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Background Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors.Methods We retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers.Results We observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations in TP53, PLXNB3, Zinc finger genes, and fewer mutations in SDC2, CDH1, PIK3CA, SVIL, and PTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA-protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, including KIAA1522, DCK, FOXO3, MYO9B, ARID1A, EPRS, ZC3HAV1, and RBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors.Conclusions This study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

HIV-specific antibodies (Abs) may contribute to the elimination of HIV reservoirs by binding to reactivated cells and targeting them for antibody-dependent cell-mediated cytotoxicity (ADCC) (1, 2). Broadly neutralizing antibodies (bNAbs) may be particularly efficacious, by mediating virus neutralization alongside ADCC for multiple HIV strains (in the context of untreated simian-human immunodeficiency virus [SHIV] infection, neutralization rather than ADCC appears to dominate the antiviral effect of bNAb infusion [3, 4]). Few studies have assessed the activities of bNAbs against reservoir-derived viruses. The relationships between neutralizing activity, ADCC function, and binding to reservoir virus in infected primary CD4+ T cells have not been comprehensively studied. In a study previously published by Ren et al. (5), we tested a panel of 14 bNAbs against 36 reservoir-derived viral isolates, demonstrating that susceptibility to neutralization by broadly neutralizing antibodies generally correlated with infected-cell binding for a panel of clade B HIV reactivated from latent reservoirs.

Abstract Introduction: Breast cancer (BC) is the second most common cause of cancer death among women in the USA. Recent proteogenomic studies of human BC have identified many potential therapeutic biomarkers for the most common types of BC. Here, we strive to comprehensively understand the proteogenomic landscape of difficult-to-treat breast cancer (DTBC) tumors. Methods: 118 primary breast tumors, with a focus on DTBC patients, were selected from the IRB-approved protocol. The study cohort included 30 triple negative, 16 HER2+, 16 Luminal A, 39 Luminal B1, and 17 Luminal B2 selected by immunohistochemistry. Breast tumors were embedded in OCT (Optimum Cutting Temperature) and processed by laser microdissection (LMD) to remove non-tumorous tissue. DNA, RNA, and protein were simultaneously extracted from each tumor using the illustra triplePrep kit. Paired-end RNA sequencing and whole genome sequencing (WGS) were performed for 118 and 100 tumors, respectively, using the Illumina HiSeq platform. Quantitative global proteomics and phosphoproteomic analyses were performed on 113 and 50 tumors, respectively, using isobaric TMT 6-plex labeling with the “universal reference” strategy. Results: Unlike other BC cohorts, our WGS cohort had an almost equal number of Estrogen(ER)+ (55.6%) and ER- (44.4%) cases, which gave us a nominally higher percentage TP53 (57%) and lower PIK3CA (32%) non-silent somatic mutation. Similar to previous reports, GATA3 was mutated in 13% of only ER+ cases. Unlike a recent report on proteomics clustering of bulk-processed tumors, our data show that a stromal-enriched cluster was not mixed with all subtypes but a majority of Luminal A (LumA) subtype, probably because LMD excluded stromal components in other tumor subtypes. Consensus clustering of proteomics data separated intrinsic Her2 patients with outcome differences. Her2 patients associated with the Basal-enriched proteome cluster had better survival than those associated with the LumA-enriched proteome cluster. Previously reported marker PGK1, a predictor for poor survival in BC, was upregulated in Her2 tumors associated with the LumA-enriched proteome cluster. The clustering of phosphoproteomics data separated intrinsic Basal patients into groups with and without relapse. We identified 3 and 18 genes with upregulated kinase activities in the relapse-free and relapsed Basal patients, respectively. In conclusion, this study provides a high-quality proteogenomic resource for DTBC investigation and identifies potential molecular markers for predicting outcomes for patients with less responsive tumors. The views expressed in this abstract are solely of the authors and do not reflect the official policy of the Departments of Army/Navy/Air Force, DoD, USUHS, HJF. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Praveen-Kumar Raj-Kumar, Tao Liu, Lori A. Sturtz, Albert J. Kovatich, Marina A. Gritsenko, Vladislav A. Petyuk, Brenda Deyarmin, Jianfang Liu, Anupama Praveen-Kumar, Jason E. McDermott, Anil K. Shukla, Ronald J. Moore, Matthew E. Monroe, Bobbie-Jo M. Webb-Robertson, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Leonid Kvecher, Jennifer Kane, Jennifer Melley, Stella Somiari, Stephen C. Benz, Justin Golovato, Shahrooz Rabizadeh, Patrick Soon-Shiong, Richard D. Smith, Richard J. Mural, Karin D. Rodland, Craig D. Shriver, Zhaoyu Li, Hai Hu. Proteogenomics of laser microdissected difficult-to-treat breast cancers identifies Basal and Her2 patients associated with outcome differences [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2172.

Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.

Clinical successes utilizing checkpoint inhibitors demonstrate the potential of immune-based therapies for the treatment of cancer. Research suggests that these successes are a result of T cells recognizing nonsynonymous mutations uniquely expressed by a patient’s tumor. The advent of comparative tumor-normal DNA sequencing and tumor RNA sequencing for accurate prediction of presented somatic mutations has made it possible to rapidly identify these neoepitopes, allowing for the development of personalized cancer vaccines. However, such vaccines are often ineffective due to the immunosuppressive nature of the tumor microenvironment, and their clinical efficacies would be improved when used in an orchestrated combination approach with additional immune-oncology agents. In the present study, we investigated the combination of three immune-oncology agents, each targeting a unique mechanism of immune therapy, with a vaccine targeting tumor neoepitopes. We combined a neoepitope vaccine, delivered as a peptide or via a novel E2b-deleted adenovirus vehicle, with N-IL15 (previously called ALT-803), an IL-15 superagonist, to enhance the development of antigen-specific immunity; NHS-IL12, a tumor-targeting IL-12 molecule, to promote the maintenance and expansion of T cells within the tumor microenvironment; and PD-L1 blockade to decrease immune cell exhaustion within the tumor microenvironment. This combination therapy resulted in the regression of established tumors, which associated with the generation of robust immunity against neoepitopes contained in the vaccine. It also showed evidence of inducing a neoepitope cascade by causing development of T-cell responses to neoepitopes expressed by the tumor but not contained within the vaccine. This report is the first demonstration of an E2b-deleted adenovirus neoantigen vaccine generating robust immunity. Together, this study demonstrates the importance of a multifaceted, orchestrated treatment regimen to promote the generation of effective antitumor immunity. This abstract is also being presented as Poster A18. Citation Format: Karin L. Lee, Claudia Palena, John Lee, Kayvan Niazi, Andrew Nguyen, Frank Jones, Shahrooz Rabizadeh, Patrick Soon-Shiong, Jeffrey Schlom, Duane H. Hamilton. A novel combination immunotherapy with a vaccine targeting tumor neoepitopes that mediates immune cascade in murine tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR20.