Breast Cancer Phenotype Research Articles

SSEA-1 Correlates With the Invasive Phenotype in Breast Cancer.

The glycan moiety Lewis X (LeX) has been implicated in defining progenitor cells as well as playing a role in the progression of solid tumors, including breast cancer. Here, we used the original stage-specific embryonic antigen-1 (SSEA-1) antibody, MC-480, targeting the LeX motif to examine the expression pattern of this marker within the context of a differentiation hierarchy as well as functional properties of breast cancer cells. Immunohistochemical staining revealed the presence of SSEA-1 in a progenitor zone in the normal breast gland. In breast cancer, 81 of 220 carcinomas (37%) were positive for SSEA-1 and a distinct pattern could be correlated to major subtypes. Specifically, estrogen receptor alpha (ERα)-negative tumors showed a higher frequency of SSEA-1 expression compared to ERα-positive tumors, which are generally considered more differentiated (56% vs 29%, p<0.005). Functional assays performed on two representative breast cancer cell lines demonstrated that SSEA-1-expressing cells exhibited cancer stem cell properties as well as having more invasive potential, regardless of ERα status. A potential role of SSEA-1 in metastasis was confirmed by pairwise staining of primary- and corresponding lymph node tumors. Altogether, our data suggest that expression of SSEA-1 in breast cancer contributes to the malignant phenotype.

Role of Cyclin D1b in Inducing Macrophages Toward a Tumor-associated Macrophage-like Phenotype in Murine Breast Cancer.

Tumor-associated macrophages (TAMs) of the M2 phenotype are frequently associated with cancer progression. Invasive cancer cells undergoing epithelial-mesenchymal transition (EMT) have a selective advantage as TAM activators. Cyclin D1b is a highly oncogenic splice variant of cyclin D1. We previously reported that cyclin D1b enhances the invasiveness of breast cancer cells by inducing EMT. However, the role of cyclin D1b in inducing macrophage differentiation toward tumor-associated macrophage-like cells remains unknown. This study aimed to explore the relationship between breast cancer cells overexpressing cyclin D1b and TAMs. Mouse breast cancer 4T1 cells were transfected with cyclin D1b variant and co-cultured with macrophage cells in a Transwell coculture system. The expression of characteristic cytokines in differentiated macrophages was detected using qRT-PCR, ELISA and zymography assay. Tumor-associated macrophage distribution in a transplanted tumor was detected by immunofluorescence staining. The proliferation and migration ability of breast cancer cells was detected using the cell counting kit-8 (CCK-8) assay, wound healing assay, Transwell invasion assay, and lung metastasis assay. Expression levels of mRNAs were detected by qRT-PCR. Protein expression levels were detected by Western blotting. The integrated analyses of The Cancer Genome Atlas (TCGA) datasets and bioinformatics methods were adopted to discover gene expression, gene coexpression, and overall survival in patients with breast cancer. After co-culture with breast cancer cells overexpressing cyclin D1b, RAW264.7 macrophages were differentiated into an M2 phenotype. Moreover, differentiated M2-like macrophages promoted the proliferation and migration of breast cancer cells in turn. Notably, these macrophages facilitated the migration of breast cancer cells in vivo. Further investigations indicated that differentiated M2-like macrophages induced EMT of breast cancer cells accompanied with upregulation of TGF-β1 and integrin β3 expression. Breast cancer cells transfected with cyclin D1b can induce the differentiation of macrophages into a tumor-associated macrophage-like phenotype, which promotes tumor metastasis in vitro and in vivo.

MRI-based breast cancer radiogenomics using RNA profiling: association with subtypes in a single-center prospective study

BackgroundThere are few prospective studies on the correlations between MRI features and whole RNA-sequencing data in breast cancer according to molecular subtypes. The purpose of our study was to explore the association between genetic profiles and MRI phenotypes of breast cancer and to identify imaging markers that influences the prognosis and treatment according to subtypes.MethodsFrom June 2017 to August 2018, MRIs of 95 women with invasive breast cancer were prospectively analyzed, using the breast imaging-reporting and data system and texture analysis. Whole RNA obtained from surgical specimens was analyzed using next-generation sequencing. The association between MRI features and gene expression profiles was analyzed in the entire tumor and subtypes. Gene networks, enriched functions, and canonical pathways were analyzed using Ingenuity Pathway Analysis. The P value for differential expression was obtained using a parametric F test comparing nested linear models and adjusted for multiple testing by reporting Q value.ResultsIn 95 participants (mean age, 53 years ± 11 [standard deviation]), mass lesion type was associated with upregulation of CCL3L1 (sevenfold) and irregular mass shape was associated with downregulation of MIR421 (sixfold). In estrogen receptor-positive cancer with mass lesion type, CCL3L1 (21-fold), SNHG12 (11-fold), and MIR206 (sevenfold) were upregulated, and MIR597 (265-fold), MIR126 (12-fold), and SOX17 (fivefold) were downregulated. In triple-negative breast cancer with increased standard deviation of texture analysis on precontrast T1-weighted imaging, CLEC3A (23-fold), SRGN (13-fold), HSPG2 (sevenfold), KMT2D (fivefold), and VMP1 (fivefold) were upregulated, and IGLC2 (73-fold) and PRDX4 (sevenfold) were downregulated (all, P < 0.05 and Q < 0.1). Gene network and functional analysis showed that mass type estrogen receptor-positive cancers were associated with cell growth, anti-estrogen resistance, and poor survival.ConclusionMRI characteristics are associated with the different expressions of genes related to metastasis, anti-drug resistance, and prognosis, depending on the molecular subtypes of breast cancer.

