Prognostic Marker In Triple Negative Breast Cancer Research Articles

Hypomethylation of ATP1A1 Is Associated with Poor Prognosis and Cancer Progression in Triple-Negative Breast Cancer.

Dysregulated DNA methylation in cancer is critical in the transcription machinery associated with cancer progression. Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, but no treatment targeting TNBC biomarkers has yet been developed. To identify specific DNA methylation patterns in TNBC, methyl-binding domain protein 2 (MBD) sequencing data were compared in TNBC and the three other major breast cancer subtypes. Integrated analysis of DNA methylation and gene expression identified a gene set showing a correlation between DNA methylation and gene expression. ATPase Na+/K+-transporting subunit alpha 1 (ATP1A1) was found to be specifically hypomethylated in the coding sequence (CDS) region and to show increased expression in TNBC. The Cancer Genome Atlas (TCGA) database also showed that hypomethylation and high expression of ATP1A1 were strongly associated with poor survival in patients with TNBC. Furthermore, ATP1A1 knockdown significantly reduced the viability and tumor-sphere formation of TNBC cells. These results suggest that the hypomethylation and overexpression of ATP1A1 could be a prognostic marker in TNBC and that the manipulation of ATP1A1 expression could be a therapeutic target in this disease.

Integrated analysis of single-cell RNA-seq and bulk RNA-seq unravels T cell-related prognostic risk model and tumor immune microenvironment modulation in triple-negative breast cancer

Triple negative breast cancer (TNBC) is an aggressive and fatal malignancy. The current success of tumor immunotherapy has focused attention on intermediate T-cell subsets and the tumor microenvironment, which are essential for activation of the anti-tumor response. Therefore, both areas require further research to accelerate progress in developing tailored immunotherapeutic approaches for patients with TNBC. We obtained scRNA-seq data of TNBC from the GEO database. A multiplex strategy was used to analyze and identify the T-cell heterogeneity of TNBC. By combining the METABRIC and GEO databases, a prognostic risk model for T-cell marker genes was constructed and validated. In addition, the immune-infiltrating cells of TNBC was analyzed using CIBERSORT, and the association between the risk model and response to immunotherapy was investigated. Based on scRNA-seq data, 25,932cells were identified for multiple analyzes. T cells were studied with a focus on 2 subtypes, including CD8+ and CD4+. There were also communication relationships between T cells and multiple cell types. The results of the enrichment analysis showed that the T-cell marker genes were focused in pathways related to the immune system. In addition, OPTN, TMEM176A, PKM and HES1 deserve attention as prognostic markers in TNBC. The immune infiltration results showed that the high-risk group had significant immune cell infiltration and immunosuppression status. This study provides a resource for understanding T-cell heterogeneity and the associated prognostic risk model for TNBC. The results show that the model helps predict prognosis and response to treatment in breast cancer.

Androgen Receptor Status in Triple Negative Breast Cancer: Does It Correlate with Clinicopathological Characteristics?

Triple negative breast cancer (TNBC) is a breast carcinoma subtype that neither expresses estrogen (ER) and progesterone receptors (PR) nor the human epidermal growth factor receptor 2 (HER2). Patients with TNBC have been shown to have poorer outcomes mainly owing to the limited treatment options available. However, some studies have shown TNBC tumors expressing androgen receptors (AR), raising hopes of its prognostic role. This retrospective study investigated the expression of AR in TNBC and its relationship with known patient demographics, tumor and survival characteristics. From the records of 205 TNBC patients, 36 had available archived tissue samples eligible for AR staining. For statistical purposes, tumors were classified as either "positive" or "negative" for AR expression. The nuclear expression of AR was scored by measuring the percentage of stained tumor cells and its staining intensity. AR was expressed by 50% of the tissue samples in our TNBC cohort. The relationship between AR status with age at the time of TNBC diagnosis was statistically significant, with all AR positive TNBC patients being greater than 50 years old (vs 72.2% in AR negative TNBC). Also, the relationship between AR status and type of surgery received was statistically significant. There were no statistically significant associations between AR status with other tumor characteristics including "TNM status", tumor grade or treatments received. There was no statistically significant difference in median survival between AR negative and AR positive TNBC patients (3.5 vs 3.1 years; p = 0.581). The relationship between OS time and AR status (p = 0.581), type of surgery (p = 0.061) and treatments (p = 0.917) were not statistically significant. The androgen receptor may be an important prognostic marker in TNBC, with further research warranted. This research may benefit future studies investigating receptor-targeted therapies in TNBC.

