Primary Triple-negative Breast Cancer Tumors Research Articles

Abstract PO2-13-04: Single cell characterization of senescent CD8+ T cell promotes immunotherapy resistance in early-stage triple-negative breast cancer

Abstract Background: Immune checkpoint blockade (ICB) has been explored in triple-negative breast cancer (TNBC) and has been proven to improve patient survival in advanced TNBCs (IMpassion130) or increase the pathological complete response rate in early TNBCs (KEYNOTE522). However, ICB could only benefit a subset of patients, highlighting the urgent need to optimize patient selection. Unlike in advanced TNBCs, a predictive biomarker for ICB in early TNBCs is still lacking. The main reason might be that previous studies neglected the heterogeneity of tumor ecosystems between early and advanced TNBCs. There is an urgent need to understand the unique ecosystem of early-stage TNBC and develop biomarkers for precision immunotherapy. Objective: This study aims to characterize the micro-loop associated with the immune efficacy of early TNBC, so as to explain the mechanisms behind the differences in early and late efficacy markers, identify efficacy markers in early TNBC immunotherapy, and search for clinical targeting strategies to sensitize TNBC for neoadjuvant therapy. Methods: We conducted an in-depth analysis of 32 single-cell transcriptomes, comprising 26 early-stage TNBC enrolled in prospective clinical trials (NCT04418154 and NCT04613674), receiving neoadjuvant chemotherapy alone (n=8) or combined with anti-PD1 (n=18), and 6 advanced TNBC. We analyzed the microenvironmental differences between early and advanced tumors, and between different treatment response groups in early stages, and identified a subpopulation of cells specific to early stages and associated with immunotherapeutic efficacy. We used mIHC, flow-through, and validated the presence of this cell in clinical samples. We also explored its predictive performance on four independent cohorts of TNBCs with ICB treatment (GSE194040, NCT04129996, GSE169246, GSE124821). Furthermore, to understand the impact of ISG+ T cells on immunotherapy efficacy and explore targeting strategies, we employed a multi-faceted approach to assess their tumor-killing ability, response to Anti-PD1 treatment, and sensitization effect of drugs targeting ISG+ T cells. ISG+ T cells were sorted using flow cytometry for further experiment. In vitro, we established a co-culture model of primary TNBC patients-derived organoid (PDO) and tumor infiltrated CD8+T cells and evaluated the tumor cell viability as well as T cell cytotoxicity. In vivo, we established TS/A tumor cell bearing BalB/c mouse and E0771 tumor cell-bearing C57BL mouse and measured tumor size and tumor microenvironment by flow cytometry. Results: We obtained 236,017 high-quality cells and identified cell subsets across 32 samples with different stages, treatments and responses. We then uncovered an enriched subset of interferon-induced (ISG+) CD8+ T cells specific to early TNBC, serving as a predictive factor for ICB resistance. Mechanistically, the upregulation of the IFN pathway in early-stage TNBC triggers a transition in ISG+ CD8+ T cells, resulting in cellular senescence, diminished cytotoxicity, and impaired response to ICB. Encouragingly, we made a noteworthy discovery that Nicotinamide mononucleotide, an anti-aging drug, effectively restores the functionality of ISG+ CD8+ T cells, amplifying immunotherapy sensitivity. Conclusions: This study provided a comprehensive single-cell transcriptome atlas of early-stage TNBC and depicted stage-related ISG+CD8+ T cell for predicting ICB response in early TNBC. We emphasize the critical role of T cell dysfunction prompted by cellular senescence in driving ICB resistance during the early stages of TNBC and propose an approach for sensitizing immunotherapy with aging drugs. Citation Format: Tong Fu, Yi-Yu Chen, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. Single cell characterization of senescent CD8+ T cell promotes immunotherapy resistance in early-stage triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-04.

Lung-derived soluble factors support stemness/plasticity and metastatic behaviour of breast cancer cells via the FGF2-DACH1 axis.

Patients with triple-negative breast cancer (TNBC) have an increased propensity to develop lung metastasis. Our previous studies demonstrated that stem-like ALDHhiCD44+ breast cancer cells interact with lung-derived soluble factors, resulting in enhanced migration and lung metastasis particularly in TNBC models. We have also observed that the presence of a primary TNBC tumor can 'prime' the lung microenvironment in preparation for metastasis. In this study, we hypothesized that soluble lung-derived factors secreted in the presence of a primary TNBC tumor can influence stemness/plasticity of breast cancer cells. Using an ex vivo pulmonary metastasis assay (PuMA), we observed that the lung microenvironment supports colonization and growth of ALDHhiCD44+ TNBC cells, potentially via interactions with lung-derived FGF2. Exposure of TNBC cells to lung-conditioned media (LCM) generated from mice bearing TNBC primary tumors (tbLCM) significantly enhanced the proportion of ALDHhiCD44+ cells compared to control or LCM from tumor-naïve mice (tnLCM). Further analysis using a human cancer stem cell qPCR array revealed that, relative to tnLCM or control, exposure of TNBC cells to tbLCM leads to downregulation of the transcription factor and putative tumor suppressor Dachshund homolog 1 (DACH1), a downstream regulator of FGF2. In addition, inhibition of DACH1 using siRNA or treatment with recombinant FGF2 enhanced the ALDHhiCD44+ phenotype. Taken together, our findings suggest that the FGF2-DACH1 signaling axis supports stemness/plasticity of TNBC cells in the lung microenvironment and lays the foundation for future evaluation of FGF2 as a potential novel therapeutic target for treatment or prevention of breast cancer metastasis to the lung.

