Prognostic Marker In Breast Cancer Research Articles

Integrated analysis of co-expression and ceRNA network identifies five lncRNAs as prognostic markers for breast cancer.

Long non‐coding RNAs (lncRNAs), which competitively bind miRNAs to regulate target mRNA expression in the competing endogenous RNAs (ceRNAs) network, have attracted increasing attention in breast cancer research. We aim to find more effective therapeutic targets and prognostic markers for breast cancer. LncRNA, mRNA and miRNA expression profiles of breast cancer were downloaded from TCGA database. We screened the top 5000 lncRNAs, top 5000 mRNAs and all miRNAs to perform weighted gene co‐expression network analysis. The correlation between modules and clinical information of breast cancer was identified by Pearson's correlation coefficient. Based on the most relevant modules, we constructed a ceRNA network of breast cancer. Additionally, the standard Kaplan‐Meier univariate curve analysis was adopted to identify the prognosis of lncRNAs. Ultimately, a total of 23 and 5 modules were generated in the lncRNAs/mRNAs and miRNAs co‐expression network, respectively. According to the Green module of lncRNAs/mRNAs and Blue module of miRNAs, our constructed ceRNA network consisted of 52 lncRNAs, 17miRNAs and 79 mRNAs. Through survival analysis, 5 lncRNAs (AL117190.1, COL4A2‐AS1, LINC00184, MEG3 and MIR22HG) were identified as crucial prognostic factors for patients with breast cancer. Taken together, we have identified five novel lncRNAs related to prognosis of breast cancer. Our study has contributed to the deeper understanding of the molecular mechanism of breast cancer and provided novel insights into the use of breast cancer drugs and prognosis.

MiR‑425‑5p is associated with poor prognosis in patients with breast cancer and promotes cancer cell progression by targeting PTEN.

Breast cancer (BC) is the most common invasive cancer in women, and it imposes a heavy burden on patients. microRNAs (miRNAs/miRs) have been found to play an important role in the development of tumors, but their role in the malignant progression of BC is unclear. In the present study, the expression level of miR-425-5p was examined in patients with BC, and its association with prognosis was investigated. In vitro experiments were performed to examine role of miR-425-5p in the development of BC cells. A downstream target gene of miR-425-5p was predicted using a miRNA target prediction tool and validated with a luciferase reporter assay. It was found that miR-425-5p expression was increased in BC tissues and cell lines, and was associated with tumor size, clinical stage, lymph node metastasis, distant metastasis and poor overall survival in patients with BC. Knockdown of miR-425-5p in BC cell lines inhibited proliferation and migration. PTEN was identified as a downstream target gene of miR-425-5p. Overexpression of PTEN was demonstrated to partially inhibit the promotional effect of miR-425-5p on cell proliferation and migration. Taken together, miR-425-5p is associated with poor prognosis, and promotes cell proliferation and migration via PTEN. Thus, miR-425-5p may serve as a therapeutic and prognostic marker for BC.

Targeting the TIGIT-PVR immune checkpoint axis as novel therapeutic option in breast cancer

ABSTRACTImmune checkpoints are intensively investigated as targets in cancer therapy. T-cell immunoreceptor with immunoglobulin (Ig) and ITIM domains (TIGIT) and its ligand poliovirus receptor (PVR) are recently emerging as novel promising targets in immunotherapy. Here, we show that high expression of PVR represents an independent prognostic marker being associated with poor outcome for breast cancer patients. Furthermore, PVR mRNA, as well as protein expression, is associated with more aggressive breast cancer subtypes such as HER2 positive and triple-negative breast cancer. In vitro, blocking TIGIT or PVR resulted in enhanced immune cell-mediated lysis of breast cancer cell lines SKBR-3, MDA-MB-231, MDA-MB-468, and BT549 and additionally increased the cytotoxic effects of a bispecific T cell engager BiTE® antibody construct targeting EGFR. Taken together, our data identify the immune checkpoint factor PVR as a novel prognostic marker in breast cancer and indicate that blocking the TIGIT-PVR axis might represent a novel therapeutic option for the treatment of breast cancer patients.

Improving patient classification and biomarker assessment using Gaussian Mixture Models and Bayes' rule.