Surface Stiffness Has No Impact on MCF-7 Sensitivity to Doxorubicin.

Resistance to the chemotherapeutic agents in the clinical management of cancer remains a significant challenge, and the mechanical environment of cancer cells is one of the major determinants of this. Stiffening of the environment is usually associated with increased chemoresistance of cancer cells, although this process depends on the type of cancer. Breast cancer is the most frequently diagnosed cancer, and more than half a million people die from it each year worldwide. In this study, we used the most frequent (70% of diagnosed cases) breast cancer phenotype, representing the MCF-7 cell line, to investigate the influence of surface stiffness on its sensitivity to one of the most commonly used anticancer drugs-doxorubicin. We showed that the mechanical environment affected MCF-7 proliferation, adhesion, and the expression and activation of mitogen-activated protein kinases (MAPKs). Furthermore, the role of MAPKs in response to doxorubicin was dependent on surface stiffness; nevertheless, surface stiffness did not affect MCF-7 resistance to doxorubicin.

Omics-Based Investigations of Breast Cancer.

Breast cancer (BC) is characterized by an extensive genotypic and phenotypic heterogeneity. In-depth investigations into the molecular bases of BC phenotypes, carcinogenesis, progression, and metastasis are necessary for accurate diagnoses, prognoses, and therapy assessments in predictive, precision, and personalized oncology. This review discusses both classic as well as several novel omics fields that are involved or should be used in modern BC investigations, which may be integrated as a holistic term, onco-breastomics. Rapid and recent advances in molecular profiling strategies and analytical techniques based on high-throughput sequencing and mass spectrometry (MS) development have generated large-scale multi-omics datasets, mainly emerging from the three "big omics", based on the central dogma of molecular biology: genomics, transcriptomics, and proteomics. Metabolomics-based approaches also reflect the dynamic response of BC cells to genetic modifications. Interactomics promotes a holistic view in BC research by constructing and characterizing protein-protein interaction (PPI) networks that provide a novel hypothesis for the pathophysiological processes involved in BC progression and subtyping. The emergence of new omics- and epiomics-based multidimensional approaches provide opportunities to gain insights into BC heterogeneity and its underlying mechanisms. The three main epiomics fields (epigenomics, epitranscriptomics, and epiproteomics) are focused on the epigenetic DNA changes, RNAs modifications, and posttranslational modifications (PTMs) affecting protein functions for an in-depth understanding of cancer cell proliferation, migration, and invasion. Novel omics fields, such as epichaperomics or epimetabolomics, could investigate the modifications in the interactome induced by stressors and provide PPI changes, as well as in metabolites, as drivers of BC-causing phenotypes. Over the last years, several proteomics-derived omics, such as matrisomics, exosomics, secretomics, kinomics, phosphoproteomics, or immunomics, provided valuable data for a deep understanding of dysregulated pathways in BC cells and their tumor microenvironment (TME) or tumor immune microenvironment (TIMW). Most of these omics datasets are still assessed individually using distinct approches and do not generate the desired and expected global-integrative knowledge with applications in clinical diagnostics. However, several hyphenated omics approaches, such as proteo-genomics, proteo-transcriptomics, and phosphoproteomics-exosomics are useful for the identification of putative BC biomarkers and therapeutic targets. To develop non-invasive diagnostic tests and to discover new biomarkers for BC, classic and novel omics-based strategies allow for significant advances in blood/plasma-based omics. Salivaomics, urinomics, and milkomics appear as integrative omics that may develop a high potential for early and non-invasive diagnoses in BC. Thus, the analysis of the tumor circulome is considered a novel frontier in liquid biopsy. Omics-based investigations have applications in BC modeling, as well as accurate BC classification and subtype characterization. The future in omics-based investigations of BC may be also focused on multi-omics single-cell analyses.

