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PD-L1 expression is a predictive biomarker for anti-PD-L1 immunotherapy in triple negative breast cancer (TNBC). In the neoadjuvant setting, immunohistochemical (IHC) evaluation of PD-L1 expression can only be performed on small tissue biopsies. In our study we investigated heterogeneity of PD-L1 expression in TNBC, and how reliably PD-L1 expression in small tissue samples reflects PD-L1 expression in larger tumor sections in TNBC. Tissue microarrays (TMAs) were constructed from surgical specimens of 110 patients with TNBC. TMAs contained 4 cores (1 mm in diameter) per patient. To evaluate PD-L1 expression, TMAs were stained with PD-L1 IHC 22C3 PharmDx. Single-core PD-L1 expression was compared to overall PD-L1 expression of each patient’s tumor, to ascertain how often small samples of tumor tissue show the same PD-L1 expression as larger tumor samples. Our study found substantial heterogeneity of PD-L1 expression between different TMA cores from the same patient. Heterogeneity was greater in immune cells (ICs) than in tumor cells, in large part due to the uneven distribution of ICs in the tumor. For IC PD-L1 expression, we found that sensitivity can be as low as 0.81 for detecting PD-L1 expression at the 1% threshold most commonly used in breast cancer. Negative predictive value for ICs was 0.7. There is substantial heterogeneity of PD-L1 expression between small tissue samples from the same TNBC tumor, especially for IC expression. This poses challenges for evaluation of PD-L1 expression in the neoadjuvant setting. Negative biopsies should prompt further investigation, and multiple biopsies might be necessary.

Secreted phospholipase A2 group IIa (sPLA2-IIa) has been shown to promote tumor genesis and cell proliferation. The properties of this group of enzymes are utilized in liposomal drug delivery of chemotherapy. sPLA2-IIa is also under investigation as a possible treatment target in itself, and as a prognostic marker. The expression of sPLA2-IIa in breast cancer has not been examined extensively, and never using immunohistochemistry. We sought to investigate the expression of sPLA2-IIa in a cohort of advanced breast cancer patients with correlation to known clinicopathologic risk factors and survival. Material from 525 breast cancer patients (426 primary tumors and 99 metastases or local recurrences) was examined for sPLA2-IIa expression using immunohistochemistry. Out of these, 262 showed expression of sPLA2-IIa. We found that there was no correlation to clinicopathologic characteristics, and no impact of sPLA2-IIa expression on prognosis. However, we found that a large proportion of patients in our study had high levels of sPLA2-IIa expression, and that sPLA2-IIa was equally expressed in primary tumors and metastases. These findings may be significant in the future development of liposomal drug delivery or targeted sPLA2-IIa treatment.

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.

Stromal tumour infiltrating lymphocytes (sTILs) are a strong prognostic marker in triple negative breast cancer (TNBC). Consistency scoring sTILs is good and was excellent when an internet-based scoring aid developed by the TIL-WG was used to score cases in a reproducibility study. This study aimed to evaluate the reproducibility of sTILs assessment using this scoring aid in cases from routine practice and to explore the potential of the tool to overcome variability in scoring. Twenty-three breast pathologists scored sTILs in digitized slides of 49 TNBC biopsies using the scoring aid. Subsequently, fields of view (FOV) from each case were selected by one pathologist and scored by the group using the tool. Inter-observer agreement was good for absolute sTILs (ICC 0.634, 95% CI 0.539–0.735, p &lt, 0.001) but was poor to fair using binary cutpoints. sTILs heterogeneity was the main contributor to disagreement. When pathologists scored the same FOV from each case, inter-observer agreement was excellent for absolute sTILs (ICC 0.798, 95% CI 0.727–0.864, p &lt, 0.001) and good for the 20% (ICC 0.657, 95% CI 0.561–0.756, p &lt, 0.001) and 40% (ICC 0.644, 95% CI 0.546–0.745, p &lt, 0.001) cutpoints. However, there was a wide range of scores for many cases. Reproducibility scoring sTILs is good when the scoring aid is used. Heterogeneity is the main contributor to variance and will need to be overcome for analytic validity to be achieved.

Simple Summary Around 15% of breast cancer patients are diagnosed as triple-negative (TNBC), which have significantly lower 5-year survival rates (77%) than other types of breast cancer (93%). Our study aimed at developing an image analysis-based biomarker to assess how the immune system interacts with the tumor and investigate the potential added value of stromal tumor-infiltrating lymphocytes (sTIL) for the prognosis of overall survival compared to the manual approach. In a large retrospective cohort of 257 patients, we found that our fully automated hematoxylin and eosin (H&E) image analysis pipeline can quantify sTIL density showing both high concordance with manual scoring and association with the prognosis of patients with TNBC. It also overcomes natural limitations of manual assessment that hinder clinical adoption of the immune biomarker. We conclude that sTIL scoring by automated image analysis has prognostic potential comparable to manual scoring and should be further investigated for future use in a clinical setting. Abstract 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.

Preclinical studies suggest that some effects of conventional chemotherapy, and in particular, gemcitabine, are mediated through enhanced antitumor immune responses. The objective of this study was to use material from a randomized clinical trial to evaluate whether patients with preexisting immune infiltrates responded better to treatment with gemcitabine + docetaxel (GD) compared to docetaxel alone. Formalin fixed, paraffin-embedded breast cancer tissues from SBG0102 phase 3 trial patients randomly assigned to treatment with GD or docetaxel were used. Immunohistochemical staining for CD8, FOXP3, LAG3, PD-1, PD-L1 and CD163 was performed. Tumor infiltrating lymphocytes (TILs) and tumor associated macrophages were evaluated. Prespecified statistical analyses were performed in a formal prospective-retrospective design. Time to progression was primary endpoint and overall survival secondary endpoint. Correlations between biomarker status and endpoints were evaluated using the Kaplan–Meier method and Cox proportional hazards models. Biomarker data was obtained for 237 patients. There was no difference in treatment effect according to biomarker status for the whole cohort. In planned subgroup analysis by PAM50 subtype, in non-luminal (basal-like and HER2E) breast cancers FOXP3 was a significant predictor of treatment effect with GD compared to docetaxel, with a HR of 0.22 (0.09–0.52) for tumors with low FOXP3 compared to HR 0.92 (0.47–1.80) for high FOXP3 TILs (Pinteraction = 0.01). Immune biomarkers were not predictive of added benefit of gemcitabine in a cohort of mixed breast cancer subtypes. However, in non-luminal breast cancers, patients with low FOXP3+ TILs may have significant benefit from added gemcitabine.

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., info:eu-repo/semantics/published

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.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'.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.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.

Introduction: There is extensive research into immune-oncology (IO) treatment targets for triple negative breast cancer (TNBC). However, little knowledge exits on variations in expression levels of IO targets between paired primary tumors (PT) and metastases (MT) in TNBC. Objectives: To compare expression levels of IO drug targets in paired PT and distant MT from TNBC patients. PD-L1 protein expression was examined immunohistochemically, as well as differences in levels of tumor infiltrating lymphocytes (TILs). Material and methods:552 patients were primarily operated for TNBC at 2 university hospitals in Denmark between 2004 and 2014. Of these, samples from 22 patients were available from both PT (22 samples) and distant MT (24 samples, with 2 chronologically separate metastases for two patients). Localizations of metastases included 9 brain, 1 skin and 1 lymph node (both not regional), 2 bone, 5 liver, 2 soft tissue, 1 lung, and 1 adrenal gland. Gene expression analyses were performed on the Ion Torrent S5XL system, using the Oncomine Immune Response Research Assay. PD-L1 expression was evaluated immunohistochemically using the 28-8 clone (pharmdX). Cut-off levels for PD-L1 expression were 1% positive tumor cells. TILs were evaluated in accordance with guidelines from the International Immuno-Oncology Biomarker Working Group. Results: Gene expression levels of 29 established IO drug targets were tested on 18 of the 22 TNBC patients with matched PTs and MTs (Table 1). 4 genes showed significant downregulation in MTs compared to PTs: IDO1 (p=0.01), CXCR4 (p=0.01), KIR2DL1 (p=0.05) and TNFRSF9 (p=0.03). However, other important IO targets such as CTLA4, STAT3, TIGIT, and TNSFRSF18 (GITR) were not significantly different between PT and MT. CD276 (B7-H3), PD-1, IL2 and LAG3 showed a tendency towards significance (p= 0.08, 0.07, 0.05, 0.08), with LAG3 being the only gene upregulated in MT compared to PT. TILs were evaluated for all PTs and MTs. TILs in PTs ranged from 1 to 40% (median=13%). TILs in MTs ranged from 1 to 60% (median=5%). There was no significant overall change in TILs from PT to MT (p=0.2). However, 9 patients had a difference in TILs between PT and MT of > ±10% (-20 - +30). PD-L1 immunohistochemical evaluation was possible for both PT and MT in 20 cases. There was no significant overall change in PD-L1 expression (p=0.39). However, 4 patients with positive PTs, had negative MTs, and 4 patients with PD-L1 negative PTs, had positive MTs. Discussion: This is, to our knowledge, one of the largest studies of gene expression of IO drug targets in TNBC matched PTs and MTs. We show significant differences in gene expression levels for IDO1, CXCR4, KIR2DL1 and TNFRSF9, pointing to a generally less immune-active environment in MTs. This was also corroborated by lower levels of TILs in MTs, although these findings were not significant. For PD-L1 protein expression, we found that PD-L1 expression can change between PT and MT. In the clinical setting, treatment decisions are often based upon expression levels in a single biopsy or surgical specimen. Intra-patient heterogeneity should be taken into account in the diagnostic setting, and when making treatment decisions. Table 1IO drug targetRatio (Primary/metastasis)P-valueIDO19.65 (1.68-54.95)0.01CXCR41.83 (1.17-2.87)0.01KIR2DL12.17 (1.01-4.69)0.05TNFRSF97.11 (1.21-41.93)0.03CD276 (B7H3)1.51 (0.94-2.39)0.08PDCD1 (PD1)3.32 (0.88-12.64)0.07IL22.87 (0.99-8.28)0.05LAG30.28 (0.07-1.16)0.08CTLA41.96 (0.45-8.51)0.35STAT31.21 (0.92-1.60)0.16TIGIT1.87 (0.25-14.03)0.53TNFRSF18 (GITR)0.38 (0.06-2.20)0.26 Citation Format: Elisabeth S Stovgaard, Tim S Poulsen, Eva Balslev, Dorte L Nielsen, Anne Roslind, Maj-Lis Talman, Iben Kümler, Ib J Christensen, Estrid Høgdall. Triple negative breast cancer: Expression of immuno-oncology treatment targets and prevalence of tumor infiltrating lymphocytes in paired primary tumors and metastases [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-13.

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

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.