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Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal form of primary breast cancer. Inflammatory signaling pathways are active in IBC, but the function of the immune response remains elusive. ErbB receptors play a role in IBC. ERBB2 is amplified in 50% of IBCs, and ERBB3 is also mutated in IBC. Currently, Lapatinib, a dual ErbB inhibitor, is used in IBC patients with ERBB2 amplification. Although immune cell inflammatory signaling may promote IBC tumor growth and metastasis, the presence of cytotoxic tumor-associated lymphocytes has also been associated with a more favorable breast cancer prognosis. The contradictory nature of the immune data highlights the need to elucidate the relationship between the immune response and subsequent treatments. Given the active IBC immune component and the recent clinical benefit of immune checkpoint inhibitors in cancer treatment, IBC’s may represent a clinically unique breast cancer population that may benefit from immunomodulating agents. Interestingly, Myc is a downstream effector of ErbB signaling, and Myc is thought to regulate the expression of immune checkpoint proteins CD47 and PD-L1. The presence of PD-L1-positive IBC immune infiltrate suggests that IBC’s may benefit from therapies that disrupt PD-L1 signaling together with ErbB inhibitors. METHODS: IHC was used to examine immune checkpoint signaling and characterize the tumor-immune infiltrate. Tumor tissues were also characterized using an RNA-seq panel that examined the expression of 377 immune-related genes. Cell lines (BT474, SKR3, AU565, and SUM225) were treated with Lapatinib and Neratinib to examine the relationship between growth factor receptors and downstream immune signaling pathways. RESULTS: RNA-seq revealed the expression of specific immunosuppressive signaling pathways in tumors. Treatment of breast cancer cells with ErbB inhibitors resulted in a decrease in the levels of PD-L1. Treatment with Lapatinib and Neratinib diminished PD-L1 in all cell lines. Levels of Phospho-Stat3 decreased in BT474 and Sum225 but not in SKBR3 and AU565, implying that PD-L1 is regulated by another mechanism (ERK-MYC). CONCLUSION: Our results provide mechanistic insight into ErbB receptor activation and the expression of downstream signaling molecules (Stat3, Myc, PD-L1, and others). In addition, our unique RNA-seq immune signature reveals the expression of several genes that may serve as biomarkers of inhibitory immune signaling pathways. Our laboratory is currently examining the correlation between PD-L1 expression and the activation of ErbB2 in model systems and clinical trials using ErbB inhibitors in combination with PD-L1 inhibitors. Our immune panel gene signature may serve as a useful diagnostic test that, in conjunction with traditional ErbB testing, can identify patients that will benefit from combination therapy of an ErbB inhibitor and an immune checkpoint inhibitor. Note: This abstract was not presented at the meeting. Citation Format: Christopher A. Hamm, Sumin Zhao, Cesar A. Santa-Maria, Massimo Cristofanilli, Neil L. Spector, Sarah Bacus. Targeted therapies to ERBB receptors downregulate expression of PD-L1: implications in combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2636. doi:10.1158/1538-7445.AM2017-2636

Background Inflammatory breast Cancer (IBC) is a rare aggressive breast cancer in which cancer cells block the lymph vessels in the skin of the breast. IBC tumors have a high rate of ERBB2 positivity and response to ERBB2 targeted therapies. Due to the rarity of this cancer, the molecular etiology of this disease is poorly understood. Materials & Methods A 208 gene next-generation sequencing (NGS) panel was used to analyze 20 IBC patient tumor and matched normal samples. Tissues were stained for cell signaling proteins and immune-markers. Cell line studies were performed to understand the impact of genomic variants on therapeutic selection. Results Common pathway alterations reoccur among IBC samples that influence genome stability, PI3K signaling, and ERBB signaling. NGS revealed alterations in both the ERBB/PI3K pathways, including: ERBB2 amplification, ERBB3 mutations, and activating PI3K mutations. Immunohistochemistry (IHC) staining for pS6 and pERBB3 identified ERBB/PI3K pathway activity in IBC samples. Cell line studies using siRNA and neutralizing antibodies demonstrate that mutant ERBB3 signaling contributes to IBC proliferation. IBC tumors with high levels of CD8+ immune infiltrate have a significantly higher somatic mutation rate than other IBC tumors. A proposed score (iScore) based on the somatic mutation rate and the average mutant allele frequency, showed greater correlation with the level of CD8+ immune infiltration. Furthermore, tumors with high CD8+ infiltrating lymphocytes were associated with a higher frequency of alterations in DNA mismatch repair (MMR) genes. IHC revealed high levels of the immune checkpoint signaling molecule PD-L1 in the inflammatory infiltrate of IBC tumors. Conclusions This study identifies a higher level of ERBB3 mutations than reported in other cancers and an important role for ERBB3 mutation in IBC. ERBB3 targeted therapies in combination with ERBB/PI3K drugs may be important for IBC treatment. Furthermore, high somatic mutations rates in a subset of IBC tumors harboring MMR mutations lead to greater levels of PD-L1+ immune infiltrates, which suggests a possible benefit from immunotherapies such as anti-PD-L1 antibodies. iScore, a more predictive value of immune infiltration in tumors, may be indicative of the level of neoantigen exposure to the immune system. The utility of the newly proposed iScore is currently being investigated as a method to predict immune cell infiltrates and immunotherapy response in other tumor types. Citation Format: Diarmuid Moran, Christopher A. Hamm, Kakuturu Rao, Patricia Bacon-Trusk, Karen Pry, Victor Velculescu, Massimo Cristofanilli, Sarah S. Bacus. Genomic analysis identifies drug targetable pathways and predicts immune infiltration in inflammatory breast cancer tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3887. doi:10.1158/1538-7445.AM2015-3887

Ependymoma is a common pediatric central nervous system (CNS) tumor that is believed to originate from ependymal cells located in the lining of ventricular surfaces in the brain. Ependymoma presents great challenges in treatment despite the advances in neurosurgical techniques and adjuvant therapy. To uncover protein interaction networks that are consistently compromised in pediatric ependymomas, we conducted a computational analysis of the array CGH data derived from 203 primary ependymomas from the St. Jude Children's Research Hospital cohort, published by Johnson and colleagues [Nature, 466(7306):632-6, 2010]. The analysis (Bischof and Soares, manuscript in preparation) revealed 15 regions of copy number variation (CNVs) that occurred in at least 50% of the tumors. Peak genes from these CNVs were selected for a confirmatory study using DNA digital PCR assays with an independent ependymoma cohort from the Ann & Robert H Lurie Children's Hospital of Chicago. In this study, all ependymomas displayed copy number alterations in at least one of the 10 genes tested by digital PCR. The digital PCR data sheds light into the biology of ependymomas as the 10 candidate genes have a wide range of cellular functions; from neural stem cell development, to tumor suppression, to the pathogenesis of glioblastoma. In conclusion, we have confirmed that the occurrence of DNA copy number variation in these 10 genes prevail in ependymomas. Indeed, all tumors exhibited at least one of these CNVs. These alterations provide insights into the protein interaction networks underlying development of ependymomas while uncovering targets that may be exploited for therapeutic intervention. Citation Format: Christopher A. Hamm, Fabricio F. Costa, Jared M. Bischof, Elio F. Vanin, Maria de Fatima Bonaldo, Steve Iannaccone, Veena Rajaram, David George, Tadanori Tomita, Stewart Goldman, Lawrence J. Jennings, Richard J. Gilbertson, Marcelo B. Soares. Identification of genes that frequently exhibit copy number alterations in pediatric ependymomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3111. doi:10.1158/1538-7445.AM2014-3111