In clinical research, determining cutoff values for continuous variables in test results remains challenging, particularly when considering candidate biomarkers or therapeutic targets for disease. Distribution of a continuous variable into two populations is known as dichotomization and has been commonly used in clinical studies. We recently reported a new method for determining multiple cutoffs for continuous variables. The development of this original approach was based on fitting Gaussian Mixture Models (GMM) onto real-world clinical data. We also explored how to leverage Bayesian probability to minimize uncertainty while classifying individual patients into respective subpopulations. In addition, we investigated the performance of the proposed method for the distribution of classical prognostic markers in breast cancer. Finally, we applied the proposed method to analyze a candidate marker and a target for cancer therapy. Here, we present an overview of this method and our prospects for its implementation in biomedical and clinical research.

349P - Polo-like kinase 1 inhibitor onvansertib synergizes with paclitaxel in breast cancer carrying p53 mutation

BackgroundSomatic mutation in TP53 gene (mutp53) is a strong prognostic marker in breast cancer. Triple negative breast cancer (TNBC) is characterized by up to 80% mutp53 and the greatest overall genomic instability among subtypes. Polo-like kinase 1 (PLK1) regulates progression of cells through the G2 phase of the cell cycle. We hypothesize that mutp53 in the context of breast cancer can predict synergy to paclitaxel [p] plus onvansertib [o], an orally available highly selective Plk1 inhibitor. MethodsDrugs were tested in a set of mutp53 breast cancer cells including TNBC SUM149 and SUM159, luminal SUM52 and T47D, and wtp53 TNBC SUM1315, luminal MCF7, and normal breast MCF10A. We performed cell proliferation assays in order to determine the IC50 (Inhibition Concentration of 50%) for each drug. Synergism was determined using the Chou-Talalay method, by determining the combination index (CI). Apoptosis was determined by PARP cleavage proteins detection in cells synchronized with double thymidine block. For the in vivo experiments, mice were injected with 5x106 SUM159 cells and randomly assigned to experimental condition (control, o, p, o+p). The primary endpoint was tumor volume (TV) measured longitudinally. We performed one-sided two-samples Wilcoxon test comparing the means of the adjusted TV measured at 10 days for p and o+p. ResultsTNBC cells were more sensitive to single agent o compared to luminal cells. Breast cell lines with mutp53 (SUM149, SUM159, SUM52 and T47D) were found to have Combination Index (CI) < 1 when treated with o+p. Conversely, in cell line with wtp53 (SUM1315, MCF7, and MCF10A), o+p did not show synergy, CI≥1. Western blot showed prolonged apoptosis for PLK1 inhibition plus taxane compared to PLK1 inhibitor alone, in SUM149 and SUM159. In the xenograft model SUM159, the adjusted 10-day TV for o+p was significantly less than TV for p, p-value = 0.0303. ConclusionsOnvansertib in combination with paclitaxel showed promising results in a subset of breast cancer cells and animal model characterized by the presence of mutp53. Our preclinical results confirm mutp53 as predictive biomarker of synergy and set the feasibility of performing a clinical trial with the combination o+p for mutp53 breast cancer patients. Legal entity responsible for the studyThe authors. FundingK12 Paul Calabresi funding, K12 CA157688. DisclosureM. Erlander: Leadership role, Chief Scientific Officer (CSO): Trovagene Oncology. All other authors have declared no conflicts of interest.

&lt;p&gt;TCDD-Inducible Poly-ADP-Ribose Polymerase (TIPARP), A Novel Therapeutic Target Of Breast Cancer&lt;/p&gt;

