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1091 Background: Tumor mutational burden (TMB) has emerged as an imperfect biomarker of immune checkpoint inhibition (ICI) outcomes in solid tumors. Despite the approval for pembrolizumab in all TMB-high (TMB-H) solid tumors, the optimal clinical approach to TMB-H or hypermutated advanced/metastatic breast cancer (MBC) is unknown with sparse prospective data. We hypothesize that TMB-H MBC will have unique genomic alterations compared to TMB-low (TMB-L) breast cancer that could inform novel therapeutic approaches. Methods: Tumor samples (N = 5621) obtained from patients with MBC were analyzed by next-generation sequencing (NGS) of DNA (592-gene panel or whole exome sequencing) and RNA (whole transcriptome sequencing) at Caris Life Sciences (Phoenix, AZ). TMB was calculated based on recommendations from the Friends of Cancer Research TMB Harmonization Project (Merino et al., 2020), with the TMB-H threshold set to ≥ 10 muts/Mb. IHC was performed for PD-L1 (Ventana SP142 ≥1% immune cells). Deficient mismatch repair (dMMR)/high microsatellite instability (MSI-H) was tested by IHC and NGS, respectively. Results: TMB-H was identified in 8.2% (n = 461) of MBC samples, with similar frequencies observed across molecular subtypes (7.8-8.6%, p = 0.85): HR+/HER2- (n = 3087) 7.8%, HR+/HER2+ (n = 266) 8.3%, HR-/HER2+ (n = 179) 7.8%, TNBC (n = 1476) 8.6%. The frequency of TMB-H was significantly increased in lobular (16%) versus ductal (5%) MBC (p < 0.01). TMB-H samples were enriched in genitourinary (42%), soft tissue (20%), and gastrointestinal non-liver (16%) biopsy specimens. Compared to TMB-L tumors, TMB-H tumors exhibited significantly higher mutation rates for TP53 (60 v 52%), PIK3CA (55 vs 31%), ARID1A (34 vs 11%), CDH1 (27 vs 11%), NF1 (22 vs 9%), RB1 (14 vs 5%), KMT2C (12 vs 7%), PTEN (12 vs 7%), ERBB2 (7 vs 2.9%), and PALB2 (3.3 vs 1%) genes (p < 0.05 each). Copy number alteration and fusion rates did not differ between TMB-H and TMB-L breast cancers. PI3K/AKT/MTOR, TP53, Histone/Chromatin remodeling, DNA damage repair (DDR), RAS, and cell cycle pathway alterations were detected in > 25% TMB-H MBCs (p < 0.05 each). dMMR/MSI-High (7.2 vs 0.3%, p < 0.01) and PD-L1 positivity (36 vs 28%, p < 0.05) frequencies were significantly increased in TMB-H tumors. DNA signature analyses including APOBEC and homologous recombination repair deficiency, as well as gene expression profiling to assess immune-related signatures and tumor microenvironment are underway. Conclusions: TMB-H breast cancers contain a unique genomic profile enriched with targetable mutations such as PIK3CA, ARID1A, NF1, PTEN, ERBB2, and PALB2. Concurrent predictive biomarkers of response to immune checkpoint inhibition such as MSI-H and PDL-1 positivity are also more prevalent in TMB-H MBC. These findings suggest novel combination strategies within TMB-H MBC could be explored.

