The presence of tumor‐infiltrating lymphocytes (TILs) is associated with favorable long‐term outcome in breast cancer. However, little is known about changes in TILs during metastatic progression. To confirm our hypothesis that malignant tumors escape from the host immune system during metastasis, we evaluated the percentage of TILs in paired samples of primary and metastatic breast tumors. We retrospectively identified 25 patients with human epidermal growth factor receptor‐2 (HER2+, n = 14) and triple negative (TN, n = 11) early breast cancer diagnosed between 1990 and 2009 at Tokai University Hospital (Isehara, Japan) and who subsequently experienced regional or distant recurrence confirmed by tumor biopsy/resection. Hematoxylin–eosin‐stained slides of these paired samples were evaluated for stromal TILs. Immunohistochemical staining was carried out using primary antibodies against CD4, CD8, Foxp3, programmed cell death ligand 1 (PD‐L1), PD‐L2, and HLA class I for characterizing the TILs and breast tumors. The percentage of TILs in the primary tumors was significantly higher (average 34.6%) than that in metastatic tumors (average 15.7%) (paired t‐test, P = 0.004) and that of CD8+ and CD4+ T cells significantly decreased from primary to metastatic tumors (paired t‐test, P = 0.008 and P = 0.026, respectively). The PD‐L1, PD‐L2, and HLA class I antibody expression changed from positive to negative and vice versa from the primary to the metastatic tumors. Tumors at first metastatic recurrence in HER2+ and TN breast cancers have a lower percentage of TILs and CD8+ and CD4+ T cells compared to primary tumors, which indicates that immune escape plays a role in tumor progression.

BackgroundImmune checkpoint inhibitors are reported to be effective in patients with brain metastases. However, detailed characteristics of the brain metastasis immune microenvironment remain unexplored.ResultsThe median tumor-infiltrating lymphocyte (TIL) category in brain metastases was 5% (1–70%). In 46 pair-matched samples, the percentages of TILs were significantly higher in primary breast tumors than in brain metastases (paired t-test, P < 0.01). The numbers of CD4/CD8/Foxp3-positive cells were significantly higher in primary breast tumors than in brain metastases (paired t-test, P < 0.05 for all antibodies). In patients with triple-negative breast cancer specifically, low TIL numbers were associated with significantly shorter overall survival compared to high TIL numbers (log-rank test, P = 0.04).Materials and MethodsWe retrospectively identified 107 patients with breast cancer and brain metastases who had undergone surgery between 2001 and 2012 at 8 institutions, and collected 191 samples including brain metastases alone and primary tumors with pair-matched brain metastasis samples. Hematoxylin and eosin-stained slides were evaluated for TILs and categorized according to the extent of staining. Immunohistochemistry for CD4, CD8, Foxp3, PD-L1, PD-L2, and HLA class I was also performed.ConclusionsThere are significantly fewer TILs in brain metastases than in primary breast tumors.

Pheochromocytomas and paragangliomas arise from the adrenal glands and extraadrenal paraganglia, respectively. Malignant behavior of these tumors is uncommon and is, in part, dependent on their sites of origin, such as extraadrenal location. Morphologic criteria for malignancy of pheochromocytoma and paragangliomas have not been clearly defined. In this study, to clarify the histologic features that distinguish the benign from malignant pheochromocytomas and paragangliomas, we examined metastatic and nonmetastatic tumors using immunohistochemical techniques. A total of eight cases, five pheochromocytomas from the adrenal glands (four benign and one malignant tumor) and three paragangliomas with invasion or metastasis, were studied. The markers used in this study were chromogranin A, synaptophysin, NCAM (CD56), SNAP25, neuron-specific enolase, S-100 protein, and MIB-1. Our results suggest that MIB-1 immunostaining is a useful adjunct marker to predict malignant behavior in these tumors.

Tumor-infiltrating lymphocytes (TILs) and programmed cell death 1 ligand 1 (PD-L1) are established prognostic and predictive biomarkers for certain breast cancer subsets. However, their association with the immune response complexity is not fully understood. Therefore, we analyzed the association between the immune cell fractions in breast cancer tissues and histologically assessed TIL (hTIL) and PD-L1 (hPD-L1). Forty-five tumor and eighteen blood samples were collected from patients with breast cancer. Total leukocyte counts, frequency of 11 immune cell populations, and PD-L1 expression in each cell fraction were evaluated by flow cytometry. TILs and PD-L1 were assessed by hematoxylin and eosin staining and immunohistochemistry, respectively. A higher hTIL score showed association with increased leukocyte infiltration, higher CD4+ and CD8+ T cell proportions, and lower natural killer and natural killer T cell proportions. PD-L1 was highly expressed in nonclassical monocytes, monocyte/macrophages, myeloid-derived suppressor cells, myeloid dendritic cells, dendritic cells, and other lineages in tumors. hPD-L1 positivity reflected PD-L1 expression accurately in these fractions, as well as increased leukocyte infiltration in tumors. These results indicate that hTILs reflect differences in the immune responses in the tumor microenvironment, and certain immune cell fractions are favorably expressed in the PD-L1 pathway in breast cancer microenvironments.

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