Alteration of DNA methyltransferases by eribulin elicits broad DNA methylation changes with potential therapeutic implications for triple-negative breast cancer.

Background: Triple-negative breast cancer (TNBC) is an aggressive disease with limited treatment options. Eribulin, a chemotherapeutic drug, induces epigenetic changes in cancer cells, suggesting a unique mechanism of action. Materials & methods: MDA-MB 231 cells were treated with eribulin and paclitaxel, and the samples from 53 patients treated with neoadjuvant eribulin were compared with those from 14 patients who received the standard-of-care treatment using immunohistochemistry. Results: Eribulin treatment caused significant DNA methylation changes in drug-tolerant persister TNBC cells, and it also elicited changes in the expression levels of epigenetic modifiers (DNMT1, TET1, DNMT3A/B) in vitro and in primary TNBC tumors. Conclusion: These findings provide new insights into eribulin's mechanism of action and potential biomarkers for predicting TNBC treatment response.

A second-generation eIF4A RNA helicase inhibitor exploits translational reprogramming as a vulnerability in triple-negative breast cancer

In this study, we aimed to address the current limitations of therapies for macro-metastatic triple-negative breast cancer (TNBC) and provide a therapeutic lead that overcomes the high degree of heterogeneity associated with this disease. Specifically, we focused on well-documented but clinically underexploited cancer-fueling perturbations in mRNA translation as a potential therapeutic vulnerability. We therefore developed an orally bioavailable rocaglate-based molecule, MG-002, which hinders ribosome recruitment and scanning via unscheduled and non-productive RNA clamping by the eukaryotic translation initiation factor (eIF) 4A RNA helicase. We demonstrate that MG-002 potently inhibits mRNA translation and primary TNBC tumor growth without causing overt toxicity in mice. Importantly, given that metastatic spread is a major cause of mortality in TNBC, we show that MG-002 attenuates metastasis in pre-clinical models. We report on MG-002, a rocaglate that shows superior properties relative to existing eIF4A inhibitors in pre-clinical models. Our study also paves the way for future clinical trials exploring the potential of MG-002 in TNBC and other oncological indications.

Core Needle Biopsies as an Alternative Source for Ex Vivo Expanded TIL for Adoptive Cell Therapy in Triple-Negative Breast Cancer.

Adoptive transfer of ex vivo expanded tumor-infiltrating lymphocytes (TILs) have produced long-term response in metastatic cancers. TILs have traditionally been expanded from surgically resected specimens. Ultrasound-guided core needle biopsy (CNB) is an alternative method that avoids the morbidity of surgery and have added benefits which may include patients not amenable to surgery as well as the potential to produce TILs from multiple lesions in the same patient. We assessed the ability to produce and expand TILs from primary triple-negative breast cancer tumors from CNB (n=7) and demonstrate comparable expansion, phenotype and cytokine secretion after phorbol myristate acetate-ionomycin stimulation to TILs expanded from surgery (n=6). T cell Receptor clonality and diversity were also comparable between the two cohorts throughout the TIL culture. CNB is a safe and feasible method to obtain tumor tissue for TIL generation in patients with triple-negative breast cancer.

Whole-Exome Sequencing Reveals High Mutational Concordance between Primary and Matched Recurrent Triple-Negative Breast Cancers.

Triple-negative breast cancer (TNBC) is a molecularly complex and heterogeneous breast cancer subtype with distinct biological features and clinical behavior. Although TNBC is associated with an increased risk of metastasis and recurrence, the molecular mechanisms underlying TNBC metastasis remain unclear. We performed whole-exome sequencing (WES) analysis of primary TNBC and paired recurrent tumors to investigate the genetic profile of TNBC. Genomic DNA extracted from 35 formalin-fixed paraffin-embedded tissue samples from 26 TNBC patients was subjected to WES. Of these, 15 were primary tumors that did not have recurrence, and 11 were primary tumors that had recurrence (nine paired primary and recurrent tumors). Tumors were analyzed for single-nucleotide variants and insertions/deletions. The tumor mutational burden (TMB) was 7.6 variants/megabase in primary tumors that recurred (n = 9); 8.2 variants/megabase in corresponding recurrent tumors (n = 9); and 7.3 variants/megabase in primary tumors that did not recur (n = 15). MUC3A was the most frequently mutated gene in all groups. Mutations in MAP3K1 and MUC16 were more common in our dataset. No alterations in PI3KCA were detected in our dataset. We found similar mutational profiles between primary and paired recurrent tumors, suggesting that genomic features may be retained during local recurrence.

