Background: Hormone receptor-positive (HR+) tumors have fewer tumor-infiltrating lymphocytes (TILs) and lower response rates to immune checkpoint inhibitors (ICI), either as single agents or in combination with chemotherapy, than triple negative cancers. However, some HR+ cancers do respond to ICI and biomarkers that accurately reflect the immune microenvironment may help guide the use of ICI therapy. Prior evidence suggests that macrophage-related immune pathways may be relevant to the pathophysiology of HR+ BC. Methods: HR+/HER2- patients were identified from a prospective trial of preoperative bevacizumab (preop bev) followed by bev with adriamycin/cyclophosphamide/paclitaxel dose-dense chemotherapy (chemo). Tumor samples were collected at diagnosis and surgery (pre-tx and post-tx), and PD-L1 expression (by immunohistochemistry), TILs, and Nanostring PanCancer Immune Profiling Panel were evaluated on both pre-tx and post-tx specimens. Pre-tx whole transcriptome sequencing was performed. Pathologic response at surgery was centrally assessed by Miller-Payne (MP) and residual cancer burden (RCB) scores. An immune score was calculated for each pre-tx specimen by integrating 10 published immune signatures. Immune cell subsets were inferred from bulk transcriptional data using CIBERSORT and immune cell-specific signatures from MSigDB. Results: 55 patients who received trial therapy and had at least 1 evaluable specimen were included for analysis. Pre-tx TILs and tumor PD-L1 (tPD-L1) scores are shown in the table. 18% of pre-tx tumors had “high” (≥10%) TILs and “high” TILs were associated with significantly higher immune signature score (p=0.004). Immune score correlated highly with proportion of CIBERSORT anti-tumor M1 macrophages as well as CD8 T-cell signatures (r>0.65 and p<0.001). Higher pre-tx TILs, tPD-L1, or immune score were each significantly associated with more favorable RCB and MP in unadjusted analyses (all Spearman p<0.01 for pathologic markers; ANOVA p<0.04 for immune score). After adjustment for age and tumor grade, higher pre-tx TILs and tPD-L1 were associated with favorable RCB (p<0.01 for both), and higher pre-tx tPD-L1 correlated with favorable MP (p=0.03). Pathologic complete response occurred in 4 pts; all 4 had high pre-tx TILs, pre-tx tPD-L1, or both. Among patients with residual disease, large changes (>5%) in TILs or tPD-L1 from pre-tx to post-tx were rare: 2 pts each had large changes in TIL or tPD-L1 score (N=38/N=31 pairs, respectively). Conclusions: High levels of tumor-lymphocyte interaction were seen in only a minority of untreated HR+ breast tumors, and did not typically change with chemo plus bev. An immune score derived from bulk RNAseq correlated with histological observations in these specimens. Nonetheless, TILs, tPD-L1, and signature-derived immune score were significantly associated with pathologic response to preop treatment in HR+ disease. Early data suggest that the role of M1 macrophages in HR+ tumors warrants further investigation. ScoreTILs (N=50 evaluable)Tumor PD-L1 (N=51)0%0 pts (0%)28 pts (55%)>0-5% (low)19 (38%)18 (35%)>5-10% (intermediate)22 (44%)3 (6%)>10% (high)9 (18%)2 (4%) Citation Format: Waks AG, Stover DG, Barry W, Dillon D, Gjini E, Rodig SJ, Brock J, Baltay M, Savoie J, Winer EP, Krop I, Tolaney SM. The immune microenvironment in hormone receptor-positive breast cancer patients and relationship to treatment outcome following preoperative chemotherapy plus bevacizumab [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 PD6-09.

