Background: In the evaluation of PD-L1 expression to select patients for anti-PD-1/PD-L1 treatment, uniform guidelines that account for different immunohistochemistry assays, different cell types and different cutoff values across tumor types are lacking. Data on how different scoring methods compare in breast cancer are scant. Methods: Using FDA-approved 22C3 diagnostic immunohistochemistry assay, we retrospectively evaluated PD-L1 expression in 496 primary invasive breast tumors that were not exposed to anti-PD-1/PD-L1 treatment and compared three scoring methods (TC: invasive tumor cells; IC: tumor-infiltrating immune cells; TCIC: a combination of tumor cells and immune cells) in expression frequency and association with clinicopathologic factors. Results: In the entire cohort, positive PD-L1 expression was observed in 20% of patients by TCIC, 16% by IC, and 10% by TC, with a concordance of 87% between the three methods. In the triple-negative breast cancer patients, positive PD-L1 expression was observed in 35% by TCIC, 31% by IC, and 16% by TC, with a concordance of 76%. Associations between PD-L1 and clinicopathologic factors were investigated according to receptor groups and whether the patients had received neoadjuvant chemotherapy. The three scoring methods showed differences in their associations with clinicopathologic factors in all subgroups studied. Positive PD-L1 expression by IC was significantly associated with worse overall survival in patients with neoadjuvant chemotherapy and showed a trend for worse overall survival and distant metastasis-free survival in triple-negative patients with neoadjuvant chemotherapy. Positive PD-L1 expression by TCIC and TC also showed trends for worse survival in different subgroups. Conclusions: Our findings indicate that the three scoring methods with a 1% cutoff are different in their sensitivity for PD-L1 expression and their associations with clinicopathologic factors. Scoring by TCIC is the most sensitive way to identify PD-L1-positive breast cancer by immunohistochemistry. As a prognostic marker, our study suggests that PD-L1 is associated with worse clinical outcome, most often shown by the IC score; however, the other scores may also have clinical implications in some subgroups. Large clinical trials are needed to test the similarities and differences of these scoring methods for their predictive values in anti-PD-1/PD-L1 therapy.

N-Myc downstream regulated gene 1 (NDRG1) suppresses metastasis in many human malignancies, including breast cancer, yet has been associated with worse survival in patients with inflammatory breast cancer. The role of NDRG1 in the pathobiology of aggressive breast cancers remains elusive. To study the role of NDRG1 in tumor growth and brain metastasis in vivo, we transplanted cells into cleared mammary fat pads or injected them in tail veins of SCID/Beige mice (n = 7-10 per group). NDRG1 protein expression in patient breast tumors (n = 216) was assessed by immunohistochemical staining. Kaplan-Meier method with 2-sided log-rank test was used to analyze the associations between NDRG1 and time-to-event outcomes. A multivariable Cox regression model was used to determine independent prognostic factors. All statistical tests were 2-sided. We generated new sublines that exhibited a distinct propensity to metastasize to the brain. NDRG1-high–expressing cells produced more prevalent brain metastases (100% vs 44.4% for NDRG1-low sublines, P = .01, Fisher’s exact test), greater tumor burden, and reduced survival in mice. In aggressive breast cancer cell lines, silencing NDRG1 led to reduced migration, invasion, and tumor-initiating cell subpopulations. In xenograft models, depleting NDRG1 inhibited primary tumor growth and brain metastasis. In patient breast tumors, NDRG1 was associated with aggressiveness: NDRG1-high expression was also associated with shorter overall survival (hazard ratio [HR] = 2.27, 95% confidence interval [95% CI] = 1.20 to 4.29, P = .009) and breast cancer–specific survival (HR = 2.19, 95% CI = 1.07 to 4.48, P = .03). Multivariable analysis showed NDRG1 to be an independent predictor of overall survival (HR = 2.17, 95% CI = 1.10 to 4.30, P = .03) and breast cancer–specific survival rates (HR = 2.27, 95% CI = 1.05 to 4.92, P = .04). We demonstrated that NDRG1 drives tumor progression and brain metastasis in aggressive breast cancers and that NDRG1-high expression correlates with worse clinical outcomes, suggesting that NDRG1 may serve as a therapeutic target and prognostic biomarker in aggressive breast cancers.