Abstract P2-11-28: Copy number alteration is an independent prognostic biomarker in triple-negative breast cancer patients

Abstract Background: Triple-negative breast cancer (TNBC) is the most fatal breast cancer subtype, which often shows aggressive progression, a high potential to metastasize, and resistance to chemotherapy. Comprehensive genomic profiling using next-generation sequencing (NGS) has been expected to identify gene alterations that are targetable by drugs. However, the significance of these genomic alterations in the cancer biology of TNBC patients has not yet been fully understood due to the lack of accurate clinical outcome data to compare with the genomic data. The aim of this study was to clarify the clinical impact of genomic profiling data, including copy number alterations (CNAs), in TNBC by comparing comprehensive genomic data with clinical outcomes. Methods: A total of 47 patients diagnosed with stage I-III TNBC (from the cohort reported in JCO Precis Oncol. 2018;2:PO.17.00211) were enrolled in this study. The genomic profiling of 435 known cancer genes by NGS with clinical outcomes were analyzed. Overall survival (OS) was evaluated for its association to gene alterations and distinctively CNAs. The cut-off values of CNA for OS were determined from the receiver operating characteristic curve using the Youden index for area under the curve (AUC). Kaplan-Meier plots and log-rank tests of OS were applied for each group. Univariate and multivariate analyses for OS were performed using a Cox proportional-hazards model to obtain the hazard ratio (HR) and 95% confidence intervals. Results: Utilizing NGS-based genomic profiling, at least one alteration was found in 82 of the 435 cancer-associated genes, and a total of 162 alterations were found in the 47 patients. Among the 82 genes with alterations, the presence or absence of TP53 and PTEN alterations was significantly associated with OS of TNBC patients; patients with TP53 alterations (n = 31) showed significantly shorter OS than those without TP53 alterations (n = 16, p = 0.023), and patients with PTEN alterations (n = 9) showed significantly shorter OS than those without PTEN alterations (n = 38, p = 0.023). The cut-off value of CNA for OS was set at 25 (AUC, 0.788; sensitivity, 0.727; specificity, 0.900). Interestingly, CNA-high patients (n = 20) showed significantly shorter OS than CNA-low patients (n = 27, p = 0.014). Univariate analysis revealed that TP53 alterations and CNAs were significant prognostic factors for OS (HR, 8.81; p = 0.008; and HR, 8.00; p = 0.014, respectively). Finally, multivariate analysis using background clinical data revealed that CNA was an independent prognostic factor for OS in TNBC patients (HR, 7.15; p = 0.044). Conclusion: Our data suggest that CNA is an independent prognostic marker in TNBC, and that can be estimated from comprehensive genomic profiling data by NGS. Further investigation is needed to clarify the mechanisms of how CNAs are associated with this lethal disease. Citation Format: Masayuki Nagahashi, Chie Toshikawa, YiWei Ling, Tetsu Hayashida, Yuko Kitagawa, Manabu Futamura, Takashi Kuwayama, Seigo Nakamura, Hideko Yamauchi, Teruo Yamauchi, Koji Kaneko, Chizuko Kanbayashi, Nobuaki Sato, Junko Tsuchida, Kazuki Moro, Masato Nakajima, Yoshifumi Shimada, Hiroshi Ichikawa, Stephen Lyle, Yasuo Miyoshi, Kazuaki Takabe, Shujiro Okuda, Toshifumi Wakai. Copy number alteration is an independent prognostic biomarker in triple-negative breast cancer patients [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 P2-11-28.

Evaluation of CDC6 Expression in Triple-Negative Breast Cancer and its Prognostic Significance

ABSTRACT Background: Triple-negative breast cancer (TNBC) leads to high morbidity and poor prognosis due to high proliferation and metastasis. Cell division cycle 6 (CDC6) is one of the genes involved in cell division and is significantly upregulated in TNBC. There is not much information available to understand whether CDC6 can be used as a prognostic marker in TNBC. Methods: In this study, BC-GenExMiner, NetworkAnalyst, GeneMania, GEPIA, COSMIC, cBioPortal, STRING, and FunRich were used to investigate the expression of CDC6 gene. Furthermore, survival analysis to correlate the gene expression was carried out using the Kaplan-Meier plotter. Results: The results showed that mRNA levels of CDC6 were significantly higher in TNBC compared to healthy and other types of breast cancer, and high expression levels were also associated with increased TNBC proliferation. The survival analysis showed that increased mRNA levels of CDC6 in all TNBC patients were associated with a poor prognosis for relapse-free survival. Conclusion: The findings of the current study indicate that CDC6 may be used as a possible prognostic biomarker for TNBC. However, further studies are required to check the effects of CDC6 on the progression and pathogenesis of TNBC.

