ONEST (Observers Needed to Evaluate Subjective Tests) Analysis of Stromal Tumour-Infiltrating Lymphocytes (sTILs) in Breast Cancer and Its Limitations.

Tumour-infiltrating lymphocytes (TILs) reflect antitumour immunity. Their evaluation of histopathology specimens is influenced by several factors and is subject to issues of reproducibility. ONEST (Observers Needed to Evaluate Subjective Tests) helps in determining the number of observers that would be sufficient for the reliable estimation of inter-observer agreement of TIL categorisation. This has not been explored previously in relation to TILs. ONEST analyses, using an open-source software developed by the first author, were performed on TIL quantification in breast cancers taken from two previous studies. These were one reproducibility study involving 49 breast cancers, 23 in the first circulation and 14 pathologists in the second circulation, and one study involving 100 cases and 9 pathologists. In addition to the estimates of the number of observers required, other factors influencing the results of ONEST were examined. The analyses reveal that between six and nine observers (range 2-11) are most commonly needed to give a robust estimate of reproducibility. In addition, the number and experience of observers, the distribution of values around or away from the extremes, and outliers in the classification also influence the results. Due to the simplicity and the potentially relevant information it may give, we propose ONEST to be a part of new reproducibility analyses.

Neither Tumor-Infiltrating Lymphocytes nor Cytotoxic T Cells Predict Enhanced Benefit from Chemotherapy in the DBCG77B Phase III Clinical Trial.

Recent studies have shown that immune infiltrates in the tumor microenvironment play a role in response to therapy, with some suggesting that patients with immunogenic tumors may receive increased benefit from chemotherapies. We evaluated this hypothesis in early breast cancer by testing the interaction between immune biomarkers and chemotherapy using materials from DBCG77B, a phase III clinical trial where high-risk premenopausal women were randomized to receive chemotherapy or no chemotherapy. Tissue microarrays were evaluated for tumor-infiltrating lymphocytes (TILs) assessed morphologically on hematoxylin and eosin-stained slides, and by immunohistochemistry for CD8, FOXP3, LAG-3, PD-1 and PD-L1. Following REMARK reporting guidelines, data analyses were performed according to a prespecified statistical plan, using 10-year invasive disease-free survival as the endpoint. Differences in survival probabilities between biomarker groups were evaluated by Kaplan-Meier and Cox proportional hazard ratio analyses and prediction for treatment benefit by an interaction test. Our results showed that stromal TILs were associated with an improved prognosis (HR = 0.93; p-value = 0.03), consistent with previous studies. However, none of the immune biomarkers predicted benefit from chemotherapy in the full study set nor within major breast cancer subtypes. Our study indicates that primary tumors with higher immune infiltration do not derive extra benefit from cyclophosphamide-based cytotoxic chemotherapy.

Automated Quantification of sTIL Density with H&E-Based Digital Image Analysis Has Prognostic Potential in Triple-Negative Breast Cancers.

Triple-negative breast cancer (TNBC) is an aggressive and difficult-to-treat cancer type that represents approximately 15% of all breast cancers. Recently, stromal tumor-infiltrating lymphocytes (sTIL) resurfaced as a strong prognostic biomarker for overall survival (OS) for TNBC patients. Manual assessment has innate limitations that hinder clinical adoption, and the International Immuno-Oncology Biomarker Working Group (TIL-WG) has therefore envisioned that computational assessment of sTIL could overcome these limitations and recommended that any algorithm should follow the manual guidelines where appropriate. However, no existing studies capture all the concepts of the guideline or have shown the same prognostic evidence as manual assessment. In this study, we present a fully automated digital image analysis pipeline and demonstrate that our hematoxylin and eosin (H&E)-based pipeline can provide a quantitative and interpretable score that correlates with the manual pathologist-derived sTIL status, and importantly, can stratify a retrospective cohort into two significant distinct prognostic groups. We found our score to be prognostic for OS (HR: 0.81 CI: 0.72-0.92 p = 0.001) independent of age, tumor size, nodal status, and tumor type in statistical modeling. While prior studies have followed fragments of the TIL-WG guideline, our approach is the first to follow all complex aspects, where appropriate, supporting the TIL-WG vision of computational assessment of sTIL in the future clinical setting.

Report on computational assessment of Tumor Infiltrating Lymphocytes from the International Immuno-Oncology Biomarker Working Group.

Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring.

PD-L1 expression in breast cancer: expression in subtypes and prognostic significance: a systematic review.

PURPOSE: To systematically review the literature on the expression of PD-L1 in primary BC, variation of expression between subtypes and effect on overall survival (OS), disease-free survival (DFS), and recurrence-free survival (RFS). Additionally, for studies in the neoadjuvant setting, we have reviewed the ability of PD-L1 to predict pathological complete response (pCR). METHODS: Articles included in this review were retrieved by searching PubMed (1966-2018) and EMBASE (1980-2018). The following search terms were used: "PD-L1 expression" and "breast cancer" (PubMed234; EMBASE 161). RESULTS: Thirty-seven articles were found relevant to this study. We summarize important findings from these works, and show that the observed PD-L1 expression in the studies varies greatly, with expression rates ranging from 0 to 83% across subtypes. PD-L1 expression in relation to prognosis both in the adjuvant and neoadjuvant chemotherapy setting remains controversial, with studies finding better, worse, or no effect on prognosis. We also show that a wide variety of strategies are used when evaluating PD-L1 immunohistochemically, e.g., different cut-off points, different cell types evaluated, and different perceptions of when a cell is positive for PD-L1 (cytoplasmic vs membrane staining). CONCLUSION: Further investigation of PD-L1 expression in breast cancer and its effect on prognosis is required. There is little consensus on the methods used to evaluate PD-L1 expression immunohistochemically, and this may contribute to the diverging results found in this study.