Abstract 6051: Dissecting unique metabolic vulnerabilities in triple-negative breast cancer metastasis

Abstract Introduction. Triple-negative breast cancer (TNBC) exhibits the worst outcome due to an aggressive clinical course, high rates of metastasis, and therapeutic resistance. Lack of effective FDA approved anti-metastatic targeted therapies has generated an unmet medical need for developing effective novel treatments. Targeting metastatic seeding and colonization remains a therapeutic challenge. Only recently, attention has been drawn to the idea that cancer cells selectively and dynamically adapt their metabolism along the changing microenvironments of the metastatic cascade. However, the precise nature of the metabolic reprograming in vivo is poorly understood and constitutes a major barrier in formulating a rationally designed therapeutic approach. Using a genetic lineage tracing approach, we identified a discrete subpopulation of highly metastatic SOX2/OCT4+ cells within primary tumors that exhibit enhanced invasion and metastasis, increased self-renewal, higher chromosomal instability, and marked resistance to chemotherapy. We posited that this lineage tracing system may allow assessment of precise metabolic alterations as a function of tumor progression directly in vivo. Methods and data. Transcriptomic analysis of sorted cellular fractions from progressing primary TNBC tumor identified metabolic heterogeneities between metastatic SOX2/OCT4+ and the SOX2/OCT4- counterparts. SOX2/OCT4+ metastatic cells exhibited elevated levels of mitochondrial respiration (OXPHOS) and fatty acid (FA) metabolism; a finding supported by proteomic analysis. Using a combination of CRISPR knockout of key pathway genes, seahorse metabolic flux analysis, metabolomics, and stable isotope tracing, we provide evidence for increased dependence of the metastatic SOX2/OCT4+ population on these unique metabolic adaptions. Importantly, we have identified upstream regulators of these pathways and demonstrate impact of both genetic and pharmacological inhibition of the metabolic regulatory networks on metastasis in vivo. Conclusion. By identifying metabolic adaptations unique to the metastatic compartment of the primary tumor, we provide evidence for using clinical grade pharmacological inhibitors including metformin, tetrathiomolybdate and FA pathway inhibitors like TVB-2640 and BMS-303141, as an anti-metastatic approach in TNBC. This knowledge of metabolic remodeling could therefore be employed as a next-generation therapeutic approach for high-risk TNBC patients. Citation Format: Divya Ramchandani, Liron Yoffe, Arshdeep Singh, Linda Vahdat, Vivek Mittal. Dissecting unique metabolic vulnerabilities in triple-negative breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6051.

Disruption of estrogen receptor beta's DNA binding domain impairs its tumor suppressive effects in triple negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive sub-type of the disease which accounts for a disproportionately high percentage of breast cancer morbidities and mortalities. For these reasons, a better understanding of TNBC biology is required and the development of novel therapeutic approaches are critically needed. Estrogen receptor beta (ERβ) is a reported tumor suppressor that is expressed in approximately 20% of primary TNBC tumors, where it is associated with favorable prognostic features and patient outcomes. Previous studies have shown that ERβ mediates the assembly of co-repressor complexes on DNA to inhibit the expression of multiple growth promoting genes and to suppress the ability of oncogenic transcription factors to drive cancer progression. To further elucidate the molecular mechanisms by which ERβ elicits its anti-cancer effects, we developed MDA-MB-231 cells that inducibly express a mutant form of ERβ incapable of directly binding DNA. We demonstrate that disruption of ERβ's direct interaction with DNA abolishes its ability to regulate the expression of well characterized immediate response genes and renders it unable to suppress TNBC cell proliferation. Loss of DNA binding also diminishes the ability of ERβ to suppress oncogenic NFκB signaling even though it still physically associates with NFκB and other critical co-factors. These findings enhance our understanding of how ERβ functions in this disease and provide a model system that can be utilized to further investigate the mechanistic processes by which ERβ elicits its anti-cancer effects.