In Silico Identification of a BRCA1:miR-29:DNMT3 Axis Involved in the Control of Hormone Receptors in BRCA1-Associated Breast Cancers.

Germline inactivating mutations in the BRCA1 gene lead to an increased lifetime risk of ovarian and breast cancer (BC). Most BRCA1-associated BC are triple-negative tumors (TNBC), aggressive forms of BC characterized by a lack of expression of estrogen and progesterone hormone receptors (HR) and HER2. How BRCA1 inactivation may favor the development of such a specific BC phenotype remains to be elucidated. To address this question, we focused on the role of miRNAs and their networks in mediating BRCA1 functions. miRNA, mRNA, and methylation data were retrieved from the BRCA cohort of the TCGA project. The cohort was divided into a discovery set (Hi-TCGA) and a validation set (GA-TCGA) based on the platform used for miRNA analyses. The METABRIC, GSE81002, and GSE59248 studies were used as additional validation data sets. BCs were differentiated into BRCA1-like and non-BRCA1-like based on an established signature of BRCA1 pathway inactivation. Differential expression of miRNAs, gene enrichment analysis, functional annotation, and methylation correlation analyses were performed. The miRNAs downregulated in BRCA1-associated BC were identified by comparing the miRNome of BRCA1-like with non-BRCA1-like tumors from the Hi-TCGA discovery cohort. miRNAs:gene-target anticorrelation analyses were then performed. The target genes of miRNAs downregulated in the Hi-TCGA series were enriched in the BRCA1-like tumors from the GA-TCGA and METABRIC validation data sets. Functional annotation of these genes revealed an over-representation of several biological processes ascribable to BRCA1 activity. The enrichment of genes related to DNA methylation was particularly intriguing, as this is an aspect of BRCA1 functions that has been poorly explored. We then focused on the miR-29:DNA methyltransferase network and showed that the miR-29 family, which was downregulated in BRCA1-like tumors, was associated with poor prognosis in these BCs and inversely correlated with the expression of the DNA methyltransferases DNMT3A and DNMT3B. This, in turn, correlated with the methylation extent of the promoter of HR genes. These results suggest that BRCA1 may control the expression of HR via a miR-29:DNMT3:HR axis and that disruption of this network may contribute to the receptor negative phenotype of tumors with dysfunctional BRCA1.

10-year mastectomy trends among breast cancer phenotypes: A national cohort study.

584 Background: Breast cancer tumor phenotype has prognostic value with triple negative (TN) cancers having higher rates of distant metastases early. While tumor phenotypes are prognostic, published data demonstrates that the choice of local therapy does not affect this predisposition or affect survival. This study was performed to determine whether mastectomy is being performed more frequently for TN or HER2+ phenotypes, relative to hormone receptor positive (HR+) phenotypes despite the lack of benefit this should provide. Methods: Data from the National Cancer Database (NCDB) was analyzed from 2010 through 2019 to assess mastectomy trends and associations with patient and tumor characteristics. Women with invasive breast cancer were included. Women with Stage IV disease were excluded. Patients were categorized as mastectomy or breast conservation surgery. Patient and tumor characteristics were compared across groups using chi-square and Wilcoxon rank sum tests, and a multivariable logistic regression model was fit to assess the association between mastectomy and tumor phenotype controlling for patient and tumor characteristics. Results: 543,590 patients were evaluated. 173,380 (31.9%) patients underwent mastectomy, and 370,210 (68.1%) patients underwent breast conservation surgery. Mean age at diagnosis was 56. There were 425,174 HR+, 64,960 HER2+, and 53,456 TN tumors. The proportion of patients undergoing mastectomy peaked in 2013 at 36.14% before declining. Compared to HR+, HER2+ patients were more likely to undergo mastectomy, OR 1.39 p &lt; 0.0001 (95% CI 1.35 – 1.43); however, there was no significant difference in mastectomy between HR+ patients and TN patients. Compared to whites, black patients were less likely to undergo mastectomy, OR 0.71 p &lt; 0.0001 (95% CI 0.69 – 0.74), and individuals of Hispanic ethnicity less likely to undergo mastectomy, OR 0.92 p &lt; 0.0001 (95% CI 0.89 – 0.95). Compared to private insurance, Medicare had a greater association with mastectomy, OR 1.2 p &lt; 0.0001 (95% CI 1.18 – 1.23). There was no significant difference between other forms of insurance (Medicaid, other government insurance, no insurance) and private insurance. Education and income were not associated with different frequencies of mastectomy. Patients with higher comorbidity scores were more likely to undergo mastectomy. Conclusions: Mastectomy rates have been declining since 2013 at CoC centers. While TN breast cancer is not associated with increased mastectomy percent, mastectomy continues to be performed more frequently for HER2+ positive phenotype when adjusting for tumor and patient characteristics. These data suggest a need for education about HER2 positive phenotype due to a possible lack of understanding about the why such tumors pose a risk, and the role of local therapy in treating them.

