Abstract P6-08-04: Creation of a robust algorithm utilizing minimal gene sets normalized against a reference gene set to identify triple-negative breast cancer (TNBC) subtypes

AbstractCancer-associated adipocytes are known to cause inflammation; however, the role of adipogenesis, the formation of adipocytes, in breast cancer is unclear. We hypothesized that intra-tumoral adipogenesis reflects a different cancer biology than abundance of intra-tumoral adipocytes. The Molecular Signatures Database Hallmark adipogenesis gene set of gene set variant analysis was used to quantify adipogenesis. Total of 5,098 breast cancer patients in multiple cohorts (training; GSE96058 (n = 3273), validation; TCGA (n = 1069), treatment response; GSE25066 (n = 508) and GSE20194 (n = 248)) were analyzed. Adipogenesis did not correlate with abundance of adipocytes. Adipogenesis was significantly lower in triple negative breast cancer (TNBC). Elevated adipogenesis was significantly associated with worse survival in TNBC, but not in the other subtypes. High adipogenesis TNBC was significantly associated with low homologous recombination deficiency, but not with mutation load. High adipogenesis TNBC enriched metabolism-related gene sets, but neither of cell proliferation- nor inflammation-related gene sets, which were enriched to adipocytes. High adipogenesis TNBC was infiltrated with low CD8+ T cells and high M2 macrophages. Although adipogenesis was not associated with neoadjuvant chemotherapy response, high adipogenesis TNBC was significantly associated with low expression of PD-L1 and PD-L2 genes, and immune checkpoint molecules index. In conclusion, adipogenesis in TNBC was associated with cancer metabolism and unfavorable tumor immune microenvironment, which is different from abundance of adipocytes.

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Erratum to: Generation of an algorithm based on minimal gene sets to clinically subtype triple negative breast cancer patients

BMC Cancer ◽

10.1186/s12885-016-2307-0 ◽

2016 ◽

Vol 16 (1) ◽

Author(s):

Brian Z. Ring ◽

David R. Hout ◽

Stephan W. Morris ◽

Kasey Lawrence ◽

Brock L. Schweitzer ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Breast Cancer Patients ◽

Gene Sets

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Generation of an algorithm based on minimal gene sets to clinically subtype triple negative breast cancer patients

BMC Cancer ◽

10.1186/s12885-016-2198-0 ◽

2016 ◽

Vol 16 (1) ◽

Cited By ~ 27

Author(s):

Brian Z. Ring ◽

David R. Hout ◽

Stephan W. Morris ◽

Kasey Lawrence ◽

Brock L. Schweitzer ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Breast Cancer Patients ◽

Gene Sets

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Nuclear ErbB-2-Induced Transcriptome Drives Triple Negative Breast Cancer Growth

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.2104 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A1028-A1029

Author(s):

Maria F Chervo ◽

Micaela Parra ◽

Nicolas Bellora ◽

Ezequiel Petrillo ◽

Santiago Madera ◽

...

Keyword(s):

Breast Cancer ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Gene Set Enrichment Analysis ◽

