CD8 T cell is an essential component of tumor-infiltrating lymphocytes (TIL) and tumor immune microenvironment (TIME). Using the xCell CD8 T cell score of whole tumor gene expression data, we estimated these cells in total of 3837 breast cancer patients from TCGA, METABRIC and various GEO cohorts. The CD8 score correlated strongly with expression of CD8 genes. The score was highest for triple-negative breast cancer (TNBC), and a high score was associated with high tumor immune cytolytic activity and better survival in TNBC but not other breast cancer subtypes. In TNBC, tumors with a high CD8 score had enriched expression of interferon (IFN)-α and IFN-γ response and allograft rejection gene sets, and greater infiltration of anti-cancerous immune cells. The score strongly correlated with CD4 memory T cells in TNBC, and tumors with both a high CD8 score and high CD4 memory T cell abundance had significantly better survival. Finally, a high CD8 score was significantly associated with high expression of multiple immune checkpoint molecules. In conclusion, a high CD8 T cell score is associated with better survival in TNBC, particularly when tumor CD4 memory T cells were elevated. Our findings also suggest a possible use of the score as a predictive biomarker for response to immune checkpoint therapy.

The vast majority of breast cancer death is a result of metastasis. Thus, accurate identification of patients who are likely to have metastasis is expected to improve survival. The G2M checkpoint plays a critical role in cell cycle. We hypothesized that breast cancer tumors with high activity of G2M pathway genes are more aggressive and likely to metastasize. To test this, we used the single-sample gene set variation analysis method to calculate the score for the Hallmark G2M checkpoint pathway using gene expression data of a total of 4626 samples from 12 human breast cancer cohorts. As expected, a high G2M pathway score correlated with enriched tumor expression of other cell proliferation-related gene sets. The score was significantly associated with clinical aggressive features of tumors and patient survival in estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Interestingly, a high G2M score of metastasis tumors was also significantly associated with worse survival. In primary as well as metastasis tumors with high scores, the infiltration of both pro- and anti-cancerous immune cells increased. Tumor G2M score was also associated with treatment response to systemic chemotherapy in ER-positive/HER2-negative cancer, and was predictive of response to cyclin-dependent kinase inhibition therapy.

Tissue biopsy is the gold standard for diagnosis and morphological and immunohistochemical analyses to characterize cancer. However, tissue biopsy usually requires an invasive procedure, and it can be challenging depending on the condition of the patient and the location of the tumor. Even liquid biopsy analysis of body fluids such as blood, saliva, gastric juice, sweat, tears and cerebrospinal fluid may require invasive procedures to obtain samples. Liquid biopsy can be applied to circulating tumor cells (CTCs) or nucleic acids (NAs) in blood. Recently, urine has gained popularity due to its less invasive sampling, ability to easily repeat samples, and ability to follow tumor evolution in real-time, making it a powerful tool for diagnosis and treatment monitoring in cancer patients. With the development and advancements in extraction methods of urinary substances, urinary NAs have been found to be closely related to carcinogenesis, metastasis, and therapeutic response, not only in urological cancers but also in non-urological cancers. This review mainly highlights the components of urine liquid biopsy and their utility and limitations in oncology, especially in non-urological cancers.

E2F transcription factors play critical roles in the cell cycle. Therefore, their activity is expected to reflect tumor aggressiveness and responsiveness to therapy. We scored 3905 tumors of nine breast cancer cohorts for this activity based on their gene expression for the Hallmark E2F targets gene set. As expected, tumors with a high score had an increased expression of cell proliferation-related genes. A high score was significantly associated with shorter patient survival, greater MKI67 expression, histological grade, stage, and genomic aberrations. Furthermore, metastatic tumors had higher E2F scores than the primary tumors from which they arose. Although tumors with a high score had greater infiltration by both pro- and anti-cancerous immune cells, they had an increased expression of immune checkpoint genes. Estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative cancer with a high E2F score achieved a significantly higher pathological complete response (pCR) rate to neoadjuvant chemotherapy. The E2F score was significantly associated with the expression of cyclin-dependent kinase (CDK)-related genes and strongly correlated with sensitivity to CDK inhibition in cell lines. In conclusion, the E2F score is a marker of breast cancer aggressiveness and predicts the responsiveness of ER-positive/HER2-negative patients to neoadjuvant chemotherapy and possibly to CDK and immune checkpoint inhibitors.

Regulatory CD4⁺ T cell (Treg), a subset of tumor-infiltrating lymphocytes (TILs), are known to suppress anticancer immunity but its clinical relevance in human breast cancer remains unclear. In this study, we estimated the relative abundance of Tregs in breast cancer of multiple patient cohorts by using the xCell algorithm on bulk tumor gene expression data. In total, 5177 breast cancer patients from five independent cohorts (TCGA-BRCA, GSE96058, GSE25066, GSE20194, and GSE110590) were analyzed. Treg abundance was not associated with cancer aggressiveness, patient survival, or immune activity markers, but it was lower in metastatic tumors when compared to matched primary tumors. Treg was associated with a high mutation rate of TP53 genes and copy number mutations as well as with increased tumor infiltration of M2 macrophages and decreased infiltration of T helper type 1 (Th1) cells. Pathological complete response (pCR) after neoadjuvant chemotherapy (NAC) was significantly associated with low Treg abundance in triple negative breast cancer (TNBC) but not in ER-positive/Her2-negative subtype. High Treg abundance was significantly associated with high tumor expression of multiple immune checkpoint inhibitor genes. In conclusion, Treg abundance may have potential as a predictive biomarker of pCR after NAC in TNBC.

Dendritic cells (DC) represent a major antigen-presenting cell type in the tumor immune microenvironment (TIME) and play an essential role in cancer immunity. Conventional DC (cDC) and plasmacytoid DC (pDC) were defined by the xCell algorithm and a total of 2968 breast cancer patients (TCGA and METABRIC) were analyzed. We found that triple-negative breast cancer (TNBC) had a high fraction of cDC and pDC compared to the other subtypes. In contrast to cDC, high pDC in TNBC was significantly associated with better disease-specific and disease-free survival consistently in both cohorts. High cDC TNBC tumors enriched not only inflammation and immune-related, but also metastasis-related gene sets in Gene Set Enrichment Analysis, whereas high pDC TNBC enriched inflammation and immune -related gene sets including IFN-γ signaling more strongly than cDC. pDC TNBC correlated with CD8⁺, CD4⁺ memory, IFN-γ score, and cytolytic activity stronger than cDC TNBC. High pDC TNBC were associated with a high fraction of anti-cancer immune cells and high expression of all the immune check point molecules examined. In conclusion, pDC levels correlated with the infiltration of immune cells and patient survival in TNBC more strongly than cDC; this is the first study suggesting the clinical relevance of pDC infiltration in TNBC.