PURPOSETriple Negative Breast Cancer (TNBC) is more common in African American (AA) than Non-AA (NAA) population. We hypothesize that Tumor Microenvironment (TME) contributes to this disparity. Here we use multiplex quantitative immunofluorescence (QIF) to characterize the expression of immunologic biomarkers in the TME in both populations.PATIENTS AND METHODSTNBC tumor resection specimen tissues from a 100-patient case: control cohort including 49 AA and 51 NAA were collected. TME markers including CD45, CD14, CD68, CD206, CD4, CD8, CD20, CD3, Ki67, GzB, Thy1, FAP, aSMA, CD34, Col4, VWF and PD-L1 we quantitatively assessed in every field of view. Mean expression levels were compared between cases and controlsRESULTSAlthough no significant differences were detected in individual lymphoid and myeloid markers, we found that infiltration with CD45+ immune cells (p=0.0102) was higher in TNBC in AA population. AA TNBC tumors also had significantly higher level of lymphocytic infiltration defined as CD45+CD14- cells (p=0.0081). CD3+T-cells in AA tumors expressed significantly higher levels of Ki67 (0.0066) compared to NAAs, indicating that a higher percentage of AA tumors contained activated T-cells. All other biomarkers showed no significant differences between the AA and NAA group.CONCLUSIONSWhile the TME in TNBC is rich in immune cells in both racial groups, there is a numerical increase in lymphoid infiltration in AA compared to NAA TNBC. Significantly higher activated T cells seen in AA patients raises the possibility that there may be a subset of AA patients with improved response to immunotherapy.

Background: Prosigna's ROR score was demonstrated as a strong predictor of response to NAC in a representative cohort of EBC patients including HR+/HER2- N0-N1 patients.1 Given that the ROR score is partially derived from the correlation of the tumor's expression profile to that of the four prototypical intrinsic subtypes, we determined the relative strength of the association between each subtype correlation and the likelihood of response to NAC. Methods: We analyzed 294 FFPE breast cancer samples from pts treated with NAC (anthracyclines and taxanes) in a multi-center Spanish cohort. The Prosigna Assay was performed on the NanoString nCounter® Dx Analysis System at HU Virgen de la Victoria de Málaga/CIMES-UMA. Pathologic complete response (pCR) was used as the primary endpoint for this study and was determined using the Miller and Payne scoring criteria. Results: Mean patient age in this population was 50 (±11yr). Apart from targeted therapy, all patients received a standard neoadjuvant treatment regimen consisting of 8-12 cycles of anthracyclines and taxanes. 58% of patients were HR+/HER2- while 24% were classified as HER2+ and 18% were TNBC patients. Of the 311 pts samples previously tested, subtype correlation data was available for 294. Overall subtype concordance between IHC and Prosigna was 72% (K=0.66). The overall pCR rate in this population was 24.9%. Prosigna subtype breakdown in the full study population was 60 Luminal A, 118 Luminal B, 69 HER2-enriched and 47 Basal-like with response rates of 7.2%, 7.2%, 46.2% and 57.4%, respectively. We found that in all study populations, subtype correlation was a strong predictor of response to NAC. Tumors with expression profiles that correlated well with the Luminal prototypical centroids were found to be largely unresponsive to NAC (Luminal A Odds ratio=0.074 per unit increase, p<0.0001; Luminal B Odds ratio=0.059 per unit increase, p<0.0001). Conversely, higher HER2E and Basal-like correlations were associated with increased probability of response (HER2E Odds ratio=11.2 per unit increase, p<0.0001; Basal-like Odds ratio=9.0 per unit increase, p<0.0001). Increased proliferation-score (p-score) was also associated with increased probability of response to NAC (Odds ratio=3.43 per unit increase, p<0.0001). Conclusions: In a representative cohort of breast cancer patients, both the magnitude of the subtype correlation to the prototypical centroids as well as p-score, as determined by the Prosigna Assay, were strong predictors of response to NAC. This data underlines the importance of molecular testing for optimal systemic therapy indications in EBC. 1Rodriguez B, Lavado Fernandez A, Ribelles N, et al. Prosigna (PAM50) to predict response to neoadjuvant chemotherapy (NAC) in HR+/HER2- early breast cancer (EBC) patients. J Clin Oncol 33, 2015 (suppl; abstr 11049). Citation Format: Chica Parrado R, Jiménez-Rodríguez B, Sánchez Rovira P, Álvarez M, Vicioso L, Fernandez AI, de Luque V, José Lozano M, Villar E, Zarcos I, Ramírez C, González-Hermoso C, Jeiranian A, Dowidar N, Schaper C, Buckingham W, Ferree S, Ribelles N, Rodrigo I, Prat AP, Alba E. Prosigna® subtype correlation is a strong predictor of response to neoadjuvant chemotherapy (NAC) in early breast cancer (EBC) study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-15.