BackgroundTIPARP (TCDD-inducible poly-ADP-ribose polymerase), a mono-ADP-ribosyltransferase and a transcriptional repressor of aryl hydrocarbon receptor (AHR), was one of the potential therapeutic targets for human cancers identified by CRISPR–Cas9 screens recently. Studies about TIPARP on cancers are scarce till now, most of which just focus on expressions, while the functions have not been widely reported yet. Moreover, the TIPARP prognostic significance and therapeutic value of breast cancer is also uncertain.MethodsThe present study was performed to comprehensively analyze the expression pattern, prognostic effect, potential therapeutic function of TIPARP in breast cancer by pooling all currently available databases online including Oncomine, UALCAN, bc-GenExMiner, Kaplan–Meier Plotter, COSMIC, UCSC Xena, STRING, DAVID and Comparative Toxicogenomics Database. Further, we also performed several cell biology experiments including RT-qPCR, Western blot and CCK-8 in cellular and clinical sample levels to confirm the conclusions from bioinformatics analysis.ResultsTIPARP was expressed lower in tumor tissues comparing with normal tissues. Meanwhile, several clinical parameters of breast cancer patients were correlated with TIPARP expression. Further, higher TIPARP expression was related to preferable survival. Moreover, the mutations and DNA methylation of TIPARP might contribute to TIPARP dysregulation in breast cancer. Interactors with TIPARP were significantly enriched in telomere maintenance, telomere organization and mainly participated in pathways in cancer. Finally, several common drugs including metformin were observed to up-regulate the expression of TIPARP.ConclusionTIPARP might act as a preferable prognostic marker of breast cancer through multiple biological processes such as DNA methylation, mutation as well as pathway related to telomere and so on. TIPARP could be considered as a potential therapeutic target for breast cancer. However, large-scale and comprehensive research is needed to clarify our results.

Role of miR-10b-5p in the prognosis of breast cancer.

Breast cancer is the leading cause of cancer-related death in women worldwide. Aberrant expression levels of miR-10b-5p in breast cancer has been reported while the molecular mechanism of miR-10b-5p in tumorigenesis remains elusive. Therefore, this study was aimed to investigate the role of miR-10b-5p in breast cancer and the network of its target genes using bioinformatics analysis. In this study, the expression profiles and prognostic value of miR-10b-5p in breast cancer were analyzed from public databases. Association between miR-10b-5p and clinicopathological parameters were analyzed by non-parametric test. Moreover, the optimal target genes of miR-10b-5p were obtained and their expression patterns were examined using starBase and HPA database. Additionally, the role of these target genes in cancer development were explored via Cancer Hallmarks Analytics Tool (CHAT). The protein–protein interaction (PPI) networks were constructed to further investigate the interactive relationships among these genes. Furthermore, GO, KEGG pathway and Reactome pathway analyses were carried out to decipher functions of these target genes. Results demonstrated that miR-10b-5p was down-regulated in breast cancer and low expression of miR-10b-5p was significantly correlated to worse outcome. Five genes, BIRC5, E2F2, KIF2C, FOXM1, and MCM5, were considered as potential key target genes of miR-10b-5p. As expected, higher expression levels of these genes were observed in breast cancer tissues than in normal tissues. Moreover, analysis from CHAT revealed that these genes were mainly involved in sustaining proliferative signaling in cancer development. In addition, PPI networks analysis revealed strong interactions between target genes. GO, KEGG, and Reactome pathway analysis suggested that these target genes of miR-10b-5p in breast cancer were significantly involved in cell cycle. Predicted target genes were further validated by qRT-PCR analysis in human breast cancer cell line MDA-MB-231 transfected with miR-10b mimic or antisense inhibitors. Taken together, our data suggest that miR-10b-5p functions to impede breast carcinoma progression via regulation of its key target genes and hopefully serves as a potential diagnostic and prognostic marker for breast cancer.

Generation of in situ sequencing based OncoMaps to spatially resolve gene expression profiles of diagnostic and prognostic markers in breast cancer.

BackgroundGene expression analysis of breast cancer largely relies on homogenized tissue samples. Due to the high degree of cellular and molecular heterogeneity of tumor tissues, bulk tissue-based analytical approaches can only provide very limited system-level information about different signaling mechanisms and cellular interactions within the complex tissue context.MethodsWe describe an analytical approach using in situ sequencing (ISS), enabling highly multiplexed, spatially and morphologically resolved gene expression profiling. Ninety-one genes including prognostic and predictive marker profiles, as well as genes involved in specific cellular pathways were mapped within whole breast cancer tissue sections, covering luminal A/B-like, HER2-positive and triple negative tumors. Finally, all these features were combined and assembled into a molecular-morphological OncoMap for each tumor tissue.FindingsOur in situ approach spatially revealed intratumoral heterogeneity with regard to tumor subtype as well as to the OncotypeDX recurrence score and even uncovered areas of minor cellular subpopulations. Since ISS-resolved molecular profiles are linked to their histological context, a deeper analysis of the core and periphery of tumor foci enabled identification of specific gene expression patterns associated with these morphologically relevant regions.InterpretationISS generated OncoMaps represent useful tools to extend our general understanding of the biological processes behind tumor progression and can further support the identification of novel therapeutical targets as well as refine tumor diagnostics.FundSwedish Cancerfonden, UCAN, Vetenskapsrådet, Cancer Genomics Netherlands, Iris, Stig och Gerry Castenbäcks Stiftelse, BRECT, PCM Program, King Gustaf V Jubilee Fund, BRO, KI and Stockholm County Council, Alice Wallenberg Foundation.

