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Background: The presence of TILs has been correlated with clinical outcomes and response to therapy in breast cancer. However, evaluation of TILs in breast cancer has largely been based on pathologic examination of tumor samples. Here, we report the relationship between invasive breast cancer locoregional recurrence (LRR) and the presence of TILs estimated by transcriptomic analysis with the deconvolution algorithm CIBERSORT. Methods: Patients were identified from an IRB-approved prospective tissue collection protocol at one academic institution and two community hospitals. 526 primary breast tumor samples were identified and gene expression profiling was assessed with high density Affymetrix microarray chips. Proportions of 22 different TIL types in samples were inferred based on the CIBERSORT algorithm, which uses gene expression data to estimate TIL presence. TIL presence was determined by dichotomization at the level of the first quartile among all samples (>Q1=TIL presence). Patient characteristics and clinical outcomes were obtained by chart review. Time to event analysis was performed using Kaplan Meier (KM) estimates and the log-rank test. Associations between patient factors, tumor factors, TIL presence, and LRR were explored with univariable (UVA) and multivariable (MVA) analyses. Factors significant on UVA (p<0.10) were included on MVA. P<0.05 was considered statistically significant on MVA. Results: 526 women with invasive breast cancer and available genomic profiling were retrospectively identified for analysis. Median age at diagnosis was 58 years. 70% of tumors were Stage I-II. 69% were luminal subtypes and 17% were triple negative. 37% received mastectomy, 25% received mastectomy + radiation, and 32% received breast conserving therapy. 64% received chemotherapy, and 62% received hormonal therapy. Median follow-up was 74.4 months. There were 61 LRRs. We found significant differences in time to LRR when comparing presence vs. no presence of resting memory CD4+ T-cells (RMCD4+) (p=0.01), activated natural killer cells (ANK) (p=0.003), and neutrophils (PMNs) (p=0.03). On UVA, factors associated with LRR were patient age at diagnosis (p=0.009), pathologic T stage (p=0.045), Estrogen receptor status (p=0.03), biologic subtype (p=0.01), lymphovascular invasion (LVI) (p=0.018), positive margins (p<0.0001), receipt of hormonal therapy (0.014), and presence of tumor infiltrating RMCD4+ (p=0.012), ANK (p=0.0004), and PMNs (p=0.033). On MVA, factors remaining significant were LVI (HR 2.16 CI 1.13-4.13, p=0.011), positive margins (HR 4.36 CI 1.57-12.11, p=0.018), receipt of hormonal therapy (HR 0.31 CI 0.12-0.77, p=0.042), and presence of RMCD4+ (HR 0.48 CI 0.26-0.88, p=0.017), ANK (HR 0.43 CI 0.23-0.83, p=0.012), and PMNs (HR 2.15 CI 1.02-4.53, p=0.043). Conclusion: In this study of 526 women with invasive breast cancer, we identified that enrichment of certain TILs is associated with LRR. These results suggest genomic-based assays of TIL presence may be useful to predict LRR in invasive breast cancer. Citation Format: Liveringhouse CL, Grass GD, Figura NB, Mills MN, Purcell JD, Rosensweig SR, Blumencranz PW, Allen KG, Ahmed KA, Harrison LB, Torres-Roca JF, Robinson TJ, Diaz R. Locoregional recurrence in invasive breast cancer and association with tumor infiltrating leukocyte (TIL) presence [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-15.

Sustained locoregional control of breast cancer is a significant issue in patients with high risk disease. We recently reported the results of a phase I clinical trial describing acute and late toxicity of PARP1 inhibition (PARPi) with concurrent veliparib and RT. The purpose of this study was to determine predictive biomarkers of acute (during treatment) and late (1, 2, and 3 year) toxicity in patients treated with PARPi and RT from that trial. We hypothesized that protein and gene expression changes in skin epithelial cells after concurrent PARPi and RT might be used as predictive biomarkers of toxicity. Materials/Methods: Acute toxicity was any dose-limiting toxicity defined in the protocol; late toxicity was defined as any CTCAE v4 Grade 3 event, regardless of attribution. Skin punch biopsies from the irradiated field were taken from patients at baseline (pretreatment TZ0) and 6 hrs after first fraction of RT alone (TZ1). Veliparib was then started and a 3rd punch taken 6 hrs after the second fraction of RT (TZ2). RNA was isolated from skin epithelial cells and gene expression assessed using Affymetrix Human Gene ST 2.1 arrays. GSEA and MSigDB software was used for analysis. Significant difference was predetermined as a log2 fold change of+/-1.5 and FDR adjusted p-value <0.05. Protein changes (pgH2AX, PAR, Ki67) were detected using IHC and quantitated using Aperio Digital Path software. Results: Severe acute and late toxicity rates with combined therapy were 15% (5/33) and 21% (7/33), respectively. Acute toxicity was not a predictor of late toxicity. Indeed, the two were almost mutually exclusive. Gene expression analysis identified 31 genes whose expression was significantly different 6 hrs after RT and 54 genes differed after combined treatment, including genes associated with DNA damage repair (ATM, MDM2, XPC) and proliferation (Ki67, TP53TG1). 67 genes were associated with acute toxicity, including overrepresentation of miRNAs associated with gene repression. Additionally, 63 genes were associated with late toxicity and were associated with metabolism, inflammation, and DNA damage response. There was no overlap in biomarkers of acute and late toxicity. Both pgH2AX and PAR protein levels increased and Ki67 decreased significantly (more than 2 fold for all proteins) with RT and combined therapy (T1 and T2) but were not significantly associated with acute or late toxicity at any time point. Conclusion: This demonstrates the feasibility of using skin punch biopsy in the irradiated field for biomarker analysis and identification of a number of putative biomarkers of early and late radiation toxicity following combined PARPi and RT treatment. Additionally, for the first time in patient skin punch samples we report (1) gene expression changes in acute responding genes shortly after RT and (2) decreased proliferation after combined treatment. Future trials will validate the utility of these biomarkers for predicting toxicity and will seek to identify biomarkers of treatment efficacy.

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