Tyrosine Kinase Receptor ◽

Alternative Transcript ◽

Promoting Effect ◽

Primary Tumors ◽

Clinical Biomarkers ◽

Gene Sets

Abstract Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2 tyrosine kinase receptor. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Gene expression analyses have categorized TNBC into distinct molecular subtypes. Up to 78% of clinical TNBCs belong to the basal-like (BL) subtype. Here we found ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing, using a PCR-sequencing approach combined with RNA interference, revealed that BL TNBC cells express the canonical ErbB-2 (WTErbB-2), encoded by transcript 1, and the non-canonical isoform c, encoded by alternative transcript 3 (T3). The latter was not previously reported in normal or malignant cells. To characterize the isoform c we designed siRNAs targeting T3 (T3 siRNAs), which silenced up to 93% of said isoform. Transfection of T3 siRNAs into BL cells expressing only isoform c or both isoform c and WTErbB-2 was sufficient to decrease cell proliferation. Intratumoral injections of T3 siRNAs into mice bearing BL TN tumors also blocked in vivo growth. To explore whether isoform c growth-promoting effect is due to its functions as a transcriptional regulator, we performed RNA-seq in BL cells expressing only this isoform. We identified a set of genes differentially regulated in BL cells where we evicted isoform c from the nucleus, as compared to control cells. In the up-regulated group, we found enrichment of pro-apoptotic and tumor suppressor genes and in the down-regulated one, genes involved in proliferation and stemness. We used gene set enrichment analysis (GSEA) to identify the biological processes associated with these isoform c-regulated genes. We found a pronounced enrichment of gene sets related to apoptosis, activation of DNA damage pathways and cell cycle arrest in response to eviction of nuclear isoform c. GSEA also revealed negative regulation of gene sets involved in cell motility, cellular differentiation and growth pathways in BL cells lacking nuclear isoform c expression. These results suggest that NErbB-2 function modulates tumor growth and promotes a metastatic phenotype in TNBC. Furthermore, our clinical findings identified NErbB-2 as an independent predictor of shorter OS (HR 2.54; 95% CI 1.22-5.28; P = 0.013), DFS (HR 2.91; 95% CI 1.44-5.87; P = 0.003), and DMFS (HR 2.59; 95% CI 1.20-5.60; P = 0.015) in 99 TN primary tumors. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type proteins, which conserve the canonical domains and are located in their classical cellular compartments, highlighting the potential of NErbB-2 isoforms as novel therapeutic targets and clinical biomarkers in TNBC.

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Molecular Classification Models for Triple Negative Breast Cancer Subtype Using Machine Learning

Journal of Personalized Medicine ◽

10.3390/jpm11090881 ◽

2021 ◽

Vol 11 (9) ◽

pp. 881

Author(s):

Rassanee Bissanum ◽

Sitthichok Chaichulee ◽

Rawikant Kamolphiwong ◽

Raphatphorn Navakanitworakul ◽

Kanyanatt Kanokwiroon

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Machine Learning ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Gene Expression Pattern ◽

Classification Models ◽

Gene Set ◽

Svm Algorithm

Triple negative breast cancer (TNBC) lacks well-defined molecular targets and is highly heterogenous, making treatment challenging. Using gene expression analysis, TNBC has been classified into four different subtypes: basal-like immune-activated (BLIA), basal-like immune-suppressed (BLIS), mesenchymal (MES), and luminal androgen receptor (LAR). However, there is currently no standardized method for classifying TNBC subtypes. We attempted to define a gene signature for each subtype, and to develop a classification method based on machine learning (ML) for TNBC subtyping. In these experiments, gene expression microarray data for TNBC patients were downloaded from the Gene Expression Omnibus database. Differentially expressed genes unique to 198 known TNBC cases were identified and selected as a training gene set to train in seven different classification models. We produced a training set consisting of 719 DEGs selected from uniquely expressed genes of all four subtypes. The highest average accuracy of classification of the BLIA, BLIS, MES, and LAR subtypes was achieved by the SVM algorithm (accuracy 95–98.8%; AUC 0.99–1.00). For model validation, we used 334 samples of unknown TNBC subtypes, of which 97 (29.04%), 73 (21.86%), 39 (11.68%) and 59 (17.66%) were predicted to be BLIA, BLIS, MES, and LAR, respectively. However, 66 TNBC samples (19.76%) could not be assigned to any subtype. These samples contained only three upregulated genes (EN1, PROM1, and CCL2). Each TNBC subtype had a unique gene expression pattern, which was confirmed by identification of DEGs and pathway analysis. These results indicated that our training gene set was suitable for development of classification models, and that the SVM algorithm could classify TNBC into four unique subtypes. Accurate and consistent classification of the TNBC subtypes is essential for personalized treatment and prognosis of TNBC.

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High MYC mRNA Expression Is More Clinically Relevant than MYC DNA Amplification in Triple-Negative Breast Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms21010217 ◽

2019 ◽

Vol 21 (1) ◽

pp. 217 ◽

Cited By ~ 15

Author(s):

Eriko Katsuta ◽

Li Yan ◽

Takashi Takeshita ◽

Kerry-Ann McDonald ◽

Subhamoy Dasgupta ◽

...

