from two-sided chi-square tests. Receiver operating characteristics (ROC) analysis for the whole patient material was used to determine optimal cutoff scores of CD3, CD8, CD56, CD68 and TLS densities for discriminating the survivors from the non-survivors. Survival was analysed by using the Kaplan-Meier method with the log-rank test. Multivariate analysis was performed using Cox regression analysis. Correlation analysis was performed using Spearman’s correlation (2-tailed) and scatter plot analysis. Probability values below 0.05 were considered significant. […]

Pipeline specifications

Software tools ImageScope, ImageJ, SPSS
Applications Miscellaneous, Whole slide imaging, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms