Morphological Evaluation of Tumor-Infiltrating Lymphocytes (TILs) to Investigate Invasive Breast Cancer Immunogenicity, Reveal Lymphocytic Networks and Help Relapse Prediction: A Retrospective Study.

Tumor-infiltrating lymphocytes (TILs) in breast cancer are a key representative of the tumor immune microenvironment and have been shown to provide prognostic and predictive biomarkers. The extent of lymphocytic infiltration in tumor tissues can be assessed by evaluating hematoxylin and eosin (H&E)-stained tumor sections. We investigated tissue microarrays of 31 invasive breast cancer patients, looking at quantity and topological distribution of CD3+, CD8+, CD20+, Ki67+, FoxP3+ TILs and CD3+/FoxP3+, CD8+/FoxP3+ cell ratios. We separately evaluated TILs at the invasive edge and at the center of the tumor, to find any clinical implications of tumor heterogeneity. No statistically significant difference was found in quantity and distribution of both TIL subsets and TIL ratios, by comparing patients who suffered from a local or distant recurrence of the tumor (relapse group: 13 patients) with patients not showing cancer relapse (non-relapse group: 18 patients). In the whole sample, we observed three main statistically significant positive correlations: (1) between CD3+ and CD8+ T-cells; (2) between FoxP3+ and Ki67+ lymphocyte infiltration; (3) between CD3+/FoxP3+ cell ratio (C3FR) and CD8+/FoxP3+ cell ratio (C8FR). Tumor heterogeneity and stronger positive TIL associations were found in the non-relapse group, where both CD3-CD8 and FoxP3-Ki67 inter-correlations were found to be significant at the center of the tumor, while the correlation between C3FR and C8FR was significant at the invasive edge. No correlations between TIL subsets were detected in the relapse group. Our findings suggest the existence of stronger inter-subtype lymphocytic networks in invasive breast cancer not showing recurrence. Further evaluations of clinical and topological correlations between and within TIL subsets are needed, in addition to the assessment of TIL quantification and distribution, in order to follow up on whether morphological evaluation of TILs might reveal the underlying lymphocytic functional connectivity and help relapse prediction.

In situ localization of tumor cells associated with the epithelial-mesenchymal transition marker Snail and the prognostic impact of lymphocytes in the tumor microenvironment in invasive ductal breast cancer.

PURPOSE: Tumor surgery is aimed at complete resection of the lesion while ensuring a sufficient tumor-specific safety distance. Nevertheless, in many cases the most peripheral part - the invasion front - remains in situ. Tumor cells at the tumor margin have been reported to lose their epithelial properties and acquire features of mesenchymal cells. The process of epithelial-to-mesenchymal transition (EMT) is believed to be of prime importance for tissue and vessel invasion. Furthermore, the detection of tumor-infiltrating lymphocytes in the microenvironment of breast cancer might serve as a reliable prognostic marker. METHODS: We investigated tissue microarrays of 352 breast cancer patients with regard to the presence and distribution of the EMT factor Snail, and the presence of FoxP3, CD3 and CD8 in the immune microenvironment. RESULTS: The expression of the transcription factor Snail is strongly associated with longer disease-free and overall survival. The presence of CD3, CD8 or FoxP3 is associated with a better outcome, although statistically significant results were noted only for FoxP3. The prognostic significance of FoxP3 and Snail were also proven in multivariate analysis. CONCLUSIONS: Based on previous studies concerning the intratumoral heterogeneity of EMT, our results suggest that Snail and FoxP3 are possible prognostic markers for breast cancer. The diverse presence of lymphocytes in the tumor microenvironment (CD3 and CD8) was confirmed. Although the importance of these markers is known, their specific role in tumor invasion and metastasis as well as their hierarchical organization in these tumors remain unclear.

Transcription factors associated with epithelial-mesenchymal transition and cancer stem cells in the tumor centre and margin of invasive breast cancer.

Although tumor surgery aims for a complete resection respecting tumor-specific safety distance, in many cases the most peripheral part, the invasion front, remains in situ. Tumor cells at the tumor margin lose epithelial properties and acquire features of mesenchymal cells. The process of epithelial-to-mesenchymal transition (EMT) has been suggested to be of prime importance for tissue and vessel invasion. Recently, features of EMT were shown to be linked to cells with tumor-founding capability, so- called cancer stem cells (CSC). In this study we show that transcription factors associated with EMT markers Snail, Slug, Twist and Zeb1 are differentially expressed between normal breast epithelium, ductal carcinoma in situ and invasive breast cancer. Both invasive and in situ carcinoma expressed less Slug and Twist and more Zeb1 compared to normal epithelium. Using fluorescence multi-staining the number of potential CSC among invasive cancer cells varied dramatically depending on the staining combination used (18.5% for CD44(+)/CD24(-) and 2.4% for CD49f(+)/CD24(+)). Interestingly, neither transcription factors associated with EMT nor potential CSC counts varied between tumor centre and invasion front. No association of these features with clinical outcome was detected. Our results suggest that reliable in situ markers for EMT are missing for invasive breast cancer. Alternatively, the process of EMT might be activated in tumor cells at the margin as well as the centre. Furthermore, our data show that the bio-markers of CSC detect very variable cell populations within breast cancer, challenging the assumption of a hierarchical organization of CSC in these tumors.