Circulating Cell Free Nuclear DNA, Mitochondrial DNA and Global DNA Methylation: Potential Noninvasive Biomarkers for Breast Cancer Diagnosis

Eighty seven women with benign breast lesion, 120 patients with breast cancer (BC) and one hundred controls were included in the study. Quantification of mtDNA and nDNA was done by qPCR. Global DNA methylation was measured using ELISA. Circulating cell-free nDNA and mtDNA were significantly elevated in BC and benign breast lesions patients. Global methylation was significantly low in BC patients. Combining the studied parameters in one panel, nDNA/mtDNA/hypomethylation, improved their sensitivity in detecting BC to reach 92.5%. Circulating cell-free nDNA, mtDNA and global DNA hypomethylation can be used as diagnostic and prognostic markers for BC.

Potential prognostic value of PD-L1 and FOXP3 as predictors of relapse in breast cancer

Background: Expression of PD-L1 detected by immunohistochemistry can represent a new hope for cancer management. The role of PD L1 in breast cancer is still unclear. Similarly, is the role of tumor-infiltrating FOXP3 +ve regulatory T (Treg) cells where literature data are conflicting. Our study aimed to evaluate the immunohistochemical expression of PD L1 and FOXP3 in breast cancer, correlate them with clinicopathological parameters as well as evaluating their relation.Methods: This is a retrospective study carried out on 136 breast cancer specimens. Only cases with proved pathological diagnosis of infiltrating duct carcinoma of no special type (NST) were included. Tissue microarray blocks were constructed and immunostained with the polyclonal antibody for PDL1 and monoclonal antibody for FOXP3.Results: Statistically significant correlation was found between high FOXP3 and nearly all adverse prognostic factors including; grade III tumors (p = .003), basal-like subtype(p = .001), high Ki67(p = .001), negative ER status(p = .001), negative PR(p = .028), HER2 expression(p = .04), advanced stage (p = .001), and LN metastases(p = .001). For PDL1, only statistically significant correlation with high Ki67 (p = .018) and advanced stage(p = .03) was found. A statistically significant positive correlation was found between PD L1 and FOXP3(p= .001). No statistically significant correlation was found between both PDL1 and FOXP3 in relation to disease-free survival (DFS) (p = .054). PDL1, age (≥ 50 years), nodal metastases were significant predictors of relapse in breast cancer.Conclusion: The current study supports PDL1 as a predictor of relapse in breast cancer. Additionally, it highlights the synergistic role between PDL1 and FOXP3 in breast cancer microenvironment. Each can be considered as a poor prognostic marker in breast cancer. This raises a concern about the benefit of breast cancer patients from blocking of PDL1 pathway.

High A20 expression negatively impacts survival in patients with breast cancer.

BackgroundA20 protein has ubiquitin-editing activities and acts as a key regulator of inflammation and immunity. Previously, our group showed that A20 promotes tumor metastasis through multi-monoubiquitylation of SNAIL1 in basal-like breast cancer. Here, we investigated survival outcomes in patients with breast cancer according to A20 expression.Patients and methodsWe retrospectively collected tumor samples from patients with breast cancer. Immunohistochemistry (IHC) with an A20-specific antibody was performed, and survival outcomes were analyzed.ResultsA20 expression was evaluated in 442 patients. High A20 expression was associated with advanced anatomical stage and young age. High A20 expression showed significantly inferior recurrence-free-survival and overall-survival (P<0.001 and P<0.001, respectively). Multivariate analysis showed that A20 was an independent prognostic marker for RFS (HRs: 2.324, 95% CIs: 1.446–3.736) and OS (HRs: 2.629, 95% CIs: 1.585–4.361). In human epidermal growth factor receptor 2 (HER2)-positive and triple negative breast cancer (TNBC) subtypes, high A20 levels were associated with poor OS.ConclusionWe found that A20 expression is a poor prognostic marker in breast cancer. The prognostic impact of A20 was pronounced in aggressive tumors, such as HER2-positive and TNBC subtypes. Our findings suggested that A20 may be a valuable target in patients with aggressive breast cancer.