Stem Cell Marker Aldehyde Dehydrogenase 1A1 Expression in Triple-negative Breast Carcinoma

Background: Increasing evidence has proposed that tumor contains tumor initiating cells or cancer stem cells (CSCs) are responsible for its progression and relapse. Aldehyde dehydrogenase 1A1 (ALDH1A1) has recently been identified as a marker for cancer stem cells in some human malignancies including breast cancer.Triple negative breast carcinomas (TNBCs) are group of primary breast tumors with aggressive clinical behavior that have no targeted therapy at present.
 Aim: The assessment of immunohistochemical expression of ALDH1A1 in triple negative breast carcinoma and its correlation with the clinicopathological features of TNBC.
 Material and Methods: This study consisted of 60 cases of TNBC. Immunohistochemical reactions were carried out by using ALDH1A1 monoclonal antibody. A total score of ALDH1A1 expression is obtained by multiplying the score of staining intensity & percentage of stained cells to obtain score ranging from 0 up to 300. Due to the relatively large number of positive cases, a statistical analysis was performed with a negative (score ≤ 10) and positive (score > 10) cutoff [1].
 Results: Evaluation of the results of immunostaining for ALDH1A1 showed 88.3% of totalcases (53 cases) having a positive cytoplasmic reactivity. Statistical analysis for a possible correlation between ALDH1A1 expression and prognostic clinicopathological parameters; age, size, tumor grade, histologic subtypes, lymphovascular invasion, intraductal components, tumor infiltrating lymphocytes and TNM stage grouping revealed a non-significant correlation.
 Conclusion: ALDH1A1 couldn’t be used solely as a diagnostic or prognostic marker In TNBCs. Further research combining with other biomarkers and with a greater number of patients is necessary to confirm the role of ALDH1A1 in TNBC.
 Key words: Cancer stem cells (CSCs), Aldehyde dehydrogenase 1A1 (ALDH1A1), Triple Negative Breast cancer (TNBC).

CXCL13 expression in mouse 4T1 breast cancer microenvironment elicits antitumor immune response by regulating immune cell infiltration.

Breast cancer is the most commonly diagnosed cancer type and the leading cause of cancer-related deaths among women worldwide. Previous studies have reported contradictory performance of chemokine CXC motif ligand 13 (CXCL13) in breast cancer. In this study, The Cancer Genome Atlas database analysis revealed that CXCL13 was overexpressed in various human cancers including breast carcinoma, and associated with good clinical prognosis in breast cancer. Flow cytometry detection also found upregulated intracellular CXCL13 expression in human breast cancer cell lines. To explore the possible role of CXCL13 in the breast cancer microenvironment, mouse triple negative breast cancer (TNBC) was lentivirally transfected to stably overexpress mouse CXCL13 (4T1-CXCL13). Both parental 4T1 and 4T1-CXCL13 strains showed no in vitro or in vivo endogenous cell surface CXCR5 expression. In immune-competent BALB/c mice, the in vivo tumor growth of 4T1-CXCL13 was significantly inhibited and even completely eradicated, accompanied with increased infiltrations of CD4+, CD8+ T lymphocytes and CD11b+CD11c+ DCs. Further investigations showed that CXCL13 expression in the 4T1 tumor microenvironment elicited long-term antitumor immune memory, and rejection of distal parental tumor. The antitumor activity of CXCL13 was remarkedly impaired in BALB/cA-nu nude mice, or in BALB/c mice with CD8+ T lymphocyte or NK cell depletion. Our investigation indicated that CXCL13 expression in TNBC triggered effective antitumor immunity by chemoattracting immune cell infiltrations and could be considered as a novel prognostic marker for TNBC.

ESM1 promotes triple-negative breast cancer cell proliferation through activating AKT/NF-κB/Cyclin D1 pathway.

