The T-cell inhibitory molecule PD-L1 (B7-H1, CD274) is expressed on tumor cells of a subset of breast cancer patients. However, the mechanism that regulates PD-L1 expression in this group of patients is still not well-identified. We have used loss and gain of function gene manipulation approach, multi-parametric flow cytometry, large scale gene expression dataset analysis and immunohistochemistry of breast cancer tissue sections. Induction of epithelial to mesenchymal transition (EMT) in human mammary epithelial cells upregulated PD-L1 expression, which was dependent mainly on the activation of the PI3K/AKT pathway. Interestingly, gene expression signatures available from large cohort of breast tumors showed a significant correlation between EMT score and the PD-L1 mRNA level (p < 0.001). Strikingly, very strong association (p < 0.0001) was found between PD-L1 expression and claudin-low subset of breast cancer, which is known to have high EMT score. On the protein level, significant correlation was found between PD-L1 expression and standard markers of EMT (p = 0.005) in 67 breast cancer patients. Importantly, specific downregulation of PD-L1 in claudin-low breast cancer cells showed signs of EMT reversal as manifested by CD44 and Vimentin downregulation and CD24 upregulation. We have demonstrated a bidirectional effect between EMT status and PD-L1 expression especially in claudin-low subtype of breast cancer cells. Our findings highlights the potential dual benefit of anti-PD-L1 particularly in this subset of breast cancer patients that will likely benefit more from anti-PD-L1 targeted therapy as well as in monitoring biological changes upon treatment.

Introduction: B7-H1 (PD-L1, CD274) is a T cell inhibitory molecule expressed in many types of cancer, leading to immune escape of tumor cells. Indeed, in previous reports we have shown an association of B7-H1 expression with high-risk breast cancer patients. Methods: In the current study, we used immunohistochemistry, immunofluorescence and Western blot techniques to investigate the effect of neoadjuvant chemotherapy on the expression of B7-H1 in breast cancer cells. Results: Among tested chemotherapeutic agents, doxorubicin was the most effective in downregulating cell surface expression of B7-H1 in vitro. These results were validated in vivo in a xenograft mouse model, as well as in murine heart tissue known to constitutively express B7-H1. The doxorubicin-dependent cell surface downregulation of B7-H1 was accompanied by an upregulation of B7-H1 in the nucleus. This re-distribution of B7-H1 was concurrent with a similar translocation of phosphorylated AKT to the nucleus. Inhibition of the PI3K/AKT pathway abrogated the doxorubicin-mediated nuclear up-regulation of B7-H1, suggesting an involvement of PI3K/AKT pathway in the nuclear up-regulation of B7-H1. Interestingly, siRNA knock down of B7-H1 lead to an increase in spontaneous apoptosis, as well as doxorubicin-induced apoptosis, which indicates an anti-apoptotic role for B7-H1 in breast cancer cells. The novel discovery of B7-H1 expression in the nuclei of breast cancer cells suggests that B7-H1 has functions other than inhibition of T cells. Conclusions: Our findings explain the previously reported immunomodulatory effect of anthracyclines on cancer cells, and provide a link between immunoresistance and chemoresistance. Finally these results suggest the use of dual combinatorial agents to inhibit B7-H1 beside chemotherapy, in breast cancer patients.

Background: Recent studies have demonstrated a direct involvement of B7-H1, PD-1 and FOXP3 molecules in the immune escape of cancer. B7-H1 is an inhibitory molecule that binds to PD-1 on T lymphocytes, while FOXP3 is a marker for regulatory T cells (T_regs). We have previously demonstrated the association of B7-H1-expressing T infiltrating lymphocytes (TIL) with high-risk breast cancer patients while other studies reported the involvement of FOXP3+ T_regsas a bad prognostic factor in breast tumors. Although the co-existence between the two types of cells has been demonstratedin vitroand animal models, their relative infiltration and correlation with the clinicopathological parameters of cancer patients have not been well studied. Therefore, we investigated TIL-expressing the B7-H1, PD-1, and FOXP3 molecules, in the microenvironment of human breast tumors and their possible association with the progression of the disease.Methods: Using immunohistochemistry, tumor sections from 62 breast cancer patients were co-stained for B7-H1, PD-1 and FOXP3 molecules and their expression was statistically correlated with factors known to be involved in the progression of the disease.Results: A co-existence of B7-H1⁺T lymphocytes and FOXP3⁺T_regswas evidenced by the highly significant correlation of these molecules (P< .0001) and their expression by different T lymphocyte subsets was clearly demonstrated. Interestingly, concomitant presence of FOXP3⁺T_regs, B7-H1⁺and PD-1⁺TIL synergistically correlated with high histological grade (III) (P< .001), estrogen receptor negative status (P= .017), and the presence of severe lymphocytic infiltration (P= .022).Conclusion: Accumulation of TIL-expressing such inhibitory molecules may deteriorate the immunity of high-risk breast cancer patients and this should encourage vigorous combinatorial immunotherapeutic approaches targeting T_regsand B7-H1/PD-1 molecules.