Abstract 758: Differential gene expression profiling of matched primary and metastatic triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is a molecularly complex and heterogeneous subtype of breast cancer with distinct biological features and clinical behavior. TNBC is associated with increased risk of metastasis and recurrence. To identify the underlying transcriptional changes and differentially expressed genes in metastatic progression of TNBC, we compared the transcriptomic profile of primary and matched metastatic tumors using massively parallel RNA sequencing. Methods: We performed gene expression profiling using paraffin embedded formalin fixed tissues of 38 TNBC patients obtained from University Hospital Zurich, Switzerland and Stavanger University Hospital, Norway. Of these, 18 were primary TNBC tumors that did not metastasize; 20 patients metastasized to lymph nodes or to distant organs (16 paired primary and metastatic tumors). Absolute fold change of ≥ 2 and a p-value <0.05 was used as the cutoff to identify significant differentially expressed genes (DEGs). Functional analysis was performed on DEGs. We also compared gene-expression profiles for hypoxia related- and EMT-related genes between our comparison groups. CIBERSORT analysis was used to estimate relative fractions of 22 immune cell types in each RNA-seq sample. Results: We identified 2442 DEGs between primary TNBC tumors and corresponding metastatic lesions and 1561 DEGs between metastasis-free and metastatic primary tumors. Of the 2442 DEGs between primary and metastatic tumors, we found 579 genes to be upregulated and 1863 genes to be downregulated in primary tumors compared to matched metastatic tumors. A total of 530 genes were found to be upregulated and 1031 genes were downregulated in metastasis-free compared to metastatic primary TNBC tumors. Of the 27 gene hypoxia signature, only 1 gene (MPRS17) was significantly different between primary and corresponding metastatic tumors and 3 hypoxia-related genes (KCTD11, FOSL1 and SDC1) showed significant differences between primary tumors with and without metastasis. We found expression for 1 EMT related gene (TWIST1) to be different between primary and matched metastatic tumors whereas 4 EMT related genes (CDH2, VIM, SNAI1 and FOXC2) were found to be significantly different between primary tumors that metastasized compared to primary tumors that remained metastasis-free. The relative fractions of 3 immune cell types, T cells CD4 memory resting, M2 macrophages and M0 macrophage were similar between the primary tumor and their matched metastatic lesions. Conclusions: Our results show that distinct patterns of gene expression exist between primary TNBCs and matched metastatic tumors. Further studies are warranted to explore whether these discrete expression profiles underlie or derive from disease status. Citation Format: Jaspreet Kaur. Differential gene expression profiling of matched primary and metastatic triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 758.

Distinct Gene Expression Profiles of Matched Primary and Metastatic Triple-Negative Breast Cancers.

Simple SummaryTriple Negative Breast Cancer (TNBC) is a molecularly complex and heterogeneous subtype of breast cancer, characterized by the lack of expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. TNBCs are often associated with an increased risk of metastasis and recurrence, however, the molecular mechanisms underlying TNBC metastasis and recurrence remail unclear. In this study, we present our findings of massively parallel RNA sequencing used to compare global gene expression profiles of primary tumors and their matched metastatic lesions. Our results shed light on the diverse genetic mechanisms underlying TNBC metastases and may provide potentially actionable therapeutic targets. Background: Although triple-negative breast cancer (TNBC) is associated with an increased risk of recurrence and metastasis, the molecular mechanisms underlying metastasis in TNBC remain unknown. To identify transcriptional changes and genes regulating metastatic progression in TNBC, we compared the transcriptomic profiles of primary and matched metastatic tumors using massively parallel RNA sequencing. Methods: We performed gene expression profiling using formalin-fixed paraffin-embedded (FFPE) TNBC tissues of patients from two cohorts: the Zurich cohort (n = 31) and the Stavanger cohort (n = 5). Among the 31 patients in the Zurich cohort, 18 had primary TNBC tumors that did not metastasize, and 13 had primary tumors that metastasized (11 paired primary and locoregional recurrences). The Stavanger cohort included five matched primary and metastatic TNBC tumors. Significantly differentially expressed genes (DEGs; absolute fold change ≥2, p < 0.05) were identified and subjected to functional analyses. We investigated if there was any overlap between DEGs from both the cohorts with epithelial-to-mesenchymal-to-amoeboid transition (EMAT) gene signature. xCell was used to estimate relative fractions of 64 immune and stromal cell types in each RNA-seq sample. Results: In the Zurich cohort, we identified 1624 DEGs between primary TNBC tumors and matched metastatic lesions. xCell analysis revealed a significantly higher immune scores for metastatic lesions compared to paired primary tumors in the Zurich cohort. We also found significant upregulation of three MammaPrint signature genes (HRASLS, TGFB3 and RASSF7) in primary tumors that metastasized compared to primary tumors that remained metastasis-free. In the Stavanger cohort, we identified 818 DEGs between primary tumors and matched metastatic lesions. No significant differences in xCell immune scores were observed. We found that 21 and 14 DEGs from Zurich and Stavanger cohort, respectively, overlapped with the EMAT gene signature. In both cohorts, genes belonging to the MMP, FGF, and PDGFR families were upregulated in primary tumors compared to matched metastatic lesions. Conclusions: Our results suggest that distinct gene expression patterns exist between primary TNBCs and matched metastatic tumors. Further studies are warranted to explore whether these discrete expression profiles underlie or result from disease status.

Abstract P3-09-14: Whole exome sequencing of matched primary and metastatic triple-negative breast cancer samples