Triple-negative breast cancer (TNBC) is a subtype of invasive breast cancer that tests negative for PR, ER and excess HER2 protein. TNBC has a greater progression potential with poorer prognosis, compared with other types of breast cancer. Endothelial cell-specific molecule 1 (ESM1), also known as endocan, is overexpressed in various cancers including breast cancer and may play an important role in cancer progression. The online resource of The Cancer Genome Atlas (TCGA) was used for analyzing the expression alteration of ESM1 in breast cancer patient tissues. We examined the changes of various malignant behaviors of TNBC cell and in vivo tumor growth after inhibiting or overexpressing ESM1 in two human TNBC cell lines, MDA-MB-468 and MDA-MB-231. When ESM1 was knocked down or overexpressed in TNBC cell, AKT and p65 phosphorylation and Cyclin D1 expression were analyzed by western blotting. The ESM1-overexpressing TNBC cell was treated with MK-2206 and BAY-117082 at various concentrations. Our analyses show that ESM1 is overexpressed in TNBC cell lines as well as patient tissues, which is correlated to poor prognosis. Our results demonstrate that ESM1 knockdown decreases while overexpression of ESM1 increases in vitro proliferation, migration and invasion of TNBC cell and knockdown of ESM1 inhibits in vivo TNBC tumor growth. Our mechanistic study further discloses that ESM1 promotes the proliferation of TNBC cell through activating an Akt-dependent NF-κB/Cyclin D1 pathway. Our results demonstrate that ESM1 knockdown decreases while overexpression of ESM1 increases in vitro migration, proliferation and invasion of TNBC cell and knockdown of ESM1 inhibits in vivo tumor growth of TNBC in the xenograft mouse model. Our mechanistic study further discloses that ESM1 promotes the proliferation of TNBC cell through activating the Akt-dependent NF-κB/CyclinD1 pathway. Our findings expand the knowledge about the molecular mechanisms underlying TNBC progression and provide rationale for using ESM1 as a therapeutic target or prognostic marker for TNBC.

ABCC9, NKAPL, and TMEM132C are potential diagnostic and prognostic markers in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly heterogeneous disease. The aim of this study is to identify the diagnostic and poor prognostic signatures in TNBC by exploring the aberrant DNA methylation and gene expression. Differential expression and methylation analysis of the TNBC and paracancer samples from The Cancer Genome Atlas were performed. Gene set enrichment and protein-protein interaction (PPI) network analysis was used to explore the mechanisms of TNBC. Methylation-gene expression correlation analysis was performed, and multivariate Cox analysis and receiver operating characteristics analysis were used to further screen the hub genes for TNBC. We identified 1,525 differentially expressed genes and 150 differentially methylated genes between TNBC and paracancer samples. About 96.64% of the methylation sites were located on the CpG island. A total of 17 Gene Ontology biological process terms and 18 signal pathways were significantly enriched. GNG4, GNG11, PENK, MAOA, and AOX1 were identified as the core genes of the PPI network. Methylation-expression correlations revealed that ABCC9 (cg06951626), NKAPL (cg18675097, cg01031101, and cg17384889), and TMEM132C (cg03530754) showed promise as diagnostic and prognostic markers in TNBC. ABCC9 (cg06951626), NKAPL (cg18675097, cg01031101, and cg17384889), and TMEM132C (cg03530754) were potential diagnostic and prognostic markers in TNBC.

Why pathologists and oncologists should know about tumour-infiltrating lymphocytes (TILs) in triple-negative breast cancer: an Australian experience of 139 cases

The presence of increased tumour infiltrating lymphocytes (TILs) is established as a positive prognostic factor in triple-negative breast cancer (TNBC). The majority of studies have examined the role of TILs in predicting response to chemotherapy, but their role as a general prognostic marker in TNBC is unclear. Moreover, there is a lack of consensus in the literature regarding a suitable cut-off point by which to stratify patients into prognostic groups. Therefore, we sought to confirm the prognostic value of TILs in an independent cohort of unselected TNBCs, and to determine an appropriate cut-off point by which to stratify TIL scores into prognostically significant categories. We used the International TILs Working Group (ITWG) methodology to assess the density of stromal TILs in our cohort of 139 TNBC patients undergoing curative resection at our institution. The percentage TILs scores were categorised first into three groups: low (0-10%), intermediate (15-50%), and high (55-100%). A second binary variable was also created by separating cases into low TILs (≤50%) and high TILs (>50%) groups. Using the three-tiered system, mean disease-free survival was 156, 99 and 94 months for the high, intermediate and low TILs groups, respectively (p=0.030). However, no statistically significant improvement was observed for overall survival. Using the two-tiered system, statistically significant improvements in both overall survival (p=0.030) and disease-free survival (p=0.010) were observed. This survival benefit remained statistically significant in multivariate analyses (p=0.010, p=0.014). We conclude that TILs scored using the ITWG system and dichotomised at a cut-off score of 50%, are a powerful predictor of all-cause and disease-free survival in TNBC regardless of chemotherapy treatment.

Next generation sequencing reveals CCNE1 amplification as an independent prognostic factor for triple negative breast cancer (TNBC) patients.