Introduction: Triple-negative breast cancer (TNBC) is a poor prognosis breast cancer subset characterized by relatively few mutations but extensive copy number alterations (CNAs). Cell-free DNA (cfDNA) offers the potential to overcome infrequent tumor biopsies in metastatic TNBC (mTNBC) and interrogate the genomics of chemotherapy resistance. Methods: 506 archival or fresh plasma samples were identified from 164 patients with mTNBC who had previously received chemotherapy. We performed low coverage whole genome sequencing to determine genome-wide copy number and estimate 'tumor fraction' of cfDNA (TFx) using our recently-developed approach, ichorCNA. In patient samples with TFx >10%, we identified regions that were significantly gained or lost using GISTIC2.0. We compared CNAs of 20 paired primary-metastatic samples and also mTNBCs from cfDNA versus primary TNBCs from TCGA and METABRIC. Results: We successfully obtained high quality, low coverage whole genome sequencing data for 478 (94.5%) plasma samples from 158 patients, with 1 to 14 samples per patient. TFx and copy number profiles were highly concordant with paired metastatic biopsy (n=10, range 0-7 days from biopsy to blood draw) with sensitivity of 0.86 and specificity of 0.90 and reproducible in independently-processed blood draws (TFx intraclass correlation coefficient 0.984). Median overall survival from time of first blood draw was 8 months, and TFx was highly correlated independent of primary stage, primary receptor status, age at primary diagnosis, BRCA status, and metastatic line of therapy: adjusted hazard ratio between 4th and 1st quartiles = 2.14 (95% CI 1.40-3.28; p=0.00049). 101/158 patients (63.9%) had at least one sample with TFx >10%, our threshold for high confidence CNA calls. Copy number profiles and percent genome altered were remarkably similar between mTNBCs and primary TNBCs in TCGA and METABRIC (n=433), suggesting that large-scale chromosomal events are infrequent in TNBC metastatic progression. We identified chromosomal gains that demonstrated significant enrichment in mTNBCs relative to paired primary TNBCs (n=20) and also TCGA/METABRIC, including driver genes (NOTCH2, AKT2, AKT3) and putative antibody-drug conjugate targets. Finally, we identify a novel association of gains of 18q11 and/or 19p13 with poor metastatic prognosis, independent of clinicopathologic factors and TFx. Conclusions: Here, we present the first large-scale genomic characterization of metastatic TNBC to our knowledge, derived exclusively from cfDNA. 'Tumor fraction' of cfDNA is an independent prognostic marker in mTNBC. Primary and metastatic TNBC have remarkably similar copy number profiles yet we identify alterations enriched and prognostic in mTNBC. Collectively, these data have potential implications in the understanding of metastasis, therapeutic resistance, and novel therapeutic targets. Citation Format: Stover DG, Parsons HA, Ha G, Freeman S, Barry B, Guo H, Choudhury A, Gydush G, Reed S, Rhoades J, Rotem D, Hughes ME, Dillon DA, Partridge AH, Wagle N, Krop IE, Getz G, Golub TA, Love JC, Winer EP, Tolaney SM, Lin NU, Adalsteinsson VA. Genome-wide copy number analysis of chemotherapy-resistant metastatic triple-negative breast cancer from cell-free DNA [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 GS3-07.

Background: Relative to other metastatic breast cancer subtypes, metastatic triple-negative breast cancer (mTNBC) has a shorter duration of response to therapy and worse overall survival. Within mTNBCs, there is a prevailing belief that inflammatory breast cancer and young women tend to have among the most aggressive phenotypes. We investigated clinical and cell-free DNA (cfDNA) characteristics of inflammatory-mTNBC and young-mTNBC. We hypothesized that inflammatory-mTNBC may have distinct clinical and cfDNA characteristics, offering potential novel biomarker and therapeutic strategies. Methods: 164 patients from the Dana-Farber metastatic triple-negative cell-free DNA cohort (Stover DG, et al J Clin Oncol 2018) were included in this secondary analysis. Patients were stratified into three groups: 1) inflammatory breast cancer ('IBC'); 2) non-IBC patients aged 45 years (yr) or younger at primary diagnosis ('non-IBC young'); and 3) non-IBC patients over age 45 yr at diagnosis. For each subset population, we evaluated clinicopathologic characteristics, sites of metastasis, survival outcomes, and cfDNA 'tumor fraction' – the fraction of DNA in circulation derived from tumor. Those patients with adequate cfDNA tumor content for high confidence copy number calls (n=101) were included in an analysis of copy number alterations. Results: Among 164 patients with metastatic TNBC, 13.4% (22/164) had IBC, 37.8% (62/164) were non-IBC young, and 