3040 Background: Pembrolizumab was recently FDA approved across solid tumors for TMB scores ≥ 10mut/Mb as assessed by next-generation sequencing (NGS) of tissue (tTMB). A prior study of advanced cancer patients treated with immunotherapy found that higher somatic TMB, as defined by the 80th percentile in each histology, was associated with better overall survival. Previously, bTMB assessed by ctDNA from patients with newly diagnosed advanced NSCLC at a score of 16 mut/MB correlated with a tTMB score of 10 mut/MB. TMB levels vary by cancer type, line of treatment, and therapy received; the distribution of bTMB scores across solid tumor types has not been well characterized. Here we report the distribution of bTMB scores in patients with advanced malignancies. Methods: We queried 5,610 samples from patients with different cancer types undergoing clinical cell-free DNA testing (Guardant360; Redwood City, CA) and assessed bTMB scores from October 2020 - January 2021. bTMB score was derived via a previously described computational algorithm examining the total number of synonymous and non-synonymous SNVs and indels across a 1.0MB genomic footprint. We assessed the success rate of bTMB evaluation, overlap with microsatellite instability (MSI) status, and defined the distribution of bTMB levels across indications in this dataset. Results: bTMB score was successfully assessed in 4,275/5,610 (76.3%) samples (Table). The majority of samples (58%) were tested at disease progression as compared to initial diagnosis (42%). The median turnaround time from sample receipt to clinical reporting was 11 days and decreased to 9 days over the course of the study. For the majority of cancer types the 80th percentile TMB was ≥ 16 mut/MB tissue equivalency. Conclusions: Our analysis demonstrates the feasibility of measuring bTMB using a commercially available liquid biopsy assay. bTMB scores trended higher than tTMB previously reported in these cancer types, reflecting the ability of ctDNA to better capture tumor heterogeneity. cfDNA may allow for exploration of bTMB evolution throughout treatment. TMB should be interpreted in the context of disease, treatment, and method; these data establish a pan-cancer benchmark for bTMB which will serve as a resource for further studies.[Table: see text]

TPS1102 Background: While immunostimulatory therapies have shown great success, a major challenge remains identification of mechanisms to effectively treat the majority of patients with so-called "non-inflamed" tumors lacking marked lymphocyte infiltration and PD-L1 expression. The DNA repair proficiency of a tumor may impact its potential for immune recognition and sensitivity to immune checkpoint blockade. Preclinically, PARP inhibition in HDR-deficient tumors has been shown to trigger antitumor immunity through a STING-dependent antitumor immune response. Effects of PARP inhibitors were augmented when combined with PD-1 blockade. We hypothesize that enhanced DNA damage and cell death induced by PARP inhibition in tumors with homology directed repair (HDR) deficiency will enhance adaptive anti-tumor immune responses and increase sensitivity to PD-1 axis blockers. Methods: This is a randomized, open-label phase II clinical trial exploring the PARP inhibitor olaparib either alone or in combination with the anti-PD-L1 human monoclonal antibody atezolizumab in BRCA1/2 mutated locally advanced or metastatic non-HER2-positive breast cancer. HDR deficiency is defined as the presence of deleterious BRCA 1/2 mutations. Randomization occurs in a 1:1 fashion to two arms: (1) olaparib 300 mg PO bid continuously in 21-day cycles or (2) olaparib 300 mg PO bid continuously in combination with atezolizumab 1200 IV every 3 weeks in 21-day cycles. Patients undergo baseline evaluations and pre-treatment biopsy within 2 weeks of starting therapy. Repeat biopsies are required at the time of first tumor assessment scan (6 weeks from the start of treatment) and in the event of disease progression. Correlative studies, including detailed analysis of the genomic profile and tumor immune contexture, will be performed at each biopsy time point. The primary objective is to compare progression free survival between the study arms. If progression occurs on the olaparib monotherapy arm, cross-over to the combination arm is allowed. This study began enrolling in August 2018; 47 of the planned 72 patients have been registered. Clinical trial information: NCT02849496 .