558 Background: Triple negative breast cancer (TNBC) has the worst prognosis among breast cancer due to the heterogeneity as well as lack of better therapeutic approach. It remains controversial whether BRCA status is the predictor of survival in TNBC. Besides, both germline and somatic mutation may contribute to the prognosis. This study is to explore the potential predictors and therapeutic targets based on genetic data and clinicopathological parameters. Methods: Seventy-five TNBC patients were enrolled with approximately 2:1 based on BRCA status. Genetic data was analysed by comprehensive genomic profiling 508 key cancer related genes. DAVID was applied to perform pathway enrichment analysis of significant enriched genetic alterations. Cox regression model was applied to evaluate disease-free survival (DFS) and overall survival (OS). Immuno-chemistry (IHC) was used to validate clinically meaningful genetic alteration. Results: In this study, 27 germline mutations were detected, including 26 homologous recombination repair (HRR) pathway gene mutations and 1 mismatch repair gene mutation among them 16 BRCA1 mutations and 5 BRCA2 mutations were found. Germline HRR including BRCA1/2 mutation marginally affected DFS ( p = 0.0624 and 0.15, respectively). We found 480 somatic genetic alterations including 110 copy number variations (CNV). The median value of TMB was determined to be 4.1 Muts/Mb which divided 74 TNBC patients into TMB-low (TMB-l) and TMB-high (TMB-h) group. TMB-l group had inferior DFS to TMB-h ( p = 0.0457). CCNE1 (with 5% frequency) copy number gain was specifically enriched in TMB-l group but mutually exclusive with BRCA1/2 mutation. TNBC with CCNE1 gain displayed worse DFS ( p< 0.0001). Cox multivariate regression analysis indicated CCNE1 gain was an independent risk factors for DFS [HR = 13.48 (95% CI 2.62-69.23), p= 0.002)]. Pathway analysis indicated CCNE1 harmed prognosis through regulation of transcription in G1/S phase. Expression of cyclin E1 was validated by IHC, which would be presented later. Conclusions: Comprehensive genomic profiling disclosed various potential prognostic markers for TNBC by integrating clinical characters. Especially, amplified CCNE1 may be a potential prognostic marker and therapeutic target. [Table: see text]

Dynamic Neutrophil-to-Lymphocyte Ratio: A Novel Prognosis Measure for Triple-Negative Breast Cancer.

The neutrophil-to-lymphocyte ratio (NLR) is a measure of systemic inflammation and a prognostic factor for multiple malignancies. This study assesses the value of the NLR as an independent prognostic marker in triple-negative breast cancer (TNBC) and explores the association between dynamic NLR changes and patient outcomes. The study retrospectively analyzed a prospectively maintained database including patients 18 to 80years old with TNBC treated at the authors' institution between 2006 to 2016. Clinical and demographic data were collected, including blood test results and treatments received. Age at diagnosis, stage of disease, and NLR scores were tested for association with overall and disease-free survival in uni- and multivariate Cox models. The inclusion criteria were met by 329 women with a median age of 58. Most of the patients had early-stage disease (30.1% with stage 1 and 47% with stage 2 malignancy). An NLR higher than 2.84 at diagnosis was associated with decreased overall survival (hazard ratio [HR], 1.8; 95% confidence interval [CI], 1.023-3.176), whereas an NLR higher than 7.82 at any time during the follow-up period was a strong predictor of 5-year mortality (HR, 10.76; 95% CI, 4.193-26.58), independent of age or stage of disease. Patients who experienced recurrence had a higher NLR than their counterparts during the 6months before recurrence. The NLR also significantly rose during the final 18months of life (p < 0.01). The NLR is an important prognostic marker in TNBC, both at diagnosis and during the course of the disease. Moreover, dynamic changes in NLR strongly correlate with disease recurrence and the time of death.

Abstract P3-10-03: Exploiting p53-dependent functional duality of estrogen receptor-beta to repurpose tamoxifen for triple negative breast cancer therapy