Reproducibility and predictive value of scoring stromal tumour infiltrating lymphocytes in triple-negative breast cancer: a multi-institutional study.

BACKGROUND: Several studies have demonstrated a prognostic role for stromal tumour infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC). The reproducibility of scoring sTILs is variable with potentially excellent concordance being achievable using a software tool. We examined agreement between breast pathologists across Europe scoring sTILs on H&E-stained sections without software, an approach that is easily applied in clinical practice. The association between sTILs and response to anthracycline-taxane NACT was also examined. METHODOLOGY: Pathologists from the European Working Group for Breast Screening Pathology scored sTILs in 84 slides from 75 TNBCs using the immune-oncology biomarker working group guidance in two circulations. There were 16 participants in the first and 19 in the second circulation. RESULTS: Moderate agreement was achieved for absolute sTILs scores (intraclass correlation coefficient (ICC) = 0.683, 95% CI 0.601-0.767, p-value < 0.001). Agreement was less when a 25% threshold was used (ICC 0.509, 95% CI 0.416-0.614, p-value < 0.001) and for lymphocyte predominant breast cancer (LPBC) (ICC 0.504, 95% CI 0.412-0.610, p-value < 0.001). Intra-observer agreement was strong for absolute sTIL values (Spearman ρ = 0.727); fair for sTILs ≥ 25% (κ = 0.53) and for LPBC (κ = 0.49), but poor for sTILs as 10% increments (κ = 0.24). Increasing sTILs was significantly associated with an increased likelihood of a pathological complete response (pCR) on multivariable analysis. CONCLUSION: Increasing sTILs in TNBCs improves the likelihood of a pCR. However, inter-observer agreement is such that H&E-based assessment is not sufficiently reproducible for clinical application. Other methodologies should be explored, but may be at the cost of ease of application.

Triple negative breast cancer - prognostic role of immune-related factors: a systematic review.

PURPOSE: Treatment of breast cancer has been increasingly successful in recent years with the advent of HER2-receptor targeted treatment and endocrine treatment. However, the triple negative subgroup of breast cancer (TNBC) (estrogen-, progesterone- and HER2-receptor negative) still lacks targeted treatment options. TNBC is a type of breast cancer that often affects younger women, and generally has a worse prognosis than other types of breast cancer. Recently, the complex role of the immune system in cancer growth, elimination and metastasis has been the object of increased attention. There is hope that a more detailed understanding of the intricate roles of the constituents of the immune system, will hold potential both as prognostic or predictive markers of cancer progression, but also as treatment targets for a wide range of tumors, including TNBC. The aim of this review is to provide an overview of the cellular immune microenvironment in TNBC, and to highlight areas in which TNBC may differ from other types of breast cancer. MATERIAL AND METHODS: A search of PubMed was made using the terms 'triple negative breast cancer' and 'tumor infiltrating lymphocytes', 'CD8', 'CD4', 'B cells', 'natural killer cells', 'macrophages', myeloid derived suppressor cells', 'dendritic cells', 'immune check point inhibitor', 'CTLA-4' and 'PD-L1'. RESULTS: We find that whilst factors such as TILs and certain subgroups of TILs (e.g., CD8 + and regulator T-cells) have been extensively researched, none of these markers are currently applicable to routine clinical practice. Also, TNBC differs from other types of breast cancer with regards to cellular composition of the immune infiltrate and PD-L1 expression, and the prognostic significance of these. CONCLUSIONS: Immune-related factors have the potential as both prognostic and predictive biomarkers for new treatments targeting the immune system in breast cancer. However, multivariate analyses, taking other well-known factors into account, are required to determine the true value of these biomarkers. Also, differences between TNBC and other types of breast cancer may have implications for treatment and use of immune-related factors as biomarkers.

Cytokeratin on frozen sections of sentinel node may spare breast cancer patients secondary axillary surgery.

Background. The feasibility and accuracy of immunohistochemistry (IHC) on frozen sections, when assessing sentinel node (SN) status intraoperatively in breast cancer, is a matter of continuing discussion. In this study, we compared a center using IHC on frozen section with a center not using this method with focus on intraoperative diagnostic values. Material and Methods. Results from 336 patients from the centre using IHC intraoperatively were compared with 343 patients from the center not using IHC on frozen section. Final evaluation on paraffin sections with haematoxylin-eosin (HE) staining supplemented with cytokeratin staining was used as gold standard. Results. Significantly more SN with isolated tumor cells (ITCs) and micrometastases (MICs) were found intraoperatively when using IHC on frozen sections. There was no significant difference in the number of macrometastases (MACs) found intraoperatively. IHC increased the sensitivity, the negative predictive value, and the accuracy of the intraoperative evaluation of SN without decreasing the specificity and positive predictive value of SN evaluation. Conclusions. IHC on frozen section leads to the detection of more ITC and MIC intraoperatively. As axillary lymph node dissection (ALND) is performed routinely in some countries when ITC and MIC are found in the SN, IHC on frozen section provides valuable information that can lead to fewer secondary ALNDs.