1031 Background: ROS1 is an important proto-oncogene involved in the development of various cancers for which we have FDA approved therapies. While activation of the ROS1 tyrosine kinase receptor has been reported in 1-2% of lung cancers, the frequency and type of ROS1 alterations in breast cancer have not been fully explored. We previously described the incidence of ROS1 alterations from breast cancer tissue. The purpose of this study was to identify the incidence of ROS1 genomic alterations occurring in cfDNA from patients with breast cancer. Methods: We queried 16,053 breast cancer samples from the Guardant Health breast cancer database between June 2015 - October 2019 to identify the incidence of ROS1 alterations detected in cfDNA in breast cancer. We identified fusion partner genes and classified each alteration type into the following categories: fusion, single nucleotide variants (SNVs), and indels. Radical amino acid changes occurring at conserved regions across the ROS1 gene were identified. In vitro analyses were used to investigate the effect of ROS1 nonsynonymous mutations on the ROS1 protein. We made associations with ROS1 alterations and co-occurring mutated genes. Results: Nonsynonymous ROS1 alterations from the Guardant Health breast cancer database were found in 162 samples from 142 patients in the 16,053-patient cohort (1%). Alterations found included: 1 (0.6%) ROS1-SLC35F1 fusion, 155 (95.7%) SNVs, and 6 (3.7%) indels. Of the 155 SNVs, we identified 23 (14.8%) mutations occurring in the ROS1 kinase, of which, 20 (12.9%) occurred at highly conserved regions and 15 (9.6%) harbored radical amino acid changes. The top 5 co-occurring mutations in samples with ROS1 alterations were TP53 (50%), PIK3CA (44%), ESR1 (27%), EGFR (21%), and FGFR1 (18%). Conclusions: A modest incidence of ROS1 genomic alterations occurs in cfDNA from patients with breast cancer. New somatic alterations in the ROS1 gene were identified from Guardant Health that were not detected in publicly available databases. A portion of mutations occurred at highly conserved regions across the ROS1 gene suggesting these may be more actionable than currently recognized. In vitro analyses of ROS1 gene activation from these newly discovered somatic alterations are being investigated with results to be reported. Co-occurring mutations reveal a unique genotype associated with ROS1 alterations that may play a biologic role in ROS1-mediated pathogenesis.

e12641Background: Breast cancers thrive in an immunosuppressive microenvironment. Checkpoint blockade has had poor results in metastatic breast cancers. Recombinant oncolytic polio:rhinovirus (PVSR...

585Background: Increased tumor infiltrating lymphocytes (TILs) are predictive and prognostic for improved outcomes in breast cancers. Increased tumor mutational burden can be immune activating. BRC...

532 Background: Metaplastic breast cancer (MBC) is a rare, aggressive variant that is often triple negative (TN). Current guidelines recommend use of standard receptor-based treatment for MBC despite evidence of chemoresistance. We sought to compare treatment patterns and outcomes of MBC and non-MBC. Methods: Women age > 18 with stage I-III MBC and non-MBC histology diagnosed from 2010-2013 were identified in the National Cancer Database. Kaplan Meier and multivariate Cox proportional hazards models were used to estimate MBC association with overall survival (OS). Subgroup analyses were conducted for (1) MBC patients only and (2) TN MBC and TN non-MBC patients. Results: 2451 MBC and 568,057 non-MBC patients were included. 70.3% of MBC were TN vs 11.3% of non-MBC (p < 0.001). 19.2% of MBC were luminal (i.e., ER+ and/or PR+, and HER2-). MBC presented with higher clinical T stage (cT4: 5.4% vs 1.8%) and grade (grade 3: 72.1% vs 29.7%) but was less frequently node-positive (19.1% vs 29.7%, all p < 0.001). A higher proportion of MBC patients were treated with mastectomy (59.0% vs 44.9%), axillary dissection (ALND, 35.2% vs 32.2%), and chemotherapy (74.1% vs 43.1%, all p≤0.001). 5-year OS was reduced among MBC vs non-MBC patients for both the entire cohort (72.7% vs 87.5%) and the TN-only analysis (71.1% vs 77.8%, both log-rank p < 0.001). Among MBC cases, TN subtype was not associated with worse OS than the luminal subtype (HR 1.16, p = 0.28). Chemotherapy (HR 0.69, p = 0.004) and/or radiotherapy (HR 0.52, p < 0.001) improved OS in MBC, and the proportional benefit of chemotherapy did not vary with pathological T or N stage (interaction p > 0.05 for both). Among TN patients, a higher proportion of TN MBC patients underwent mastectomy (58.4% vs 49.5%, p < 0.001), but in contrast to the full cohort, a lower proportion of TN MBC patients received chemotherapy (76.6% vs. 78.7%, p = 0.008) and ALND (35.2% vs. 38.2%, p = 0.01) vs TN non-MBC patients. Conclusions: MBC had worse OS vs non-MBC, and unlike other histologies, outcome was not driven by receptor status. Multimodal therapy improved outcomes. Further investigation into MBC tumor biology and the development of MBC-specific guidelines could potentially improve treatment standardization and outcomes.