Abstract Whether estrogen receptor beta (ERβ/ESR2) is a pro- or anti-oncogenic protein in breast cancer has been controversial. ERβ levels are generally high in triple-negative breast cancer (TNBC). Reports including the Cancer Genome Atlas (TCGA) show that about 80% of TNBC express mutant p53 and it is a major driver of these cancers. We tested the hypothesis that WT versus mutant status of p53 will have an important role in determining the duality of ERβ functions. We showed that ERβ directly binds to TP53 in human breast cancer cells. Using glutathione-S-transferase (GST)-pull down and co-imunoprecipitation assays, we have delineated the DNA binding domain (DBD) along with the hinge domain of ERβ and the C-terminal regulatory domain of p53 to be essential for the interaction. The highly sensitive in situ proximity ligation assay (PLA) showed that ERβ is capable of interacting with both wild type (WT) and mutant p53 in breast cancer cells and tumor tissues. ERβ and p53 antibodies validated for specificity were used for all experiments. Combination of proliferation, cell cycle, and apoptosis assays, RNAi technology, quantitative ChIP (qChIP), and real-time PCR (qRT-PCR) showed that ERβ is pro-proliferative in the context of WTp53, whereas it is anti-proliferative in the context of mutant p53 in multiple breast cancer cell lines. The p53-dependent diametrically opposite functions of ERβ were recapitulated in isogenic MDA-MB-231 TNBC cells (generated by CRISPR) that differ only in the presence of WT versus mutant p53. A major gain-of-function of mutant p53 is its ability to bind and inactivate tumor suppressor p73. We show that ERβ binds and sequesters mutant p53 from mutant p53−p73 complex leading to reactivation of p73. Consistent with these observations, immunohistochemistry (IHC) in TNBC patient tumor tissue microarray (TMA) showed that patients with tumors expressing mutant p53 along with high levels of ERβ were of smaller size and lower stage. Complementing these findings, our analysis of the subgroup of the basal-like/TNBC tumors expressing mutant p53 (but not WTp53) in the METABRIC dataset showed that patients with tumors expressing higher levels of ERβ RNA (ESR2) had significantly better breast cancer-specific survival. The finding that ERβ–mutant p53 combination prognosticates survival in TNBC is important as to date there are no effective prognostic markers for TNBC and suggest the potential for using ERβ−mutant p53 combination in stratification of TNBC into therapeutically actionable subgroups.Furthermore, our findings provide a mechanistic explanation for the functional duality of ERβ and the controversy over its pro-versus anti-tumorigenic role.Surprisingly, Tamoxifen (Tam) increased ERβ-mut-p53 interaction in TNBC cells leading to increased transcription of anti-proliferation genes and knockdown experiments showed that the effect on transcription was dependent on both p73 and ERβ. Importantly, when combined with doxorubicin (Adriamycin) Tam decreased several fold the IC50 of doxorubicin resulting in increased apoptosis. The combination was more effective in inhibiting TNBC xenograft tumor growth in vivo compared to either treatment alone. Significant clinical implications of these findings include the potential for treating patients with doxorubicin at much lower dose than what is currently used in the management of TNBC, thereby reducing its major cumulative dose side effects. Importantly, although at present Tam is not indicated for TNBC, our data suggest the possibility for repurposing Tam therapy alone or in combination with chemotherapy to treat TNBC stratified based on ERβ and p53 status. If validated in a clinical trial, our findings could lead to a therapy that is fundamentally better in terms of effectiveness, cost and time needed to reach the TNBC patients. Citation Format: Gokul M Das, Utpal K Mukhopadhyay, Chetan C Oturkar, Christina Adams, Nadi Wickramasekera, Sanjay Bansal, Rajesh Medisetty, Austin Miller, Wendy M Swetzig, Laxmi Silwal-Pandit, Anne-Lise Borresen-Dale, Creighton J Creighton, Jun Hyoung Park, Santhi D Konduri, Alka Mukhopadhyay, Alexander Caradori, Benny Abraham Kaipparettu. Exploiting p53-dependent functional duality of estrogen receptor-beta to repurpose tamoxifen for triple negative breast cancer therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-03.

Genomic Characteristics of Triple-Negative Breast Cancer Nominate Molecular Subtypes That Predict Chemotherapy Response.

The heterogeneity of triple-negative breast cancer (TNBC) poses difficulties for suitable treatment and leads to poor outcome. This study aimed to define a consensus molecular subtype (CMS) of TNBC and thus elucidate genomic characteristics and relevant therapy. We integrated the expression profiles of 957 TNBC samples from published datasets. We identified genomic characteristics of subtype by exploring the pathway activity, microenvironment, and clinical relevance. In addition, drug response (DR) scores (n = 181) were computationally investigated using chemical perturbation gene signatures and validated in our own patient with TNBC (n = 38) who received chemotherapy and organoid biobank data (n = 64). Subsequently, cooperative functions with drugs were also explored. Finally, we classified TNBC into four CMSs: stem-like; mesenchymal-like; immunomodulatory; luminal-androgen receptor. CMSs also elucidated distinct tumor-associated microenvironment and pathway activities. Furthermore, we discovered metastasis-promoting genes, such as secreted phosphoprotein 1 by comparing with primary. Computational DR scores associated with CMS revealed drug candidates (n = 18), and it was successfully evaluated in cisplatin response of both patients and organoids. Our CMS recapitulated in-depth functional and cellular heterogeneity encompassing primary and metastatic TNBC. We suggest DR scores to predict CMS-specific DRs and to be successfully validated. Finally, our approach systemically proposes a relevant therapeutic prediction model as well as prognostic markers for TNBC. IMPLICATIONS: We delineated the genomic characteristic and computational DR prediction for TNBC CMS from gene expression profile. Our systematic approach provides diagnostic markers for subtype and metastasis verified by machine-learning and novel therapeutic candidates for patients with TNBC.