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer subtype with distinct biological features and clinical behavior. TNBC is associated with an increased risk of metastasis and recurrence. In this whole exome study, we investigated the genetic profiles of primary TNBC tumors and paired metastases using next-generation sequencing (NGS). Methods: Genomic DNA extracted from 35 paraffin-embedded formalin-fixed (FFPE) tissues (15 primary tumors that did not metastasize, 11 primary tumors that metastasized, and nine paired metastatic tumors to the lymph nodes) was analyzed by whole exome sequencing (WES). Reads were trimmed using Trim-Galore and were aligned to the reference genome GRCh38 using Burrows-Wheeler Aligner (BWA). BAM files were preprocessed to optimize variant calling, and variant calling was carried out using the GATK pipeline. Variants were annotated using SnpEff. Tumors were analyzed for single nucleotide variants (SNV), point mutations, and insertions/deletions (indels). Results: Primary tumors that did not metastasize had a higher number of variants (~13,500) than primary tumors that metastasized (~12,900). However, the number of variants was similar between the primary tumors (~12,900 variants) and their matched metastases (~12,500 variants). MUC3A was the top mutated gene both in primary tumors that did not metastasize and in those that metastasized. MUC3A was also the top mutated gene in matched metastatic lesions. Moreover, we compared the mutational status of the most frequently mutated genes in TNBC samples from the TCGA and METABRIC datasets. We found that in our dataset, TP53, MAP3K1, and PTEN were mutated in 60%, 93%, and 7% of primary TNBC tumors without metastases and in 36%, 0%, and 36% of primary TNBC tumors that metastasized. Mutations in TP53 and PTEN were found in 2/9 and 1/9 of primary and metastases pairs, respectively. No mutations in PI3KCA were observed in any of the primary or matched metastatic tumors. Conclusions: We used WES to compare the genomic landscapes of primary TNBC tumors and matched metastatic tumors, as well as of primary TNBC tumors that metastasized and those that did not metastasize. We found very similar genomic alterations between the primary and paired metastatic tumors, indicating that genomic features may be retained during metastasis. Primary tumors that did not metastasize showed a greater extent of genomic alterations than primary tumors that metastasized. Citation Format: Jaspreet Kaur, Darshan S. Chandrashekar, Zsuzsanna Varga, Emiel Janssen, Khanjan Gandhi, Karuna Mittal, Umay Kiraz, Sooryanarayana Varambally, Ritu Aneja. Whole exome sequencing of matched primary and metastatic triple-negative breast cancer samples [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-14.

Abstract PO-141: The role of African Duffy-null allele related inflammation on the tumor microenvironment

Abstract DARC/ACKR1 erythrocyte expression, also known as the “Duffy blood group,” is understood to sequester pro-inflammatory chemokines and thereby regulate circulating gradients that direct immune cell infiltration. We hypothesize that this function also determines immune cell landscapes in the tumor microenvironment. Due to evolutionary selection pressures of malaria, individuals with sub-Saharan African ancestry typically carry the Duffy-null allele (rs4849887) and this lack of DARC/ACKR1 expression gives immunity to malaria while also allowing chronically high inflammation levels. Over 68% of African Americans (AA) have been found to have the Duffy-null genotype, compared to a rare 1-3% in European American individuals and we have shown it increases predisposition for Triple-Negative Breast Cancer (TNBC). In addition, we are currently studying if Duffy-null may contribute to higher breast cancer mortality that disproportionately affects AA women. This may be in part due to the role that low-DARC/ACKR1 expression plays in chronic inflammation, altering levels of several chemokines that modulate the migration and differentiation of specific immune cells. This role will impact tumor immune cell infiltration as well as the immune cell population composition overall, depending upon levels of DARC/ACKR1. Using RNA sequencing, our initial results indicated that for breast cancer tumors with high DARC/ACKR1 expression there was a higher estimated presence of CD8+ T cells, CD4+ T cells, regulatory T cells, follicular helper T cells, and memory B cells. Whereas with low DARC/ACKR1 expression, there was markedly less expression of resting dendritic cells and memory B cells. Therefore, in order to ascertain the influence DARC status has on spatial deposition and functional status of immune cell landscapes across the tumor microenvironment, we performed imaging mass cytometry on primary TNBC tumors. The panel contained tumor, structural, and immune markers, and was used to characterize the spatial differences between samples that had been verified to be DARC-high or DARC-low through immunohistochemistry. Our imaging analyses indicated that high DARC/ACKR1 expression correlates with infiltration of monocytes, macrophages, and cytotoxic T cells into the solid tumor microenvironment. Conversely, tumors with low DARC/ACKR1 expression showed monocytes and cytotoxic T cells contained in the tumor stroma. Using single-cell phenotyping, we were also able to identify distinct cell populations between DARC-high and -low. The tSNE analysis and heatmaps performed using Histology Topography Cytometry Analysis Toolbox (histoCAT), allowed us to visualize the spatial distribution of these cell populations, indicating an immune-suppressive tumor microenvironment in DARC-low tumors. These differences may be implicated in the causality of tumor progression as well as how to approach treatment given the cell heterogeneity of TNBC. This work provides greater context on the role that Duffy-null plays in chronic inflammation on the tumor microenvironment. Citation Format: Yanira Guerra, Rachel Martini, Jessica Bensenhaver, Yalei Chen, Joseph K. Oppong, Ishmael Kyei, Frances S. Aitpillah, Michael O. Adinku, Joseph K. Oppong, Ernest K. Adjei, Aisha Jibril, Baffour Awuah, Mahteme Bekele, Engida Abebe, Kwasi Ankomah, Ernest B. Osei-Bonsu, Kofi K. Gyan, Clayton Yates, Kim Blenman, Olivier Elemento, Lisa Newman, Melissa B. Davis. The role of African Duffy-null allele related inflammation on the tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-141.