1570 Background: Metaplastic breast cancer (MBC) is a rare, aggressive, sarcomatoid breast cancer that was first described in 1973 but only became recognized as a histologically distinct entity in 2000. Given the paucity of data on the epidemiology of MBC, we performed a population-based analysis to delineate sociodemographic and clinicopathological characteristics associated with increased likelihood of MBC diagnosis. Methods: Adult female breast cancer patients with stage I-III MBC and non-MBC histology diagnosed between 2010 and 2013 were identified in the National Cancer Database (NCDB). Multivariate logistic regression was used to identify factors associated with diagnosis of MBC, and Cox proportional hazards modeling was used to estimate the effect of MBC on overall survival. Results: 2,451 MBC and 568,057 non-MBC patients were identified. After adjusting for receptor status (ER, PR, HER2), age, stage, grade, and treatment variables, MBC patients had worse survival than non-MBC patients (HR 1.45, p < 0.001). Compared to non-MBC patients, a higher proportion of MBC patients were non-Hispanic black (16.7% vs 10.5%), had an annual income < $35k (29.0% vs 25.5%), had lower high school completion rates (36.7% vs 33.9%), were treated at academic centers (35.5% vs 30.8%), and had government-sponsored insurance (48.8% vs 43.7%, all p < 0.01). MBC diagnosis was more likely in patients with triple-negative breast cancer (OR 20.71), higher clinical T stage (cT4 vs cT1: OR 6.18), and lower clinical N stage (cN1 vs cN0: OR 0.38, all p < 0.001). MBC patients were also more likely to be diagnosed based on pathology from their first operation rather than preoperatively (OR 1.41, p < 0.001). Conclusions: Black women and women of low socioeconomic status were at increased risk for diagnosis with MBC. Though MBC was more likely to be treated at academic centers, MBC was less likely to be diagnosed prior to surgical intervention. Many of the sociodemographic factors associated with MBC have also been associated with triple-negative breast cancer. Additional research is needed to determine the contribution of sociodemographic factors to the epidemiology of MBC independent of receptor status.

602Background: Increased stromal tumor infiltrating lymphocytes (STILs) may predict for pathologic complete response (pCR) in HER2+ breast cancer patients treated with neoadjuvant trastuzumab (H). Taxanes (T) and carboplatin (C) have immune modulating effects. Antibody-dependent cytotoxicity is a major mechanism of action of H, which may be enhanced by T, C, and pertuzumab (P). Pre and posttreatment tumor microenvironment immune markers in subjects treated with neoadjuvant HER2-targeted regimens have not been thoroughly described. This study quantifies baseline and changes in STILs, CD4+, CD8+, FoxP3+, and PD-L1+ cells in thirty HER2+ patients receiving neoadjuvant H-P regimens. Methods: Thirty patients with pre and post neoadjuvant TCH (n = 4) or TCHP (n = 26) tissue available were identified. STILs were measured manually by H&E. CD4, CD8, FoxP3, and PD-L1 were stained by immunohistochemistry (IHC). Immune infiltrates were compared between the pCR and non-pCR groups using the Wilcoxon rank sums test. The...

7096 Background: Measurement of tumor response is challenging in thymic malignancies especially when the pleura is involved as it often is in stage IV disease. In this study we aimed to determine t...

7626 Background: S is an oral multikinase inhibitor that targets the Ras/Raf/MEK/ERK pathway at the level of Raf kinase. At present there are no predictive biomarkers to improve patient selection a...