48.8% (80/164) were non-IBC and over 45 yr. Race and primary receptor status were similar. IBC patients were diagnosed at a higher stage (Chi-square p=0.0009) while non-IBC young patients were significantly more likely to harbor a BRCA mutation (Chi-square p=0.03). Analysis of metastatic sites revealed that IBC patients had significantly greater frequency of ipsilateral and contralateral breast chest wall recurrences (p=0.04 and p=0.046, respectively) while non-IBC young patients had the most frequent lung metastases (p=0.002). There were no significant differences in frequency of bone, brain, or liver metastases. cfDNA analyses showed that cfDNA 'tumor fraction' was highest in non-IBC young patients (ANOVA p=0.03 for maximum tumor fraction). Median overall survival from metastatic diagnosis was 22.9 months. IBC and non-IBC young patients had a worse prognosis relative to non-IBC patients over 45 yr (hazard ratio IBC=1.97, 95% CI 1.09-3.57; HR non-IBC young=1.60 95% CI 1.07-2.41; log-rank p=0.023). By subgroup, median overall survival from metastatic diagnosis for IBC was 15.2 months, non-IBC young 21.2 months, and non-IBC over 45 yr 31.2 months. Analyses of genome-wide copy number alterations from cell-free DNA will be presented. Conclusions: Among metastatic TNBCs, IBC patients and non-IBC young patients have a significantly worse overall survival compared with non-IBC patients over 45 yr of age. Young patients have more frequent lung metastases and higher 'tumor fraction' of cfDNA. Confirmation of the reported findings is limited due to cohort size and may reflect referral bias. Citation Format: Singh J, Asad S, Nock W, Zhang Y, Adams E, Damicis A, Parsons HA, Adalsteinsson VA, Winer EP, Lin NU, Partridge AH, Overmoyer B, Stover DG. Aggressive subgroups of metastatic triple-negative breast cancer: Inflammatory breast cancer and young patients in the Dana-Farber cell-free DNA cohort [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 P4-01-17.

Background: Proliferation is the strongest predictor of response to neoadjuvant chemotherapy in estrogen receptor-positive (ER+) breast cancer. Evidence of immune activation has also been associated with improved response to neoadjuvant chemotherapy in breast cancer. We hypothesized that immune signatures may be associated with response independent of proliferation in ER+ breast cancers. Approach: We compiled microarray expression data from breast cancer biopsies obtained prior to neoadjuvant chemotherapy on 465 ER-positive/HER2-negative patients by reported pathologic receptor status. We evaluated the association of 118 published gene expression signatures with response to neoadjuvant chemotherapy, based on study-defined pathologic complete response (pCR) versus residual disease (RD). Results: Overall, 42 of 118 signatures were significantly associated with response to neoadjuvant chemotherapy in ER+ breast cancer (FDR-corrected p<0.05, simple logistic regression). Of those signatures that achieved significance, 52% (22/42) of signatures were proliferation-associated based on correlation to the 11-gene PAM50 proliferation index (Pearson's R2>0.30, p<1e-10). Among signatures that were NOT proliferation-associated, 50% (10/20) were immune-related. Using unsupervised hierarchical clustering of all 118 signatures, these ten immune signatures formed a distinct cluster. Of the 10 signatures, nine were designed to reflect "immune activation" and were highly correlated with each other in ER+ tumors (R2>0.4, p<0.001). The mean of each of these nine signatures was significantly higher in patients with pCR versus RD (FDR-corrected p<0.05, t-test). Patients with higher "immune activation" signatures had increased likelihood of pCR within multiple subgroups of ER+ breast cancer, including luminal B and non-luminal PAM50 subgroups, as well as intermediate- and high-proliferation ER+ breast cancers. For luminal A or low-proliferation breast cancers, "immune activation" signatures were not significantly associated with response, though very few patients achieved pCR in these two subgroups. Conclusions: Gene expression signatures associated with "immune activation" identify a subset of ER+ breast cancers with higher rates of pCR to neoadjuvant chemotherapy. These "immune activation" signatures appear to be proliferation-independent and may provide additional predictive information to existing gene expression-based approaches for ER+ breast cancer. Citation Format: Stover DG, Waks AG, Erica ML, Brugge JS, Winer EP, Selfors LM. Immune activation signatures identify a subset of ER+ breast cancers with increased pathologic complete response to neoadjuvant chemotherapy. [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 P3-07-31.

This abstract was not presented at the symposium.