Association of the Duffy-null allele and endometrial cancer characteristics: An effort to explore racial disparity in Black women with endometrial cancer.

5603 Background: Efforts are needed to identify factors that account for the disparately high mortality in Black women with endometrial cancer. The Duffy antigen receptor for chemokines (DARC) is expressed on erythrocytes and endothelial cells and modulates levels of inflammatory chemokines in circulation and in tissues. The African ancestry-specific Duffy-null allele, in which erythrocyte expression and function of DARC is lost, is associated with worse breast cancer phenotypes and survival. Here, we characterize Duffy-null status and associated prognostic and clinical outcomes in patients with endometrial cancer. Methods: Patients with endometrial cancer were prospectively consented, and germline DNA was extracted from saliva or blood samples. Duffy-null genotype was determined using an allelic discrimination assay. Associations between Duffy-null genotype status and clinicopathologic characteristics were performed using Fisher’s Exact analysis. Results: 33 patients were enrolled (Table); 17 (51.5%) were Duffy-null, 5 (15.2%) were Duffy-null allele carriers, and 11 (33.3%) were non-carriers. Compared with non-carriers, Duffy-null patients were more likely to be Black (100% v 9.1%, p&lt;0.0001) and have non-endometrioid histology (64.7% v 10%, p=0.0173). There was a trend towards higher rates of grade 3 histology (70.6% v 36.6%), intact tumor mismatch repair (MMR) (86.7% v 54.6%), estrogen receptor (ER) negative tumor status (25% v 10%), advanced stage (37.5% v 18.2%), and positive lymph nodes (25% v 0%) (Table). Over a minimum of 12 months of follow up, only Duffy-null individuals died from disease (4 v 0, p=0.0006). Conclusions: Duffy-null status was associated Black race, aggressive histologic subtypes, intact MMR mechanisms, and worse survival. Further investigation is needed to explore the underlying mechanism of the Duffy-null phenotype on endometrial cancer outcomes to better elucidate risk factors and underlying causes that may contribute to endometrial cancer disparities.[Table: see text]

Simultaneous expression of androgen receptor and tumor-associated neutrophil in breast cancer subtypes.

e12539 Background: Considering the heterogeneity of breast cancer phenotypes and different response rates to treatment, it is essential to determine prognostic markers that could confer therapeutic role as well. In this study, androgen receptor (AR) and tumor-associated neutrophil (TAN) in female breast cancer were investigated and their coherency with different subtypes of breastcancer and pathology findings were assessed. Methods: This cross-sectional study was conducted on patients with Stage I-IV breast cancer who were referred to Borna Pathology Center in Zanjan, Iran during 2018-2021. The tumoral paraffine blocks were evaluated for AR (by the IHC method) and TAN (by morphology and the IHC staining for myeloperoxidase). Relationships in different subtypes and stages were assessed.Using univariate logistic regression between 5 subtypes (defended by ER, PR, HER2, and Ki67), stage, age, metastasis (M), and grade with AR and TAN were studied separately and simultaneously. Results: We collected data from 83 women with Stage I-IV breast cancer with mean ± SD age of diagnosis 49.3±11.2 years. Percentage of AR+, TAN+, and AR+&amp;TAN+ were 71.1, 43.4, and26.5, respectively. The frequency of AR+ and AR-&amp;TAN- was high in hormone-positive subtypes, while TAN+ was frequent in the triple-negative group and AR+&amp;TAN+ in Luminal A and B groups. The relation between age, M, and Stage with AR, TAN, and AR &amp; TAN was not significant. AR was significantly related to subtype and grade. Conclusions: AR+ probability was significantly lower in Triple Negative subtype and poorly differentiated grade. Based on the heterogeneity of the presentation of these markers, survival evaluation can be important in risk assessment of them. Relationship between AR, TAN, and AR&amp;TAN with study variables. [Table: see text]

Application of single-cell RNA sequencing analysis of novel breast cancer phenotypes based on the activation of ferroptosis-related genes