Tumor-infiltrating lymphocytes and CD8+ T cells predict survival of triple-negative breast cancer

PurposeTumor inflammatory response was evaluated as a prognostic feature in triple-negative breast cancer (TNBC) and compared with the clinical prognosticators of breast cancer and selected biomarkers of cancer cell proliferation.MethodsTNBC patients (n = 179) with complete clinical data and up to 18-year follow-up were obtained from Auria biobank, Turku University Hospital, Turku, Finland. Tumor-infiltrating lymphocytes (TILs) and several subtypes of inflammatory cells detected with immunohistochemistry were evaluated in different tumor compartments in full tissue sections and tissue microarrays.ResultsDeficiency of stromal TILs and low number of CD8+ T cells independently predicted mortality in TNBC (HR 2.4, p 0.02 and HR 2.1, p 0.02, respectively). Each 10% decrease in stromal TILs resulted in 20% increased risk of mortality. An average of 13.2-year survival difference was observed between the majority (> 75%) of patients with low (< 14% of TILs) vs high (≥ 14% of TILs) frequency of CD8+ T cells. The prognostic value of TILs and CD8+ T cells varied when evaluated in different tumor compartments. TILs and CD8+ T cells were significantly associated with Securin and Separase, essential regulators of metaphase–anaphase transition of the cell cycle.DiscussionTILs and CD8+ T cells provide additional prognostic value to the established clinical prognostic markers in TNBC. However, possible clinical applications would still benefit from systematic guidelines for evaluating tumor inflammatory response. Increasing understanding on the interactions between the regulation of cancer cell proliferation and inflammatory response may in future advance treatment of TNBC.

Nectin-4 Expression Is an Independent Prognostic Biomarker and Associated With Better Survival in Triple-Negative Breast Cancer.

Background: Triple-negative breast cancer (TNBC) represents about 10-20% of all invasive breast cancers and is associated with a poor prognosis. The nectin cell adhesion protein 4 (Nectin-4) is a junction protein involved in the formation and maintenance of cell junctions. Nectin-4 has previously shown to be expressed in about 60% of TNBC as well as in TNBC metastases, but to be absent in normal breast tissue, which makes it a potential specific target for TNBC therapy. Previous studies have shown an association of Nectin-4 protein expression with worse prognosis in TNBC in a small patient cohort. The aim of our study was to explore the role of Nectin-4 in TNBC and confirm its impact on survival in a larger TNBC patient cohort.Material and Methods: We performed immunohistochemical staining for Nectin-4 on a tissue microarray encompassing 148 TNBC cases with detailed clinical annotation and outcomes data.Results: A high expression of Nectin-4 was present in 86 (58%) of the 148 TNBC cases. In multivariate survival analysis, high expression of Nectin-4 was associated with a significantly better overall survival when compared with low expression of Nectin-4 (p < 0.001). Nectin-4-high expression was also significantly associated with a lower tumor stage (p = 0.025) and pN0 lymph node stage (p = 0.034).Conclusion: Our results confirm that expression of Nectin-4 serves as a potential prognostic marker in TNBC and is associated with a significantly better overall survival. In addition, Nectin-4 represents a potential target in TNBC, and its role in molecular defined breast cancer subtype should be investigated in larger patient cohorts.

The Hedgehog Signaling Pathway: A Viable Target in Breast Cancer?

The hedgehog (Hh) pathway plays a key role in embryonic development and stem cell programs. Deregulation of the Hh pathway is a key driver of basal cell carcinoma, and therapeutic targeting led to approval of Hh inhibitor, vismodegib, in the management of this cancer. The Hh pathway is implicated in other malignancies including hormone receptor (HR+) positive and triple negative breast cancer (TNBC). Hh signaling, which is activated in human mammary stem cells, results in activation of glioma-associated oncogene (GLI) transcription factors. High GLI1 expression correlates with worse outcomes in breast cancer. Non-canonical GLI1 activation is one mechanism by which estrogen exposure promotes breast cancer stem cell proliferation and epithelial–mesenchymal transition. Tamoxifen resistant cell lines show aberrant activation of Hh signaling, and knockdown of Hh pathway inhibited growth of tamoxifen resistant cells. As in other cancers Hh signaling is activated by the PI3K/AKT pathway in these endocrine resistant cell lines. Hh pathway activation has also been reported to mediate chemotherapy resistance in TNBC via various mechanisms including paracrine signaling to tumor micro-environment and selective proliferation of cancer stem cells. Co-activation of Hh and Wnt signaling pathways is a poor prognostic marker in TNBC. Early phase clinical trials are evaluating the combination of smoothened (SMO) inhibitors and chemotherapy in TNBC. In addition to SMO inhibitors like vismodegib and sonidegib, which are in clinical use for basal cell carcinoma, GLI1 inhibitors like GANT58 and GANT61 are in preclinical drug development and might be an effective mechanism to overcome drug resistance in breast cancer. Gene signatures predictive of Hh pathway activation could enrich for patients likely to respond to these agents.