EP3 receptor antagonist L798,106 reduces proliferation and migration of SK-BR-3 breast cancer cells.

Purpose: COX-2 overexpression and elevated levels of prostaglandin E2 (PGE2) play an important role in breast cancer carcinogenesis. Recently, expression of the PGE2 receptor EP3 has been shown to be a positive prognostic factor in breast cancer. This study analyzes the functional aspects of targeting EP3 in breast cancer cell lines.Material and methods: EP3 and EP1 expressions were determined in five breast cancer cell lines on the mRNA- and the protein-level. The selected cell lines were subsequently stimulated for 24–72 hrs with 10–1,000 nM of PGE2, the EP1/EP3 agonist sulprostone and the EP3 antagonist L798,106. Cell proliferation was determined via BrdU-assay, migration via scratch assay, EP3, Gi-protein and p-ERK1/2 expressions via Western blot and cAMP concentrations via ELISA. The Mann–Whitney-U-test was used to test for statistical significance.Results: The cell lines T-47D (EP3 expression 77.7%) and SK-BR-3 (EP3 expression 48.7%) were chosen. EP3 antagonism reduced its expression on SK-BR-3 significantly, while no effect was observed on T-47D. The proliferation and migration of SK-BR-3 cells were significantly reduced due to treatment with the EP1/3 agonist, the EP3 antagonist or a combination of both. Neither agonism nor antagonism influenced cell proliferation or migration in T-47D. In SK-BR-3, EP3 antagonism showed a significant decrease in Gi-protein levels, an increase in cAMP levels, and no significant change in p-ERK1/2 expression.Conclusion: Antagonism of the EP3 receptor results in a reduced proliferation and migration of SK-BR-3 breast cancer cells, potentially mediated via a Gi-protein-cAMP pathway. The results suggest that EP3 plays a role in tumorigenesis. This is in accordance with the cell culture data of other gynecological tumors, but it is conflicting in so far, as positive EP3 expression is clinically a positive prognostic marker in breast cancer. Therefore, other factors may be important in explaining this contradiction.

Abstract 4926: Loss of deiodinase type 3 expression distinguishes patients with poor prognosis in breast cancer

Abstract Background: Breast cancer is a highly heterogeneous disease and the identification of biomarkers that predict tumor biological behavior is warranted in improving patient survival. Thyroid hormones (THs) are critical regulators of cellular processes, and TH status alterations are known to contribute to cancer progression through all the hallmarks of cancer. Clinical studies associate THs levels with breast cancer mortality, and THs have been shown to influence breast cancer proliferation, apoptosis, and migration in in vitro models. Type 3 deiodinase (DIO3) is the main enzyme responsible for TH inactivation and disturbed DIO3 expression has been demonstrated in several human neoplasias. Here, we aimed to evaluate expression patterns and the prognostic significance of DIO3 in breast cancer. Methods: The expression of DIO3 was assessed by immunohistochemistry in a primary cohort of 53 samples of breast tissue and was validated in a second cohort using the RNA-Seq data from TCGA database (BRCA study). We assessed DIO3 expression in both populations according to retrieved clinicopathological information. For methylation analysis, DNA methylation data from the TCGA-BRCA study were obtained. Regulation of DIO3 gene was investigated in MCF-7 and MDA-MB-231 cell lines. Results: DIO3 expression was present in normal and tumoral breast glandular tissue. DIO3 mRNA expression was found to be reduced in tumor samples when compared to normal tissue (p&amp;lt;0.0001). Unexpectedly, loss of DIO3 expression was associated with increased mortality (HR 4.29; 95% CI 1.24-14.7) in the primary cohort. We then validated this finding in the secondary cohort. Patients with low DIO3 expression exhibited reduced overall survival (OS) when compared to those higher levels of DIO3 expression. Five-year OS rates were 90.4% and 77.4% in the high and low expression groups, respectively (p=0.0002). Among patients with metastasis, five-year OS rate was 70% in the high DIO3 group and 0% in the low DIO3 group (p=0.038). Additional experiments were performed to determine DIO3 regulation in breast cancer. In MCF-7 and MDA-MD-231 cells, DIO3 was subject to T3 stimulation and was under the regulation of MAPK pathway, as MAPK inhibition resulted in a 50% reduction of DIO3 expression (p=0.004). Methylation analysis revealed that DIO3 gene promoter is hypermethylated in tumoral cells when compared to normal tissue (p &amp;lt;0.0001). Conclusions: DIO3 is expressed in normal and tumoral breast tissue. Here, we demonstrate a role for DIO3 as a prognostic marker in breast cancer, as loss of DIO3 expression distinguishes breast cancer patients with poor overall survival. Moreover, our fındings suggest the utility of DIO3 expression for stratifıcation of patients with metastasis. The finding that DIO3 gene is regulated by the MAPK pathway and that gene promoter is hypermethylated in breast cancer might have therapeutic implications. Citation Format: Iuri M. Goemann, Vicente R. Marczyk, Mariana Recamonde-Mendoza, Marcia S. Graudenz, Simone M. Wajner, Ana Luiza Maia. Loss of deiodinase type 3 expression distinguishes patients with poor prognosis in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4926.