Introduction: Triple negative breast cancer (TNBC) is a highly aggressive form of breast cancer prevalent in African-American (AA) women defined as estrogen receptor- (ER), progesterone receptor- (PR), and human epidermal growth factor receptor 2- (HER2) negative. Because ER- and HER2-targeted therapies are ineffective in TNBC, systemic chemotherapy is the standard of care and there is a tremendous need for new effective therapies with less toxicity. Steroid hormone receptors are highly druggable targets, and orphan nuclear receptors, members of the nuclear receptor superfamily, are emerging as targets for cancer therapy. In fact, we have previously shown that treatment of TNBC cells with a small molecule agonist ligand (DY131) for estrogen related receptor beta (ERRβ), has growth inhibitory and anti-mitotic activity. We have also shown that increased mRNA expression of ERRβ, correlates with better recurrence- and distant metastasis-free survival in TNBC/basal-like breast cancer. The goal of our current work is to comprehensively characterize ERRβ copy number and mRNA status in TNBC and determine its association with patients' prognosis. Methods: ESRRB copy number was determined in 106 primary breast tumors (TNBC n=56, nonTNBC n=50) by array-CGH, using the Agilent SurePrint G3 Human CGH platform. ESRRB mRNA data and its association with overall survival was determined in systemically untreated patients from METABRIC using Illumina gene expression array data (probe ID ILMN_1707398). Results: Copy number alterations (CNAs). Copy number losses at the ESRRB locus (14q24.3) were observed in 10/56 (17.8%) of TNBC vs. 10/50 (20%) of nonTNBC, while copy number gains were detected in 43/56 (76.8%) of TNBC vs. 29/50 (58%) of nonTNBC (c2 *p=0.036). Interestingly, in both TNBC and non-TNBC, ESRRB loss was seen with markedly higher frequency in AA patients when compared to Caucasian (CA) patients (c2 *p=0.012 for TNBC, p=0.052 for non-TNBC). mRNA expression. Among patients not treated with systemic chemotherapy in the METABRIC dataset, low ESRRB mRNA was significantly associated with shorter overall survival in TNBC, but not ER+ or HER2+ patients (TNBC hazard ratio 0.24, 95% confidence interval 0.07-0.85, *p=0.016). Low ESRRB also correlated with reduced overall survival in TP53 mutant (but not wild type) tumors (hazard ratio 0.28, 95% confidence interval 0.1-0.82, *p=0.013). Conclusions: ESRRB presents significantly high levels of copy number losses in TNBC when compared to non-TNBC tumors. In breast tumors from AA women, both the TNBC and non-TNBC subtypes are significantly more likely to have reduced ESRRB copy number vs. CA women. Low ESRRB mRNA expression predicts for poor overall survival in TNBC and TP53 mutant tumors. These data advocate that ERRβ expression has prognostic value in breast cancer, particularly TNBC. Future goals include immunohistochemistry staining, and analysis, of a tissue microarray consisting of 150 primary breast tumors (50 TNBC, 50 ER+, 50 HER2+); as well as ERRβ overexpression and knock-down studies in TNBC cell lines to define the role it plays in TNBC. Citation Format: Fernandez AI, Graham G, Győrffy B, Cavalli L, Mahajan A, Riggins RB. ERRβ copy number and expression in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-09.

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