Keyword(s):

Breast Cancer ◽

Mrna Expression ◽

Triple Negative Breast Cancer ◽

Target Genes ◽

Triple Negative ◽

Dna Amplification ◽

High Expression ◽

Inclusion Criteria ◽

Gene Sets ◽

The Difference

DNA abnormalities are used in inclusion criteria of clinical trials for treatments with specific targeted molecules. MYC is one of the most powerful oncogenes and is known to be associated with triple-negative breast cancer (TNBC). Its DNA amplification is often part of the targeted DNA-sequencing panels under the assumption of reflecting upregulated signaling. However, it remains unclear if MYC DNA amplification is a surrogate of its upregulated signaling. Thus, we investigated the difference between MYC DNA amplification and mRNA high expression in TNBCs utilizing publicly available cohorts. MYC DNA amplified tumors were found to have various mRNA expression levels, suggesting that MYC DNA amplification does not always result in elevated MYC mRNA expression. Compared to other subtypes, both MYC DNA amplification and mRNA high expression were more frequent in the TNBCs. MYC mRNA high expression, but not DNA amplification, was significantly associated with worse overall survival in the TNBCs. The TNBCs with MYC mRNA high expression enriched MYC target genes, cell cycle related genes, and WNT/β-catenin gene sets, whereas none of them were enriched in MYC DNA amplified TNBCs. In conclusion, MYC mRNA high expression, but not DNA amplification, reflects not only its upregulated signaling pathway, but also clinical significance in TNBCs.

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Differential expression between African-ancestry and White patients diagnosed with Triple-Negative Breast Cancer: EGFR, Myc, Bcl2 and β-Catenin as ancestry-associated markers

10.1101/2020.11.13.381608 ◽

2020 ◽

Author(s):

Ana T. Matias ◽

Ana Jacinta-Fernandes ◽

Ana-Teresa Maia ◽

Sofia Braga ◽

António Jacinto ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

African American ◽

Protein Expression ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

African Ancestry ◽

Gene Set ◽

Gene And Protein Expression ◽

Differential Gene

AbstractPurposeTriple-negative breast cancer (TNBC) has a higher incidence, a younger age of onset, and a more aggressive behavior in African-ancestry women. Biological disparities have been suggested as an important factor influencing the ancestry-associated TNBC discrepancy. In this study, we sought to identify ancestry-associated differential gene and protein expression between African-ancestry and White TNBC patients, controlling for patients’ menopause status and pathological staging at diagnosis.MethodsDifferential gene expression analyses (DGEA) were performed using RNA-sequencing data from The Cancer Genome Atlas (TCGA). Gene set enrichment analysis (GSEA) and Ingenuity Pathway Analysis (IPA), with focus on network design, were performed to highlight candidate genes for further validation through immunohistochemistry of TNBC samples from patients followed in Portugal.ResultsWith 52 African-American and 90 White TNBC patients included, TCGA’s data corroborate that African-American patients have a higher TNBC incidence (28.42% vs 11.89%, p<0.0001). Particularly, premenopausal and stage II disease African-American patients also have significantly lower survival probability, comparing with White patients (log-rank p=0.019 and 0.0038, respectively). DGEA results suggest that expression profile differences are more associated with TNBC staging than with patient’s menopause status. Hippo pathway and cellular community gene sets are downregulated, while breast cancer gene set is upregulated in African-Americans, comparing with White TNBC patients. Furthermore, MAPK pathway gene set is upregulated when controlling for stage II disease. Due to their central role in highly scored networks resulted from IPA’s network design, EGFR, Myc and Bcl2 genes were selected for further validation through immunohistochemistry. We also included β-Catenin in the validation study as it is consensually reported to be required in TNBC tumorigenesis. Although patients used in the DGEA and in the immunohistochemistry experiments are geographically and culturally distinct, both groups of African-ancestry patients are mostly of western-African ancestry and, interesting, differential gene and protein expression matched.ConclusionsWe found ancestry-associated gene expression patterns between African-ancestry and White TNBCs, particularly when controlling for menopause status or staging. EGFR, Myc, Bcl2 and β-catenin gene and protein differential expression matching results in distinct populations suggest these markers as being important indicators of TNBC’s ancestry-associated development.

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Gene set analysis of post-lactational mammary gland involution gene signatures in inflammatory and triple-negative breast cancer

PLoS ONE ◽

10.1371/journal.pone.0192689 ◽

2018 ◽

Vol 13 (4) ◽

pp. e0192689 ◽

Cited By ~ 7

Author(s):

Arvind Bambhroliya ◽

Renae D. Van Wyhe ◽

Swaminathan Kumar ◽

Bisrat G. Debeb ◽

Jay P. Reddy ◽

...