Breast cancer in young Saudi women is a crucial problem. According to the 2002 annual report of Saudi National Cancer Registry, breast cancers that developed before the age of 40 comprise 26.4% of all female breast cancers comparing to 6.5% in the USA. Breast cancer in young patients is often associated with a poorer prognosis, but there has been a scarcity of published data in the Middle East population.

Breast cancer (BC) is the most common malignancy and the leading cause of cancer-related death amongst women worldwide. The risk factors of this disease are numerous, and their prevalence varies between racial and ethnic groups as well as geographical regions. Therefore, we sought to delineate the association of socio-demographic, reproductive and life-style related risk factors with breast cancer in the Arab population. Unmatched case-control study was conducted in the kingdom of Saudi Arabia using 534 cases of histologically confirmed breast cancer and 638 controls. Controls were randomly selected from primary health care visits and were free of breast cancer. Unconditional logistic regression analysis was performed to estimate odds ratios (ORs) and to examine the predictive effect of each factor on risk for BC. All study participants were interviewed by trained interviewers at hospital (cases) or at primary health care centers (controls). A total of 1172 women were eligible for this study, of which 281 (24.0%) were aged ≤35 years, 22.9% illiterate, 43.6% employed, 89.5% married, and 38.1% were obese. Grade III tumors constituted 38.4% of cases. Tumor stage I was 7.5%; II, 50.7%; II, 30.9%; IV, 11.1%. We have shown strong association between breast cancer among Arab females and obesity (OR =2.29, 95% CI 1.68-3.13), positive family history of breast cancer (OR =2.31, 95% CI 1.60 – 3.32), the use of hormonal replacement therapy (OR =2.25, 95% CI 1.65 – 3.08), post-menopause (OR =1.72, 95% CI 1.25 – 2.38), lack of education (OR =9.09, 95% CI 5.88 – 14.29), and never breastfeed (OR =1.89, 95% CI 1.19 – 2.94). These results indicate the presence of classical risk factors established in the western countries, and also some specific ones, which may result from genetic and/or environmental factors. Thereby, these findings will be of great value to establish adequate evidence-based awareness and preventative measures in the Arab world.

Female breast cancer is frequently diagnosed at a later stage and the leading cause of cancer deaths world-wide. Levels of cell-free circulating microRNAs (miRNAs) can potentially be used as biomarkers to measure disease progression in breast cancer patients in a non-invasive way and are therefore of high clinical value. Using quantitative RT-PCR, circulating miRNAs were measured in blood samples collected from disease-free individuals (n = 34), triple-negative breast tumours (TNBC) (n = 36) and luminal tumours (n = 57). In addition to intergroup comparisons, plasma miRNA expression levels of all groups were analyzed against RNASeq data from cancerous breast tissue via The Cancer Genome Atlas (TCGA). A differential set of 18 miRNAs were identified in the plasma of breast cancer patients and 10 miRNAs were uniquely identified based on ROC analysis. The most striking findings revealed elevated tumor suppressor let-7 miRNA in luminal breast cancer patients, irrespective of subtype, and elevated miR-195 in plasma of TNBC breast cancer patients. In contrast, hsa-miR-195 and let-7 miRNAs were absent from cancerous TCGA tissue and strongly expressed in surrounding non-tumor tissue indicating that cancerous cells may selectively export tumor suppressor hsa-miR-195 and let-7 miRNAs in order to maintain oncogenesis. While studies have indicated that the restoration of let-7 and miR-195 may be a potential therapy for cancer, these results suggested that tumor cells may selectively export hsa-miR-195 and let-7 miRNAs thereby neutralizing their potential therapeutic effect. However, in order to facilitate earlier detection of breast cancer, blood based screening of hsa-miR-195 and let-7 may be beneficial in a female patient cohort.