Ferroptosis is distinct from classic apoptotic cell death characterized by the accumulation of reactive oxygen species (ROS) and lipid peroxides on the cell membrane. Increasing findings have demonstrated that ferroptosis plays an important role in cancer development, but the exploration of ferroptosis in breast cancer is limited. In our study, we aimed to establish a ferroptosis activation-related model based on the differentially expressed genes between a group exhibiting high ferroptosis activation and a group exhibiting low ferroptosis activation. By using machine learning to establish the model, we verified the accuracy and efficiency of our model in The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) set and gene expression omnibus (GEO) dataset. Additionally, our research innovatively utilized single-cell RNA sequencing data to systematically reveal the microenvironment in the high and low FeAS groups, which demonstrated differences between the two groups from comprehensive aspects, including the activation condition of transcription factors, cell pseudotime features, cell communication, immune infiltration, chemotherapy efficiency, and potential drug resistance. In conclusion, different ferroptosis activation levels play a vital role in influencing the outcome of breast cancer patients and altering the tumor microenvironment in different molecular aspects. By analyzing differences in ferroptosis activation levels, our risk model is characterized by a good prognostic capacity in assessing the outcome of breast cancer patients, and the risk score can be used to prompt clinical treatment to prevent potential drug resistance. By identifying the different tumor microenvironment landscapes between the high- and low-risk groups, our risk model provides molecular insight into ferroptosis in breast cancer patients.

Metaplastic breast cancer: an all-round multidisciplinary consensus.

Metaplastic breast cancer (MpBC) is a rare and aggressive histologic subtype of breast cancer (BC) characterized by the presence of at least two cellular types, commonly epithelial and mesenchymal components. Despite growing evidence that MpBC is a unique entity, it has long been treated as a variant of nonspecial type (NST) BC. MpBC typically shows the phenotype of triple-negative breast cancer (TNBC), but compared to NST-TNBC, it is a relatively chemorefractory tumor associated with worse outcomes. Therefore, there is an urgent need to develop management guidelines specifically for MpBC to improve the prognosis of patients with early MpBC. This expert consensus aims to guide diagnosis and standardize clinical management of early MpBC among treating physicians. We provide guidance on the challenging radiological and pathological diagnosis of MpBC. Evidence on the involvement of genetic predisposition in the development of MpBC is also explored. We emphasize the importance of a multidisciplinary approach for the treatment of patients with early MpBC. The optimal surgery and radiotherapy approach is presented, as well as the opportunity offered by novel therapeutic approaches to increase treatment response in this chemoresistant subtype. Appropriate management of patients with MpBC is critical to reduce the high risk of local and distant recurrence that characterizes this disease.

PALB2 germline mutations in a large cohort of Middle Eastern breast-ovarian cancer patients

The PALB2 gene is a breast cancer (BC) and ovarian cancer (OC) predisposition gene involved in the homologous recombination repair pathway. However, the prevalence and clinicopathological association of PALB2 pathogenic/likely pathogenic (PV/LPV) variants in Middle East is still not fully explored. Total 918 BC/OC patients from Saudi Arabia were selected for PALB2 mutations screening using capture sequencing technology. Five heterozygous PVs or LPVs were identified in six cases, accounting for 0.65% (6/918) of entire cohort. Two cases (33.3%) harbored PVs and four cases (66.7%) carried LPVs. Four PVs/LPVs (80%) were frameshift along with one novel splicing LPV (c.2835-2_2835-1delinsTT). One recurrent LPV (c.3425delT: p.L1142fs) was identified in two cases. All six affected carriers have breast cancer diagnosis with median age of 39.5 years (range 34–49 years). Only two cases (33%) have documented family history of cancer. Breast cancer phenotype was invasive ductal unilateral cancer in all cases with 66.7% of hormone receptor positive and 16% of triple negative tumors. Germline PVs/LPVs in the PALB2 gene were observed in low frequency of 0.65% in Saudi BC and/or OC. Our study confirms one recurrent LPV and one novel LPV in Saudi breast cancer patients.

72P Correlation of serum cytokine patterns and clinicopathological factors in breast carcinoma patients

