Spatial distribution of B cells and lymphocyte clusters as a predictor of triple-negative breast cancer outcome.

While tumor infiltration by CD8+ T cells is now widely accepted to predict outcomes, the clinical significance of intratumoral B cells is less clear. We hypothesized that spatial distribution rather than density of B cells within tumors may provide prognostic significance. We developed statistical techniques (fractal dimension differences and a box-counting method 'occupancy') to analyze the spatial distribution of tumor-infiltrating lymphocytes (TILs) in human triple-negative breast cancer (TNBC). Our results indicate that B cells in good outcome tumors (no recurrence within 5 years) are spatially dispersed, while B cells in poor outcome tumors (recurrence within 3 years) are more confined. While most TILs are located within the stroma, increased numbers of spatially dispersed lymphocytes within cancer cell islands are associated with a good prognosis. B cells and T cells often form lymphocyte clusters (LCs) identified via density-based clustering. LCs consist either of T cells only or heterotypic mixtures of B and T cells. Pure B cell LCs were negligible in number. Compared to tertiary lymphoid structures (TLS), LCs have fewer lymphocytes at lower densities. Both types of LCs are more abundant and more spatially dispersed in good outcomes compared to poor outcome tumors. Heterotypic LCs in good outcome tumors are smaller and more numerous compared to poor outcome. Heterotypic LCs are also closer to cancer islands in a good outcome, with LC size decreasing as they get closer to cancer cell islands. These results illuminate the significance of the spatial distribution of B cells and LCs within tumors.

Physics approaches to the spatial distribution of immune cells in tumors.

The goal of immunotherapy is to mobilize the immune system to kill cancer cells. Immunotherapy is more effective and, in general, the prognosis is better, when more immune cells infiltrate the tumor. We explore the question of whether the spatial distribution rather than just the density of immune cells in the tumor is important in forecasting whether cancer recurs. After reviewing previous work on this issue, we introduce a novel application of maximum entropy to quantify the spatial distribution of discrete point-like objects. We apply our approach to B and T cells in images of tumor tissue taken from triple negative breast cancer patients. We find that the immune cells are more spatially dispersed in good clinical outcome (no recurrence of cancer within at least 5 years of diagnosis) compared to poor clinical outcome (recurrence within 3 years of diagnosis). Our results highlight the importance of spatial distribution of immune cells within tumors with regard to clinical outcome, and raise new questions on their role in cancer recurrence.

Multi-panel immunofluorescence analysis of tumor infiltrating lymphocytes in triple negative breast cancer: Evolution of tumor immune profiles and patient prognosis.

The evolutionary changes in immune profiles of triple negative breast cancer (TNBC) are not well understood, although it is known that immune checkpoint inhibitors have diminished activity in heavily pre-treated TNBC patients. This study was designed to characterize immune profile changes of longitudinal tumor specimens by studying immune subsets of tumor infiltrating lymphocytes (TILs) in paired primary and metastatic TNBC in a cohort of "poor outcome" (relapsed within 5 years) patients. Immune profiles of TNBCs in a cohort of "good outcome" (no relapse within 5 years) patients were also analyzed. Immune subsets were characterized for CD4, CD8, FOXP3, CD20, CD33, and PD1 using immuno-fluorescence staining in stroma, tumor, and combined stroma and tumor tissue. TIL subsets in "good outcome" versus "poor outcome" patients were also analyzed. Compared with primary, metastatic TNBCs had significantly lower TILs by hematoxylin and eosin (H&E) staining. Stromal TILs (sTILs), but not tumoral TILs (tTILs) had significantly reduced cytotoxic CD8+ T cells (CTLs), PD1+ CTLs, and total PD1+ TILs in metastatic compared with matched primary TNBCs. Higher PD1+ CTLs, PD1+CD4+ helper T cells (PD1+TCONV) and all PD1+ T cells in sTILs, tTILs and total stromal and tumor TILS (s+tTIL) were all associated with better prognosis. In summary, TIL subsets decrease significantly in metastatic TNBCs compared with matched primary. Higher PD1+ TILs are associated with better prognosis in early stage TNBCs. This finding supports the application of immune checkpoint inhibitors early in the treatment of TNBCs.

Resident memory CD8+ T cells within cancer islands mediate survival in breast cancer patients.

CD8+ tumor-infiltrating lymphocytes (TILs) correlate with relapse-free survival (RFS) in most cancer types, including breast cancer. However, subset composition, functional status, and spatial location of CD8+ TILs in relation to RFS in human breast tumors remain unclear. Spatial tissue analysis via quantitative immunofluorescence showed that infiltration of CD8+ T cells into cancer islands was more significantly associated with RFS than CD8+ T cell infiltration into either tumor stroma or total tumor. Localization into cancer islands within tumors is mediated by expression of the integrin CD103, which is a marker for tissue-resident memory T cells (TRMs). Analysis of fresh tumor samples revealed that CD8+ TRMs are functionally similar to other CD8+ TILs, suggesting that the basis of their protective effect is their spatial distribution rather than functional differences. Indeed, CD103+ TRMs, as compared with CD103-CD8+ TILs, are enriched within cancer islands, and CD8+ TRM proximity to cancer cells drives the association of CD8+ TIL densities with RFS. Together, these findings reveal the importance of cancer island-localized CD8+ TRMs in surveillance of the breast tumor microenvironment and as a critical determinant of RFS in patients with breast cancer.

Connecting blood and intratumoral Treg cell activity in predicting future relapse in breast cancer.

Regulatory T (Treg) cells play a major role in the development of an immunosuppressive tumor microenvironment. The origin of intratumoral Treg cells and their relationship with peripheral blood Treg cells remain unclear. Treg cells consist of at least three functionally distinct subpopulations. Here we show that peripheral blood CD45RA-FOXP3hi Treg cells (Treg II cells) are phenotypically closest to intratumoral Treg cells, including in their expression of CCR8. Analyses of T cell antigen receptor repertoires further support the hypothesis that intratumoral Treg cells may originate primarily from peripheral blood Treg II cells. Moreover, the signaling responsiveness of peripheral blood Treg II cells to immunosuppressive, T helper type 1 (TH1) and T helper type 2 (TH2) cytokines reflects intratumoral immunosuppressive potential, and predicts future relapse in two independent cohorts of patients with breast cancer. Together, our findings give important insights into the relationship between peripheral blood Treg cells and intratumoral Treg cells, and highlight cytokine signaling responsiveness as a key determinant of intratumoral immunosuppressive potential and clinical outcome.