Tumor budding in invasive carcinoma of breast of No Special Type (NST): Value as a prognostic factor

Context: Tumor budding refers to single or small cluster of tumor cells detached from the main tumor mass in histological sections. In colon cancer high tumor budding is associated with worse prognosis, correlates with metastatic lymph nodes and is included in the protocol for reporting colectomy specimens. We studied tumor budding in mastectomy specimens of cases of breast carcinoma of no special type (NST)to evaluate its utility as a prognostic factor by correlating tumor budding, both intratumoral and peripheral tumoral, with other known prognostic markers of breast cancer like lymph nodal metastasis, tumor size, lymphovascular emboli and grading. Aims:To evaluate tumor budding in invasive ductal carcinoma (IDC) NST with: l.ymph node metastasis; 2.lymphovascular invasion (LVI); 3.Grading by Nottingham modification of Bloom-Richardson (NBR) and other prognostic parameters like age, tumor size, and ER status. Methods and Material: The present study is a prospective study of all Modified Radical Mastectomy (MRM) specimens received and examined from 1st January 2018 to 30thJuly 2018. Along with tumor budding various prognostic parameters were evaluated. Immunohistochemical marker Pancytokeratin was utilized for counting the tumor buds, wherever necessary. Statistical Analysis: Chi Square test was utilized to study significant differences between variables. A p value < 0> Results: A greater number of tumor buds both intratumoral (ITB) and peritumoral (PTB) were associated with lymph node metastasis and lymphatic invasion whereas ITB also correlated with NBR grading. Conclusion: In our study we detected the association of high tumor budding, both ITB and PTB in invasive ductal carcinoma (NST) and also noted association with known poor prognostic markers. Hence our results highlight the importance of tumor budding as a prognostic factor and submit that this histological feature could be included indiagnostic protocols just as in carcinoma of the colon. Keywords:

Screening of differentially expressed genes and identification of NUF2 as a prognostic marker in breast cancer.

The aims of the present study were to screen differentially expressed genes (DEGs) in breast cancer (BC) and investigate NDC80 kinetochore complex component (NUF2) as a prognostic marker of BC in detail. A total of four BC microarray datasets, downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases, were used to screen DEGs. A total of 190 DEGs with the same expression trends were identified in the 4 data-sets, including 65 upregulated and 125 downregulated DEGs. Functional and pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery. The upregulated DEGs were enriched for 10 Gene Ontology (GO) terms and 7 pathways, and the downregulated DEGs were enriched for 10 GO terms and 10 pathways. A protein-protein interaction network containing 149 nodes and 930 edges was constructed using the Search Tool for the Retrieval of Interacting Genes, and 2 functional modules were identified using the MCODE plugin of Cytoscape. Based on an in-depth analysis of module 1 and literature mining, NUF2 was selected for further research. Oncomine database analysis and reverse transcription-quantitative PCR showed that NUF2 is significantly upregulated in BC tissues. In analyses of correlations between NUF2 and clinical pathological characteristics, NUF2 was significantly associated with the malignant features of BC. Using 5 additional datasets from GEO, it was demonstrated that NUF2 has a significant prognostic role in both ER-positive and ER-negative BC. A Gene Set Enrichment Analysis indicated that NUF2 may regulate breast carcinogenesis and progression via cell cycle-related pathways. The results of the present study demonstrated that NUF2 is overexpressed in BC and is significantly associated with its multiple pathological features and prognosis.