Faisability pilot study to explore inflammation with 18F-DPA-714 PET CT versus immunochemistry in triple negative breast cancer: Design protocol

The Triple Negative Breast Cancer (TNBC), despite a good initial response to conventional chemotherapy, relapses frequently and has a poor prognosis after the onset of metastases. It is therefore interesting to develop new relevant targets to establish a prognosis but also potentially to propose a targeted therapy for a theranostic approach. A high density of M2-type macrophages presence in the primary TNBC tumor predicted an unfavorable prognosis. The presence of activated M2-type macrophages can be evaluated by measuring the expression of a translocating protein (TSPO) with [18F]-DPA-714 PETCT. This proof-of-concept study with [18F]-DPA-714 PET-CT has a design to establish the correlation between immunohistochemistry tumor characterization and in vivo imaging. If a valid correlation will be established, [18F]-DPA-714 PET-CT could be a based image prognosis biomarker, apart from pathological data, which can be fragmented as biopsy or modified by previous treatments. It would allow adapting early the type/dose-intensity of treatment and considering developments of treatments targeting M2-type macrophages

Ganglioside GD2: a novel therapeutic target in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by lack of hormone receptor expression and is known for high rates of recurrence, distant metastases, and poor clinical outcomes. TNBC cells lack targetable receptors; hence, there is an urgent need for targetable markers for the disease. Breast cancer stem-like cells (BCSCs) are a fraction of cells in primary tumors that are associated with tumorigenesis, metastasis, and resistance to chemotherapy. Targeting BCSCs is thus an effective strategy for preventing cancer metastatic spread and sensitizing tumors to chemotherapy. The CD44hi CD24lo phenotype is a well-established phenotype for identification of BCSCs, but CD44 and CD24 are not targetable markers owing to their expression in normal tissues. The ganglioside GD2 has been shown to be upregulated in primary TNBC tumors compared with normal breast tissue and has been shown to identify BCSCs. In this review, we discuss GD2 as a BCSC- and tumor-specific marker in TNBC; epithelial-to-mesenchymal transition and the signaling pathways that are upstream and downstream of GD2 and the role of these pathways in tumorigenesis and metastasis in TNBC; direct and indirect approaches for targeting GD2; and ongoing clinical trials and treatments directed against GD2 as well as future directions for these strategies.

HMGA1, Moonlighting Protein Function, and Cellular Real Estate: Location, Location, Location!

The gene encoding the High Mobility Group A1 (HMGA1) chromatin remodeling protein is upregulated in diverse cancers where high levels portend adverse clinical outcomes. Until recently, HMGA1 was assumed to be a nuclear protein exerting its role in cancer by transcriptionally modulating gene expression and downstream signaling pathways. However, the discovery of an extracellular HMGA1-RAGE autocrine loop in invasive triple-negative breast cancer (TNBC) cell lines implicates HMGA1 as a “moonlighting protein” with different functions depending upon cellular location. Here, we review the role of HMGA1, not only as a chromatin regulator in cancer and stem cells, but also as a potential secreted factor that drives tumor progression. Prior work found that HMGA1 is secreted from TNBC cell lines where it signals through the receptor for advanced glycation end products (RAGE) to foster phenotypes involved in tumor invasion and metastatic progression. Studies in primary TNBC tumors also suggest that HMGA1 secretion associates with distant metastasis in TNBC. Given the therapeutic potential to target extracellular proteins, further work to confirm this role in other contexts is warranted. Indeed, crosstalk between nuclear and secreted HMGA1 could change our understanding of tumor development and reveal novel therapeutic opportunities relevant to diverse human cancers overexpressing HMGA1.

Abstract PD7-06: Unstable mutational profile and heterogeneity of residual breast tumor following neoadjuvant therapy from comprehensive genomic and transcriptomic sequencing

