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Background: Prior studies in breast cancer (BC) have demonstrated variability in the immune microenvironment of BCs across subtypes, with higher levels of immune infiltration observed in triple-negative (TN) BC. Clinical trials of immunotherapy in BC have largely targeted patients with TNBC, however many of these patients do not respond to treatment, suggesting heterogeneity across TN tumors. Further, immune infiltrated hormone receptor–positive, HER2-negative (HR+/HER2-) tumors have been observed. Patients with these tumors may benefit from immune checkpoint blockade (ICB). Identification of patients with immunogenic tumors would be valuable for future patient selection and stratification in clinical trials. In this study, we conducted comprehensive molecular profiling of a set of commercially procured BC samples to characterize the tumor microenvironment (TME) of HR+/HER2- and TN tumors and to identify patient or tumor features associated with immune infiltration. Methods: 163 surgically resected BC samples were used for this study (93 HR+/HER2-, 70 TN). Clinically validated immunohistochemistry (IHC) assays were used to evaluate the expression of two immune checkpoint (IC) molecules, PD-L1 (by VENTANA PD-L1 (SP142) Assay and Agilent PD-L1 IHC 28-8 pharmDx) and LAG3 (antibody clone 17B4). The presence of CD8+ cells was measured by IHC (Dako/Agilent clone C8/144B) and used to derive the spatial location and density (topology) of CD8+ cells within the TME. RNA sequencing (RNA-seq) was used to construct gene expression signatures representative of immune cell types. Furthermore, RNAseq data was used to identify expressed somatic mutations and to calculate the total expressed tumor mutational burden (‘eTMB’). Results: As previously reported, the prevalence of PD-L1 + tumors was higher in TN vs HR+/HER2− BC, however close to half (44.1% SP142 IC> 1%) of HR+/HER2- tumors showed some degree of PD-L1 expression. Similar patterns were observed for LAG3 IHC expression (32.3% and 64.7% of HR+ and TN tumors respectively with >1% expression). Concordant expression of the two markers (SP142 IC> 1% and LAG3 >=1% or < 1% and < 1%) was observed in approximately 80% of tumors in both subtypes. Less than 10% of tumors in both subtypes had high LAG3 (>1%) and low PD-L1(< 1%) expression. Most (82.4%) HR+/HER2- tumors in the dataset were deficient in CD8+ cells in both stroma and tumor parenchyma, however a subset (12.9%) were identified as having an ‘excluded’ CD8 pattern, and four (4.7%) were classified as ‘inflamed’. The frequency of both the excluded and inflamed states was slightly higher among the TN tumors (20.6% and 15.9% respectively). Expression of PD-L1+ occurred more frequently among tumors with higher CD8 expression. Among both TN and HR+ tumors, most ‘inflamed’ tumors were grade 3. We also examined a range of gene expression signatures representative of various immune cell subpopulations, their distribution within each subtype, and relationships with other molecular data. For example, expression of a CXCL13+ T cell signature, a cell type that has been shown to be predictive of response to anti-PD-L1 + chemo combo therapy in TNBC, was significantly higher in TN vs HR+ tumors, however, a large subset of HR+ tumors had levels of expression of the signature above the median observed in TN tumors. In addition, we noted that eTMB, while low overall, was positively correlated with certain immune-related expression signatures across HR+/HER2− samples, including signatures previously associated with improved response to ICB, as well as PD-L1 positivity. Conclusions: We observed heterogeneity in the immune profile of the real-world HR+ and TN tumors in this cohort. A subset of tumors in both subtypes expressed markers or signatures previously reported to be associated with response to ICB. (1) Cancer Cell. 2021 Dec 13;39(12):1578-1593.e8. Citation Format: Aparna Chhibber, Lloye M. Dillon, John B. Wojcik, Vishantie Dostal, George Lee, Fayaz Seifuddin, Scott Ely, Mark D. Stern, Charlie Benson-Garnett, Mustimbo Roberts, Jenny Wu. Characterization of Immune Contexture in HR+/HER2- and Triple Negative Breast Cancer in a Real-world Cohort [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-07.

Tumor-derived Copy Number Variations (CNVs) are among the most important genomic aberrations in cancers. Since a CNV amplification is often associated with oncogene activation, and a CNV deletion is frequently attributed to the gene inactivation, an accurate detection of CNVs plays a crucial role in cancer diagnosis and treatment. In this study, using the next generation sequencing, we systematically interrogated somatic CNVs in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy. Whole-genome and whole-exome sequencing were carried out at six sequencing centers followed by processing with six CNV callers to evaluate their reproducibility. Different types of samples with varying input amount and tumor purity were processed using multiple library construction protocols and read depths. We compared the consistency of reference replicates, the effect of experimental, technical, and biological confounding factors, and benchmarked our callers using cytogenetic array. Our evaluation was based on two approaches; bin-based and gene-based approach. The bin-based comparison used a sliding window of 10 Kb, while gene-based comparison used segments at the gene level. Our evaluations indicate variations among CNV calls are mainly driven by callers and less by the sequencing centers. In addition to caller effect, PCA analyses also show site-specific effect, largely pairwise distances on the PC plot for pairs between different centers than for those within the same centers. Among confounding factors, namely FFPE, library concentration, and tumor purity with several sequencing depths, we report that tumor purity has a dominant and major effect in caller’s performance. PCA analysis shows low tumor purity ( Citation Format: Yun-Ching Chen, Fayaz Seifuddin, Cu Nguyen, Chunhua Yan, Qingrong Chen, Wenming Xiao, Mehdi Pirooznia, Daoud Meerzaman. Critical Assessment of CNV Calling Using Next Generation Sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3521.