Recent evidence support that the immune system has both positive and negative effects on tumorigenesis. Systemic inflammation has been linked to aggressive tumor growth, metastasis, and drug resistance in breast cancer (BC) patients. Therefore, serum cytokines (SC) may represent an exciting biomarker in the monitoring of BC pathogenesis. Pretreatment serum from 204 BC patients (23% Luminal A, 23.6% Luminal B/HER2-, 22.5% Luminal B/HER2+, 10.3% HER2-enriched, and 20.6% Triple-Negative/Basal-like -TNBL-) and 50 healthy donors was collected. Measurement of 62 SC was performed using LEGENDplex immunoassay. Fluorescence intensity was quantified by flow cytometry. The results were correlated with age, tumor size and grade, vascular invasion, necrosis, phenotype, tumor-infiltrating lymphocytes, lymph node status, and Ki67. Statistical analysis was done by Student’s t-test and Mann–Whitney U test. BC patients showed higher levels of SCF, MIP3α, EPO and 4-1BB but lower levels of IL-2RA, IL-8, IL-12p40, IL-18, IL-23, IL-27, PDGF-AA, Galectin 9, PDGF-BB, B7.2, MIP1β and PD1, compared to the healthy group. Furthermore, IL-23, IL-27, and EPO correlated with younger age (p≤0.041), in contrast to Galectin 9, MCP1, IL-2RA, and MIP1β (p≤0.003). IL-12p40 and IFNγ were elevated in cases with grade I tumors (p≤0.05) and, in addition to IL-11 and IL-27 in low Ki67 tumors (p≤0.030). Moreover, IL-12p40 and IL-23 were associated with positive lymph nodes (p≤0.031). In Luminal tumors, we detected high IL-12p40, IL-15, IL-23, IL-27, and IFNγ (p≤0.048). However, only PDGF-BB was seen in non-Luminal tumor patients (p=0.040). IL-12p40, IL-18, IL-23, IL-27, SCF, and EPO were mainly higher in Luminal A, while PDGF-AA in Luminal B/HER2-. Likewise, MIP1β, MIP3α, and EPO were elevated in Luminal B/HER2+ serum, whereas Galectin9, PDGF-BB, IL-2RA, B7.2, SCF, 4-1BB, and PD1 were found in TNBL, with no specific profile for HER2-enriched. Our results showed specific serum profiles of SC among BC phenotypes. Moreover, IL-12p40 and IL-23 were correlated with lymph node involvement in Luminal tumors, especially in Luminal A.

PO-2244 The effect of hyperthermia and radiation therapy sequence on the immune phenotype of breast cancer

PO-2244 The effect of hyperthermia and radiation therapy sequence on the immune phenotype of breast cancer

MiR-22-enriched breast cancer cells display repressed glycolytic metabolism, increased glycogen synthesis, and reduced survival in low glucose conditions.

Breast cancer (BC) is the second leading cause of cancer-related mortality among women. Beyond the established tumourigenic role of genetic mutations, metabolic reprogramming is another key cancer hallmark. Glucose metabolism in particular is known to be prominently altered in tumours, in order to support biomass accumulation and cancer cell survival. The tumor suppressor microRNA (miRNA) miR-22 has been previously associated with a plethora of BC phenotypes such as growth, invasion-metastasis, and regulation of metabolic phenotypes such as lipid and folate metabolism. In this study, we aimed to investigate the role of miR-22 in the regulation of glucose metabolism in BC cells. Here we examined how miR-22 affects glucose metabolism in the MCF-7 BC cells. We found that over-expression of miR-22 caused a reduced glycolytic rate in these cells. Moreover, the miRNA also rendered MCF-7 cells more sensitive to lower glucose levels. We next unbiasedly screened the transcript levels of 84 genes relevant to glucose metabolism using the Human Glucose RT2 Profiler PCR Array. Interestingly, the strongest effect identified by this screen was the upregulation of genes involved in glycogen synthesis and the repression of gene involved in glycogen catabolism. Examination of publicly available transcriptomic datasets confirmed the correlations between expression of miR-22 and these glycogen metabolism genes in BC cells. This study has generated evidence for a regulatory role of miR-22 in glucose and glycogen metabolism, expanding the involvement of this miRNA in BC metabolic reprogramming.

Regional Variation in the Tumor Microenvironment, Immune Escape and Prognostic Factors in Breast Cancer in Sub-Saharan Africa.

The low overall survival rates of patients with breast cancer in sub-Saharan Africa (SSA) are driven by regionally differing tumor biology, advanced tumor stages at diagnosis, and limited access to therapy. However, it is not known whether regional differences in the composition of the tumor microenvironment (TME) exist and affect patients' prognosis. In this international, multicentre cohort study, 1,237 formalin-fixed, paraffin-embedded breast cancer samples, including samples of the "African Breast Cancer-Disparities in Outcomes (ABC-DO) Study," were analyzed. The immune cell phenotypes, their spatial distribution in the TME, and immune escape mechanisms of breast cancer samples from SSA and Germany (n = 117) were investigated using histomorphology, conventional and multiplex IHC, and RNA expression analysis. The data revealed no regional differences in the number of tumor-infiltrating lymphocytes (TIL) in the 1,237 SSA breast cancer samples, while the distribution of TILs in different breast cancer IHC subtypes showed regional diversity, particularly when compared with German samples. Higher TIL densities were associated with better survival in the SSA cohort (n = 400), but regional differences concerning the predictive value of TILs existed. High numbers of CD163+ macrophages and CD3+CD8+ T cells accompanied by reduced cytotoxicity, altered IL10 and IFNγ levels and downregulation of MHC class I components were predominantly detected in breast cancer samples from Western SSA. Features of nonimmunogenic breast cancer phenotypes were associated with reduced patient survival (n = 131). We therefore conclude that regional diversity in the distribution of breast cancer subtypes, TME composition, and immune escape mechanisms should be considered for therapy decisions in SSA and the design of personalized therapies. See related Spotlight by Bergin et al., p. 705.