Abstract Background: In patients with early breast cancer, neoadjuvant therapy is widely performed as standard of care. While the molecular targeting strategy makes progress in HER2 positive breast cancer, the optimal regimens for ER+/HER2- breast cancer (BC) and triple negative breast cancer (TNBC) remain undecided. Furthermore, there are few strategies for patients who do not achieve complete pathological response (pCR) who have worse prognosis. To address these unmet clinical needs, there is urgent need to examine the genomic alterations and immune microenvironment of residual tumors after neoadjuvant therapy. Here, we conducted a comprehensive analysis integrating genomic, transcriptomic, and clinical data to investigate the difference between primary breast cancer and residual disease. Materials and Methods: Using the large prospectively ascertained ethnically diverse Chicago Multi-Ethnic (ChiMEC) cohort of 562 participants with integrated genomic data, we identified 176 patients with breast cancer who underwent sequencing with Tempus xT next-generation sequencing panel including DNA- and whole-transcriptome RNA-sequencing. These included 131 primary breast tumors and 45 residual tumors after neoadjuvant therapy. We compared mutation rates between primary tumor and residual tumor in ER+/HER2- BC and TNBC. We also investigated homologous recombination deficiency (HRD) scores, tumor mutational burden (TMB), degree of immune infiltration, and microsatellite instability (MSI), and their association with survival. Results: Out of the 176 patients, there were 72 ER+/HER2- BC, 42 TNBC, and 44 HER2 positive cases. Among patients with HR+/HER2- BC, residual tumors had higher mutation rates in PIK3CA (61% vs 33%), CDH1 (33% vs 17%), CCND1 (39% vs 7%), FGF3 (28% vs 0%), FGF4 (33% vs 2%), FGF19 (33% vs 2%), and GATA3 (44% vs 7%) than primary tumors, but lower rates in TP53 (28% vs 48%), MAP3K1 (6% vs 40%), KMT2D (0% vs 33%), MCL1 (0% vs 30%), SPEN (6% vs 20%), ZFHX3 (0% vs 20%), ARID1A (11% vs 16%), KMT2C (0% vs 19%), LZTR1 (0% vs 19%), BCORL1 (6% vs 17%), NOTCH3 (6% vs 17%), FAT1 (0% vs 17%) and LPR1B (0% vs 17%). Relative to primary TNBC tumors, residual TNBC tumors exhibited higher mutation rates in PTEN (10% vs 3%), CCNE1 (10% vs 3%), CIC (10% vs 0%), and KMT2D (10% vs 3%). Conversely, residual TNBC tumors had relatively lower rates of MCL1 (0% vs 21%), RB1 (0% vs 12%), CDH1 (0% vs 12%), KMT2C (11% vs 18%), and PIK3CA (0% vs 15%), CDKN1B (0% vs 12%) and ETV6 (0% vs 12%). There was a significant trend of higher TMB (&amp;gt;5.0 mutations/megabase, m/MB) associated with improved disease-free survival among the 176 patients (P=0.031). TMB was not significantly different between primary tumors and residual tumors, or across subtypes. Microsatellite instability (MSI) status was high in 3 patients (1.6%) and equivocal in 5 patients (2.7%). TMB in MSI-high or MSI-equivocal tumors was significantly higher than TMB in tumors with microsatellite stability (37.7m/MB vs 5.3m/MB, P&amp;lt;0.001). HRD scores were highest in TNBC, and lowest in ER+/HER2- breast cancers (P=0.022). There was no significant association between the HRD scores and survival. To date, tumors from 106 patients have undergone immune profiling, with estimation of immune cell infiltration, macrophages, B cells, CD4, CD8, and NK cells. Initial analysis showed no association between immune profiling and survival. Conclusion: These comprehensive analyses demonstrated that mutation status in HR+/HER2- breast cancers and TNBC differs between primary and residual tumors after neoadjuvant therapy. We identified genomic alterations and pathways in residual tumors that could be further explored as potential targets in the adjuvant setting to improve long term outcomes for patients who do not achieve pCR. Citation Format: Minoru Miyashita, Masaya Hattori, Yonglan Zheng, Toshio Yoshimatsu, Joshua SK Bell, Padma Sheila Rajagopal, Anna Woodard, Jean Baptiste Reynier, Elisabeth Sveen, Galina Khramtsova, Fang Liu, Abiola Ibraheem, Gini Fleming, Nora Jaskowiak, Rita Nanda, Benjamin Leibowitz, Nike Beaubier, Kevin White, Dezheng Huo, Olufunmilayo I Olopade. Unstable mutational profile and heterogeneity of residual breast tumor following neoadjuvant therapy from comprehensive genomic and transcriptomic sequencing [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-06.

An Intelligent Biomimetic Nanoplatform for Holistic Treatment of Metastatic Triple-Negative Breast Cancer via Photothermal Ablation and Immune Remodeling.

Metastasis is one of the main causes of failure in the treatment of triple-negative breast cancer (TNBC). Immunotherapy brings hope and opportunity to solve this challenge, while its clinical applications are greatly inhibited by the tumor immunosuppressive environment. Here, an intelligent biomimetic nanoplatform was designed based on dendritic large-pore mesoporous silica nanoparticles (DLMSNs) for suppressing metastatic TNBC by combining photothermal ablation and immune remodeling. Taking advantage of the ordered large-pore structure and easily chemically modified property of DLMSNs, the copper sulfide (CuS) nanoparticles with high photothermal conversion efficiency were in situ deposited inside the large pores of DLMSNs, and the immune adjuvant resiquimod (R848) was loaded controllably. A homogenous cancer cell membrane was coated on the surfaces of these DLMSNs, followed by conjugation with the anti-PD-1 peptide AUNP-12 through a polyethylene glycol linker with an acid-labile benzoic-imine bond. The thus-obtained AM@DLMSN@CuS/R848 was applied to holistically treat metastatic TNBC in vitro and in vivo. The data showed that AM@DLMSN@CuS/R848 had a high TNBC-targeting ability and induced efficient photothermal ablation on primary TNBC tumors under 980 nm laser irradiation. Tumor antigens thus generated and increasingly released R848 by response to the photothermal effect, combined with AUNP-12 detached from AM@DLMSN@CuS/R848 in the weakly acidic tumor microenvironment, synergistically exerted tumor vaccination, and T lymphocyte activation functions on immune remodeling to prevent TNBC recurrence and metastasis. Taken together, this study provides an intelligent biomimetic nanoplatform to enhance therapeutic outcomes in metastatic TNBC.