High GD2 expression defines breast cancer cells with enhanced invasiveness

IntroductionBreast cancer is the most frequently diagnosed cancer among women. Cancer stem cells (CSCs) are suggested to be responsible for tumor initiation, progression, metastasis, recurrence and drug resistance. This study was conducted to evaluate the clinical significance of GD2, a newly suggested CSC marker and two other traditional CSC markers, CD44 and CD24 in breast cancer patients. Material and methodsA total of 168 primary breast cancer tissues were evaluated in terms of GD2, CD44 and CD24 expression using tissue microarray. Then, the correlation of expression levels of these markers with patients' clinicopathological characteristics was assessed. ResultsHigher GD2 expression was mainly found in patients with advanced histological grade (p = 0.02), presence of lymph node invasion (p = 0.04), larger size of tumors (p = 0.04) and older age (p = 0.04). Breast cancer samples with advanced histological grade also showed higher CD44 (p = 0.03) and CD24 expression (p = 0.05). A significant positive association was found between increased CD24 expression and lymph node involvement (p = 0.01). Furthermore, GD2-high/CD44-high/CD24-low phenotype was frequently seen in breast cancer samples with positive lymph node involvement (p = 0.05). ConclusionIn summary, increased expression of GD2 may define more aggressive tumor behavior in breast cancer. GD2 can well be considered as a diagnostic and prognostic marker in breast cancer.

Role of Sentinel Lymph Node Biopsy and Axillary Node Sampling as a Prognostic Marker in Breast Cancer

Role of Sentinel Lymph Node Biopsy and Axillary Node Sampling as a Prognostic Marker in Breast Cancer

Roadmap of DNA methylation in breast cancer identifies novel prognostic biomarkers

BackgroundBreast cancer is a highly heterogeneous disease resulting in diverse clinical behaviours and therapeutic responses. DNA methylation is a major epigenetic alteration that is commonly perturbed in cancers. The aim of this study is to characterize the relationship between DNA methylation and aberrant gene expression in breast cancer.MethodsWe analysed DNA methylation and gene expression profiles from breast cancer tissue and matched normal tissue in The Cancer Genome Atlas (TCGA). Genome-wide differential methylation analysis and methylation-gene expression correlation was performed. Gene expression changes were subsequently validated in the METABRIC dataset. The Oncoscore tool was used to identify genes that had previously been associated with cancer in the literature. A subset of genes that had not previously been studied in cancer was chosen for further analysis.ResultsWe identified 368 CpGs that were differentially methylated between tumor and normal breast tissue (∆β > 0.4). Hypermethylated CpGs were overrepresented in tumor tissue and were found predominantly (56%) in upstream promoter regions. Conversely, hypomethylated CpG sites were found primarily in the gene body (66%). Expression analysis revealed that 209 of the differentially-methylated CpGs were located in 169 genes that were differently expressed between normal and breast tumor tissue. Methylation-expression correlations were predominantly negative (70%) for promoter CpG sites and positive (74%) for gene body CpG sites. Among these differentially-methylated and differentially-expressed genes, we identified 7 that had not previously been studied in any form of cancer. Three of these, TDRD10, PRAC2 and TMEM132C, contained CpG sites that showed diagnostic and prognostic value in breast cancer, particularly in estrogen-receptor (ER)-positive samples. A pan-cancer analysis confirmed differential expression of these genes together with diagnostic and prognostic value of their respective CpG sites in multiple cancer types.ConclusionWe have identified 368 DNA methylation changes that characterize breast cancer tumor tissue, of which 209 are associated with genes that are differentially-expressed in the same samples. Novel DNA methylation markers were identified, of which cg12374721 (PRAC2), cg18081940 (TDRD10) and cg04475027 (TMEM132C) show promise as diagnostic and prognostic markers in breast cancer as well as other cancer types.