A Novel Derivative of Curcumol, HCL-23, Inhibits the Malignant Phenotype of Triple-Negative Breast Cancer and Induces Apoptosis and HO-1-Dependent Ferroptosis.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype of breast cancer. Curcumol, as a natural small molecule compound, has potential anti-breast cancer activity. In this study, we chemically synthesized a derivative of curcumol, named HCL-23, by structural modification and explored its effect on and underlying mechanism regarding TNBC progression. MTT and colony formation assays demonstrated that HCL-23 significantly inhibited TNBC cells proliferation. HCL-23 induced G2/M phase cell cycle arrest and repressed the capability of migration, invasion, and adhesion in MDA-MB-231 cells. RNA-seq results identified 990 differentially expressed genes including 366 upregulated and 624 downregulated genes. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) revealed that these differentially expressed genes were obviously enriched in adhesion, cell migration, apoptosis, and ferroptosis. Furthermore, HCL-23 induced apoptosis via the loss of mitochondrial membrane potential and the activation of the caspase family in TNBC cells. In addition, HCL-23 was verified to trigger ferroptosis through increasing cellular reactive oxygen species (ROS), labile iron pool (LIP), and lipid peroxidation levels. Mechanistically, HCL-23 markedly upregulated the expression of heme oxygenase 1 (HO-1), and the knockdown of HO-1 could attenuate ferroptosis induced by HCL-23. In animal experiments, we found that HCL-23 inhibited tumor growth and weight. Consistently, the upregulation of Cleaved Caspase-3, Cleaved PARP, and HO-1 expression was also observed in tumor tissues treated with HCL-23. In summary, the above results suggest that HCL-23 can promote cell death through activating caspases-mediated apoptosis and HO-1-dependent ferroptosis in TNBC. Therefore, our findings provide a new potential agent against TNBC.

XPERT® breast cancer STRAT4 as an alternative method of identifying breast cancer phenotype in Cape Verde (preliminary results).

Breast cancer (BC) is a public health problem in developing countries, including Cape Verde. Immunohistochemistry (IHC) is the gold standard technique used for BC phenotypic characterisation to support efficient therapeutic decisions. However, IHC is a demanding technique that requires knowledge, trained technicians, expensive antibodies and reagents, controls, and results validation. The low number of cases in Cape Verde increases the risk of expiring the validity of the antibodies, and manual procedures often jeopardise the quality of the results. Thus, IHC is limited in Cape Verde, and an alternative technically easy solution is needed. A point-of-care messenger RNA (mRNA) STRAT4 BC assay to assess estrogen (ER), progesterone (PR), hormone growth factor 2 receptor (HER2), and Ki67, using the GeneXpert platform, has been recently validated on tissues from internationally accredited laboratories, showing excellent concordance with IHC results.To assess whether this technology can be implemented in Cape Verde to guide BC treatment we decided to study the level of agreement between the findings yielded by BC STRAT4 and the results are the same cases obtained by IHC. Formalin-fixed and paraffin-embedded (FFPE) tissue samples from 29 Cabo Verdean BC patients diagnosed in Agostinho Neto University Hospital were analysed by applying IHC and BC STRAT4 assay. The time between sample collection and pre-analytic procedures is unknown. All the samples were pre-processed in Cabo Verde (fixed in formalin and embedded in paraffin). IHC studies were performed in referenced laboratories in Portugal. STRAT4 and IHC result concordance was assessed by calculating the percentage of results agreement and Cohen's Kappa (K) statistics. STRAT4 assay failed in 2 out of the 29 analysed samples. Of the 27 successfully analysed samples, STRAT4/IHC results for ER, PR, HER2, and Ki67 were concordant in 25, 24, 25, and 18 cases, respectively. Ki67 was indeterminate in three cases, and PR was indeterminate once.The percentage of agreement between STRAT4 and IHC results for ER, PR, HER2, and Ki67 was 92.59%, 92.31%, 92.59% and 81.82%, respectively. The Cohen's K statistic coefficients for each biomarker were 0.809, 0.845, 0.757 and 0.506, respectively. According to our preliminary results, a point-of-care mRNA STRAT4 BC assay may be an alternative in laboratories unable to provide quality and/or cost-efficient IHC services. However, more data and improvement on sample pre-analytic processes are required to implement this BC STRAT4 Assay in Cape Verde.