Doxorubicin Conjugation to Reovirus Improves Oncolytic Efficacy in Triple-Negative Breast Cancer

Breast cancer is the second leading cause of cancer-related deaths in women in the United States. The triple-negative breast cancer (TNBC) subtype associates with higher rates of relapse, shorter overall survival, and aggressive metastatic disease. Hormone therapy is ineffective against TNBC, leaving patients with limited therapeutic options. Mammalian orthoreovirus (reovirus) preferentially infects and kills transformed cells, and a genetically engineered reassortant reovirus infects and kills TNBC cells more efficiently than prototypical strains. Reovirus oncolytic efficacy is further augmented by combination with topoisomerase inhibitors, including the frontline chemotherapeutic doxorubicin. However, long-term doxorubicin use correlates with toxicity to healthy tissues. Here, we conjugated doxorubicin to reovirus (reo-dox) to control drug delivery and enhance reovirus-mediated oncolysis. Our data indicate that conjugation does not impair viral biology and enhances reovirus oncolytic capacity in TNBC cells. Reo-dox infection promotes innate immune activation, and crosslinked doxorubicin retains DNA-damaging properties within infected cells. Importantly, reovirus and reo-dox significantly reduce primary TNBC tumor burden in vivo, with greater reduction in metastatic burden after reo-dox inoculation. Together, these data demonstrate that crosslinking chemotherapeutic agents to oncolytic viruses facilitates functional drug delivery to cells targeted by the virus, making it a viable approach for combination therapy against TNBC.

Triple Combination of Ascorbate, Menadione and the Inhibition of Peroxiredoxin-1 Produces Synergistic Cytotoxic Effects in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is an aggressive form of mammary malignancy currently without satisfactory systemic treatment options. Agents generating reactive oxygen species (ROS), such as ascorbate (Asc) and menadione (Men), especially applied in combination, have been proposed as an alternative anticancer modality. However, their effectiveness can be hampered by the cytoprotective effects of elevated antioxidant enzymes (e.g., peroxiredoxins, PRDX) in cancer. In this study, PRDX1 mRNA and protein expression were assessed in TNBC tissues by analysis of the online RNA-seq datasets and immunohistochemical staining of tissue microarray, respectively. We demonstrated that PRDX1 mRNA expression was markedly elevated in primary TNBC tumors as compared to non-malignant controls, with PRDX1 protein staining intensity correlating with favorable survival parameters. Subsequently, PRDX1 functionality in TNBC cell lines or non-malignant mammary cells was targeted by genetic silencing or chemically by auranofin (AUR). The PRDX1-knockdown or AUR treatment resulted in inhibition of the growth of TNBC cells in vitro. These cytotoxic effects were further synergistically potentiated by the incubation with a combination of the prooxidant agents, Asc and Men. In conclusion, we report that the PRDX1-related antioxidant system is essential for maintaining redox homeostasis in TNBC cells and can be an attractive therapeutic target in combination with ROS-generating agents.

A Multigene Assay Determines Risk of Recurrence in Patients with Triple-Negative Breast Cancer.

Approximately 40% of patients with stage I-III triple-negative breast cancer (TNBC) recur after standard treatment, whereas the remaining 60% experience long-term disease-free survival (DFS). There are currently no clinical tests to assess the risk of recurrence in TNBC patients. We previously determined that TNBC patients with MHC class II (MHCII) pathway expression in their tumors experienced significantly longer DFS. To translate this discovery into a clinical test, we developed an MHCII Immune Activation assay, which measures expression of 36 genes using NanoString technology. Preanalytical testing confirmed that the assay is accurate and reproducible in formalin-fixed paraffin-embedded (FFPE) tumor specimens. The assay measurements were concordant with RNA-seq, MHCII protein expression, and tumor-infiltrating lymphocyte counts. In a training set of 44 primary TNBC tumors, the MHCII Immune Activation Score was significantly associated with longer DFS (HR = 0.17; P = 0.015). In an independent validation cohort of 56 primary FFPE TNBC tumors, the Immune Activation Score was significantly associated with longer DFS (HR = 0.19; P = 0.011) independent of clinical stage. An Immune Activation Score threshold for identifying patients with very low risk of relapse in the training set provided 100% specificity in the validation cohort. The assay format enables adoption as a standardized clinical prognostic test for identifying TNBC patients with a low risk of recurrence. Correlative data support future studies to determine if the assay can identify patients in whom chemotherapy can be safely deescalated and patients likely to respond to immunotherapy. SIGNIFICANCE: The MHCII Immune Activation assay identifies TNBC patients with a low risk of recurrence, addressing a critical need for prognostic biomarker tests that enable precision medicine for TNBC patients.