Integrating Germline and Somatic Mutation Information for the Discovery of Biomarkers in Triple-Negative Breast Cancer.

Recent advances in high-throughput genotyping and the recent surge of next generation sequencing of the cancer genomes have enabled discovery of germline mutations associated with an increased risk of developing breast cancer and acquired somatic mutations driving the disease. Emerging evidence indicates that germline mutations may interact with somatic mutations to drive carcinogenesis. However, the possible oncogenic interactions and cooperation between germline and somatic alterations in triple-negative breast cancer (TNBC) have not been characterized. The objective of this study was to investigate the possible oncogenic interactions and cooperation between genes containing germline and somatic mutations in TNBC. Our working hypothesis was that genes containing germline mutations associated with an increased risk developing breast cancer also harbor somatic mutations acquired during tumorigenesis, and that these genes are functionally related. We further hypothesized that TNBC originates from a complex interplay among and between genes containing germline and somatic mutations, and that these complex array of interacting genetic factors affect entire molecular networks and biological pathways which in turn drive the disease. We tested this hypothesis by integrating germline mutation information from genome-wide association studies (GWAS) with somatic mutation information on TNBC from The Cancer Genome Atlas (TCGA) using gene expression data from 110 patients with TNBC and 113 controls. We discovered a signature of 237 functionally related genes containing both germline and somatic mutations. We discovered molecular networks and biological pathways enriched for germline and somatic mutations. The top pathways included the hereditary breast cancer and role of BRCA1 in DNA damage response signaling pathways. In conclusion, this is the first large-scale and comprehensive analysis delineating possible oncogenic interactions and cooperation among and between genes containing germline and somatic mutations in TNBC. Genetic and somatic mutations, along with the genes discovered in this study, will require experimental functional validation in different ethnic populations. Functionally validated genetic and somatic variants will have important implications for the development of novel precision prevention strategies and discovery of prognostic markers in TNBC.

Prognostic value of CD8\u2009+\u2009PD-1+ immune infiltrates and PDCD1 gene expression in triple negative breast cancer

The role of programmed cell death protein-1 (PD-1)/programmed cell death ligand 1 (PD-L1) in triple negative breast cancer (TNBC) remains to be fully understood. In this study, we investigated the role of PD-1 as a prognostic marker for TNBC in an Asian cohort (n = 269). Samples from patients with TNBC were labeled with antibodies against PD-L1 and PD-1, and subjected to NanoString assays to measure the expression of immune-related genes. Associations between disease-free survival (DFS), overall survival (OS) and biomarker expression were investigated. Multivariate analysis showed that tumors with high PD-1+ immune infiltrates harbored significantly increased DFS, and this increase was significant even after controlling for clinicopathological parameters (HR 0.95; P = 0.030). In addition, the density of cells expressing both CD8 and PD-1, but not the density of CD8−PD-1+ immune infiltrates, was associated with improved DFS. Notably, this prognostic significance was independent of clinicopathological parameters and the densities of total CD8+ cell (HR 0.43, P = 0.011). At the transcriptional level, high expression of PDCD1 within the tumor was significantly associated with improved DFS (HR 0.38; P = 0.027). In line with these findings, high expression of IFNG (HR 0.38; P = 0.001) and IFN signaling genes (HR 0.46; p = 0.027) was also associated with favorable DFS. Inclusion of PD-1 immune infiltrates and PDCD1 gene expression added significant prognostic value for DFS (ΔLRχ2 = 6.35; P = 0.041) and OS (ΔLRχ2 = 9.53; P = 0.008), beyond that provided by classical clinicopathological variables. Thus, PD-1 mRNA and protein expression status represent a promising, independent indicator of prognosis in TNBC.

Correction: The prognostic significance of combined androgen receptor, E-Cadherin, Ki67 and CK5/6 expression in patients with triple negative breast cancer.

[This corrects the article DOI: 10.18632/oncotarget.20293.].