Abstract 3598: Cooperative NF-KappaB and Notch1 signaling promotes a macrophage-mediated metastatic phenotype in breast cancer

Abstract Metastasis is a multistep process that leads to the formation of clinically detectable tumor foci at distant organs and, frequently, to patient demise. Only a subpopulation of breast cancer cells within the primary tumor can disseminate systemically and cause metastasis. To disseminate, cancer cells must express MenaINV, an isoform of the actin-regulatory protein, Mena, encoded by the ENAH gene that endows tumor cells with transendothelial migration activity allowing them to enter and exit the blood circulation. We have previously demonstrated that MenaINV mRNA and protein expression is induced in cancer cells by macrophage contact. Here, we report on the discovery of the precise mechanism by which macrophages induce MenaINV expression in tumor cells. We examined the promoter of the human and mouse ENAH gene and discovered a conserved NF-κB transcription factor binding site. Using live imaging of an NF-κB activity reporter and staining of fixed tissues from mouse and human breast cancer we further determined that, for maximal induction of MenaINV in cancer cells, NF-κB needs to cooperate with the Notch1 signaling pathway. Mechanistically, Notch1 signaling does not directly increase MenaINV expression, but instead enhances and sustains NF-κB signaling through retention of p65, an NF-κB transcription factor, in the nucleus of tumor cells, leading to increased MenaINV expression. In mice, these signals are augmented following chemotherapy treatment and abrogated upon macrophage depletion. Targeting Notch1 signaling in vivo decreased NF-κB signaling and MenaINV expression in the primary tumor and decreased metastasis. Altogether, these data uncover mechanistic targets for blocking MenaINV induction that should be explored clinically to decrease cancer cell dissemination and improve survival of patients with metastatic disease. Citation Format: Camille L. Duran, George S. Karagiannis, Xiaoming Chen, Ved P. Sharma, David Entenberg, John S. Condeelis, Maja H. Oktay. Cooperative NF-KappaB and Notch1 signaling promotes a macrophage-mediated metastatic phenotype in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3598.

Abstract 6086: PALB2 germline mutations in a large cohort Middle Eastern breast-ovarian cancer patients

Abstract Background: The PALB2 gene encoding protein is involved in the homologous recombination repair pathway and plays an important role in maintaining genomic integrity. PALB2 heterozygous pathological variants (PV) or likely pathogenic variants (LPV) are associated with increased risk of breast and ovarian cancer. However, the prevalence and clinicopathological association of PALB2 germline PV/LPV in Middle Eastern breast and ovarian cancers is not fully identified. Purpose: We retrospectively screened a cohort of 918 cancer patients (791 breast cancer and 127 ovarian cancer) from Saudi Arabia using targeted capture-based next generation sequencing (NGS) method for PALB2 germline variants regardless the family history of these patients or the presence of other hereditary cancer susceptibility genes mutations. Clinicopathological criteria were also fully analyzed. Results: Five heterozygous PVs or LPVs were identified in six cases, accounting for 0.65% (6/918) of entire cohort. Two cases (33.3%) harbored PVs and four cases (66.7%) carried LPVs. Four PVs/LPVs (80%) were frameshift along with one novel splicing LPV (c.2835-1 C&amp;gt;A). One recurrent LPV (c.3425delT) was identified in two cases. All six affected carriers have breast cancer diagnosis with median age of 39.5 years (range 34-49 years). Only two cases (33%) have documented family history of cancer. Breast cancer phenotype was invasive ductal unilateral cancer in all cases with 66.7% of hormone receptor positive and 16% of triple negative tumors. Conclusion: Germline PVs/LPVs in the PALB2 gene were observed in low frequency of 0.65% in Saudi breast and/or ovarian cancer. Our study confirms one recurrent LPV and one novel LPV in Saudi breast cancer patients. Citation Format: Rong Bu, Abdul K. Sira, Sandeep Parvathareddy, Kaleem Iqbal, Saud Azam, Zeeshan Qadri, Maha Al-Rasheed, Wael Haqawi, Mark Diaz, Ingrid G. Victoria, Khawla S. Al-Kuraya. PALB2 germline mutations in a large cohort Middle Eastern breast-ovarian cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6086.