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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.

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 + T CONV ) 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.

While the density of tumor-infiltrating lymphocytes (TILs) is now well known to correlate with clinical outcome, the clinical significance of spatial distribution of TILs is not well characterized. We have developed novel statistical techniques (including fractal dimension differences, a hotspot analysis, a box counting method that we call 'occupancy' and a way to normalize cell density that we call 'thinning') to analyze the spatial distribution (at different length scales) of various types of TILs in triple negative breast tumors. Consistent with prior reports, the density of CD20 + B cells within tumors is not correlated with clinical outcome. However, we found that their spatial distribution differs significantly between good clinical outcome (no recurrence within at least 5 years of diagnosis) and poor clinical outcome (recurrence with 3 years of diagnosis). Furthermore, CD20 + B cells are more spatially dispersed in good outcome tumors and are more likely to infiltrate into cancer cell islands. Lastly, we found significant correlation between the spatial distributions of CD20 + B cells and CD8 + (cytotoxic) T cells (as well as CD3 + T cells), regardless of outcome. These results highlight the significance of the spatial distribution of TILs, especially B cells, within tumors. Significance StatementImmune cells can fight cancer. For example, a patient has a good prognosis when a high density of killer T cells, a type of immune cell that can kill cancer cells, infiltrates into a tumor. However, there is no clear association between prognosis and the density of B cells, another type of immune cell, in a tumor. We developed several statistical techniques to go beyond cell density and look at the spatial distribution, i.e., the pattern or arrangement of immune cells, in tumors that have been removed from patients with triple negative breast cancer. We find that B cells and killer T cells tend to be more spread out in the tumors of patients whose cancer did not recur.

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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.

Aim:To analyze the antioxidants and anti-inflammatory potential of ethanolic crude extract of Cassia auriculata flowers. Study Design: 2 kg of fresh flowers were soaked with 90% ethanol at room temperature (25ºC-30ºC). After 72 hrs the ethanolic extract was filtered. This extract was distilled and finally the crude was obtained. This ethanolic crude to get required concentrations and were used for screening antioxidants and anti-inflammatory activities.