High expression of Linc00959 predicts poor prognosis in breast cancer

BackgroundAccumulating studies have focused on the oncogenic roles of the newly identified lncRNAs in human cancers. The aim of this study was to examine the expression pattern of Linc00959 in BC and to evaluate its biological role and clinical significance in prediction of prognosis.MethodsExpression of Linc00959 was detected in 290 BC tissues by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). We analyzed the relationship between Linc00959 expression and clinic pathological features of BC patients. The correlation was calculated by SPSS software.ResultsOur results revealed that Linc00959 expression was correlated with ER status (p = 0.005), PR status (p = 0.036), Ki67 (p = 0.025) and HER2 status (p = 0.009). The Kaplan–Meier survival curves indicated that the overall survival (OS) (p = 0.022) and relapse-free survival (RFS) (p = 0.002) were significantly poor in high Linc00959 expression BC patients (p = 0.023). Furthermore, the survival analysis by Cox regression showed that Linc00959 served as an independent prognostic marker in breast cancer (p = 0.004).ConclusionOur studies indicate that Linc00959 is significantly associated with poor prognosis and may represent a new marker of prognosis in breast cancer.

Abstract PD2-11: Ki-67 (30-9) scoring and differentiation in Luminal A and Luminal B breast cancer subtypes

Abstract Introduction Ki-67 labeling index is a powerful prognostic marker in breast cancer (BC). It is especially useful in assessing the risk of recurrence for estrogen receptor-positive (ER+) BC, where it may be considered a surrogate of the molecular assays for distinguishing Luminal A-like from Luminal B-like BCs. We evaluated the performance of the VENTANA anti Ki-67 (30-9) rabbit monoclonal antibody in assessing the risk of distant relapses for a large series of patients with ER+ BC treated and followed up in a single Institution. Patients and Methods The initial cohort (9415 patients) comprised all women operated on for early ER+, HER2-negative (HER2-) BC at the European Institute of Oncology (IEO), who did not receive neo-adjuvant treatment1. We subsequently restricted the cohort to 3986 patients operated on between 1998-2002 and for whom long-term follow-up data was available. A case-cohort was built by randomly selecting 17% of the above cohort (679 patients, including 84 with events). Additional 303 patients who developed an event (metastasis in distant organs or death due to BC as primary events) were added to this cohort. Ki-67 was evaluated using the anti-Ki-67 (30-9) antibody (Ventana Medical Systems, Inc., Tucson, AZ) using OptiView IHC DAB detection on the BenchMark ULTRA advanced staining platform. The stained slides were evaluated using the scoring method described by the International Ki-67 in BC Working Group. We considered “Luminal A-like” tumors that were ER+, HER2-, with Ki-67 &amp;lt;14% or with Ki-67 14-19% and PgR ≥20%, and “Luminal B-like” ER+, HER2- tumors with Ki-67 14-19% and PgR &amp;lt;20% or with Ki-67 ≥20%1. The main outcome was distant disease-free survival (DDFS) and was calculated from the date of surgery to the date of any first event or last contact with the patient. Cumulative incidence curves were drawn for patients in the sub-cohort and differences between BC subtypes were assessed using the log-rank test. Multivariable Cox regression with inverse sub-cohort sampling probability weighting was used to evaluate the risk of metastasis or death from BC across groups. Results In the sub-cohort, 400 (58.9%) patients had “luminal A-like” and 279 (41.1%) “luminal B-like” BC. The 10-year cumulative incidence of distant metastasis (or BC related death as first event) in the two groups were respectively 8.2% and 24.5% (log rank P&amp;lt;0.0001) In the whole case-cohort, multivariable analysis confirmed statistically significant increased risk of events for women with “Luminal B-Like” BC compared to women with “Luminal A-Like “BC (HR=1.97; 95% CI 1.38-2.79), after adjustment for pT, pN, PVI and menopausal status. Conclusion Ki-67 evaluated using the VENTANA anti-Ki67 (30-9) antibody, was able to stratify patients with endocrine responsive BC, maximizing the number of those classified as having 'Luminal A-like' intrinsic subtype for whom the use of cytotoxic drugs could be at large avoided. Funding source: Ventana Medical Systems, Inc. References Maisonneuve P, Disalvatore D, Rotmensz N, et al. (2014) Proposed new clinicopathological surrogate definitions of luminal A and luminal B (HER2-negative) intrinsic breast cancer subtypes. Breast Cancer Res 16:R65 Citation Format: Viale G, Hanlon Newell AE, Walker E, Bai I, Russo L, Dell'Orto P, Maisonneuve P. Ki-67 (30-9) scoring and differentiation in Luminal A and Luminal B breast cancer subtypes [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-11.