Keyword(s):

Breast Cancer ◽

Mammary Gland ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Gene Set Analysis ◽

Gene Signatures ◽

Gene Set ◽

Mammary Gland Involution

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Plasmacytoid Dendritic Cell (pDC) Infiltration Correlate with Tumor Infiltrating Lymphocytes, Cancer Immunity, and Better Survival in Triple Negative Breast Cancer (TNBC) More Strongly than Conventional Dendritic Cell (cDC)

Cancers ◽

10.3390/cancers12113342 ◽

2020 ◽

Vol 12 (11) ◽

pp. 3342

Author(s):

Masanori Oshi ◽

Stephanie Newman ◽

Yoshihisa Tokumaru ◽

Li Yan ◽

Ryusei Matsuyama ◽

...

Keyword(s):

Breast Cancer ◽

Dendritic Cell ◽

Triple Negative Breast Cancer ◽

Immune Cells ◽

Triple Negative ◽

Related Gene ◽

Cancer Immunity ◽

Gene Sets ◽

High Fraction ◽

Ifn Γ

Dendritic cells (DC) represent a major antigen-presenting cell type in the tumor immune microenvironment (TIME) and play an essential role in cancer immunity. Conventional DC (cDC) and plasmacytoid DC (pDC) were defined by the xCell algorithm and a total of 2968 breast cancer patients (TCGA and METABRIC) were analyzed. We found that triple-negative breast cancer (TNBC) had a high fraction of cDC and pDC compared to the other subtypes. In contrast to cDC, high pDC in TNBC was significantly associated with better disease-specific and disease-free survival consistently in both cohorts. High cDC TNBC tumors enriched not only inflammation and immune-related, but also metastasis-related gene sets in Gene Set Enrichment Analysis, whereas high pDC TNBC enriched inflammation and immune -related gene sets including IFN-γ signaling more strongly than cDC. pDC TNBC correlated with CD8+, CD4+ memory, IFN-γ score, and cytolytic activity stronger than cDC TNBC. High pDC TNBC were associated with a high fraction of anti-cancer immune cells and high expression of all the immune check point molecules examined. In conclusion, pDC levels correlated with the infiltration of immune cells and patient survival in TNBC more strongly than cDC; this is the first study suggesting the clinical relevance of pDC infiltration in TNBC.

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Enhanced Thermogenesis in Triple-Negative Breast Cancer Is Associated with Pro-Tumor Immune Microenvironment

Cancers ◽

10.3390/cancers13112559 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2559

Author(s):

Shipra Gandhi ◽

Masanori Oshi ◽

Vijayashree Murthy ◽

Elizabeth A. Repasky ◽

Kazuaki Takabe

Keyword(s):

Breast Cancer ◽

T Cells ◽

Cold Stress ◽

Triple Negative Breast Cancer ◽

Cancer Biology ◽

Triple Negative ◽

Related Gene ◽

Immune Microenvironment ◽

Gene Sets ◽

Tumor Immune Microenvironment

Mild cold stress induced by housing mice with a 4T1 triple-negative breast cancer (TNBC) cell implantation model at 22 °C increases tumor growth rate with a pro-tumorigenic immune microenvironment (lower CD8 +T cells, higher myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs)). Since cold stress also activates thermogenesis, we hypothesized that enhanced thermogenesis is associated with more aggressive cancer biology and unfavorable tumor microenvironment (TME) in TNBC patients. A total of 6479 breast cancer patients from METABRIC, TCGA, GSE96058, GSE20194, and GSE25066 cohorts were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) thermogenesis score. High-thermogenesis TNBC was associated with a trend towards worse survival and with angiogenesis, adipogenesis, and fatty acid metabolism pathways. On the other hand, low-thermogenesis TNBC enriched most of the hallmark cell-proliferation-related gene sets (i.e., mitotic spindle, E2F targets, G2M checkpoint, MYC targets), as well as immune-related gene sets (i.e., IFN-α and IFN-γ response). Favorable cytotoxic T-cell-attracting chemokines CCL5, CXCL9, CXCL10, and CXCL11 were lower; while the MDSC- and Treg-attracting chemokine CXCL12 was higher. There were higher M2 but lower M1 macrophages and Tregs. In conclusion, high-thermogenesis TNBC is associated with pro-tumor immune microenvironment and may serve as biomarker for testing strategies to overcome this immunosuppression.

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