Comprehensive evaluation of methods to assess overall and cell-specific immune infiltrates in breast cancer

Abstract

Background: Breast cancer (BC) immune infiltrates play a critical role in tumor progression and response to treatment. Besides stromal tumor infiltrating lymphocytes (sTILs) which have recently reached level 1B evidence as a prognostic marker in triple negative BC, a plethora of methods to assess immune infiltration exists, and it is unclear how these compare to each other and if they can be used interchangeably. Methods: Two experienced pathologists scored sTIL, intra-tumoral TIL (itTIL), and 6 immune cell types (CD3 + , CD4 + , CD8 + , CD20 + , CD68 + , FOXP3 +) in the International Cancer Genomics Consortium breast cancer cohort using hematoxylin and eosin-stained (n = 243) and immunohistochemistry-stained tissue microarrays (n = 254) and whole slides (n = 82). The same traits were evaluated using transcriptomic-and methylomic-based deconvolution methods or signatures. Results: The concordance correlation coefficient (CCC) between pathologists for sTIL was very good (0.84) and for cellspecific immune infiltrates slightly lower (0.63-0.66). Comparison between tissue microarray and whole slide pathology scores revealed systematically higher values in whole slides (ratio 2.60-5.98). The Spearman correlations between microscopic sTIL and transcriptomic-or methylomic-based assessment of immune infiltrates were highly variable (r = 0.01-0.56). Similar observations were made for cell type-specific quantifications (r = 0.001-0.54). We observed a strong inter-method variability between the omics-derived estimations, which is further cell type dependent. Finally, we demonstrated that most methods more accurately identify highly infiltrated (sTIL ≥ 60%; area under the curve, AUC, 0.64-0.99) as compared to lowly infiltrated tumors (sTIL ≤ 10%; AUC 0.52-0.82).

Quantitative multiplex immunofluorescence analysis identifies infiltrating PD1+CD8+ and CD8+ T cells as predictive of response to neoadjuvant chemotherapy in breast cancer

“…AI‐based analysis of multiple markers in full slides is feasible; however, it requires further validation and standardization to be translated into clinical use. Whole tissue slides would be preferable for further validation as significant variations among assessment methods have been reported to occur in tissue microarray‐based TIL analysis 22 . Interaction and linearity tests of T cells should be included in future studies to determine and validate biomarker thresholds.…”

“…21 However, different methods may have varied accuracy in special quantification, and full slides may be preferable to TMA-based analysis. 22 The International TILs Working Group recommended analyses of TILs or CD8 + T cells using AIbased approaches for further investigation. 12,23 Other new technologies such as mass cytometry, single-cell sequencing, and multicolor IHC are being optimized to assess the level and role of different immune cell types in TME.…”

“…Computational pathology can provide objective, quantitative, and reproducible tissue metrics and represents a viable means of outcome prediction in BC 21 . However, different methods may have varied accuracy in special quantification, and full slides may be preferable to TMA‐based analysis 22 . The International TILs Working Group recommended analyses of TILs or CD8 + T cells using AI‐based approaches for further investigation 12,23 .…”

TILs Immunophenotype in Breast Cancer Predicts Local Failure and Overall Survival: Analysis in a Large Radiotherapy Trial with Long-Term Follow-Up

“…Several studies investigated the number of TMA cores required for accurate representation of whole slide TILs assessment in breast cancer, with one study finding that 4 × 0.6 mm cores (=1.12 mm 2 ) was adequate for accurate TILs assessment [ 43 ], which compares favourably with our study containing 3 × 1 mm (=2.35 mm 2 ), whereas another study suggested that a single 2 mm core from a tumour had 98.9% accuracy in representing the TILs population of the tumour [ 44 ]. A comparison of sTILs H/E scoring on WSI and TMA assessment showed only a moderate correlation (Spearman 0.56), with higher levels present in WSI [ 45 ] and higher values for specific immune subpopulations on WSI. They also found that human observers tended to score higher numbers of immune subtypes compared to automated image analysis.…”

Immune Cell Infiltration-Based Characterization of Triple-Negative Breast Cancer Predicts Prognosis and Chemotherapy Response Markers

“…Increasing evidence highlights the critical role of the immune system in the initiation and progression of cancers ( Wang et al, 2018 ; Cai et al, 2019 ; Tu et al, 2020 ). TNBC is associated with a high density of tumor-infiltrating lymphocytes (TILs) defined by histopathology evaluation, which represents a robust intratumoral inflammatory response describing triple negative tumors as an immunogenic neoplasia ( Nederlof et al, 2019 ; Romero-Cordoba et al, 2019 ). In addition to TIL count by immune pathological evaluation, other methods recently emerged to assess the tumor immune landscape such as deconvolution algorithm to define the proportion of immune cells using genomic profiling ( Loi et al, 2019 ), to identify the gene expression signatures that distinguish the immune-state, and then to be a potential prognostic factor ( Romero-Cordoba et al, 2019 ).…”