Clinical Outcome In Breast Cancer Research Articles

Management and Clinical Outcomes of Breast Cancer in Women Diagnosed with Hereditary Cancer Syndromes in a Clinic-Based Sample from Colombia

Management and Clinical Outcomes of Breast Cancer in Women Diagnosed with Hereditary Cancer Syndromes in a Clinic-Based Sample from Colombia

The Impact of the Coexpression of MET and ESR Genes on Prognosticators and Clinical Outcomes of Breast Cancer: An Analysis for the METABRIC Dataset

Purpose. Breast cancer is a heterogeneous disease. Exploring new prognostic and therapeutic targets in patients with breast cancer is essential. This study investigated the expression of MET, ESR1, and ESR2 genes and their association with clinicopathologic characteristics and clinical outcomes in patients with breast cancer. Methods. The METABRIC dataset for breast cancer was obtained from the cBioPortal public domain. Gene expression data for MET, ESR1, and ESR2, as well as the putative copy number alterations (CNAs) for MET were retrieved. Results. The MET mRNA expression levels correlated inversely with the expression levels of ESR1 and positively with the expression levels of ESR2 (r = −0.379, p<0.001 and r = 0.066, and p=0.004, respectively). The ESR1 mRNA expression was significantly different among MET CNAs groups p<0.001. Patients with high MET/ESR1 coexpression had favorable clinicopathologic tumor characteristics and prognosticators compared to low MET/ESR1 coexpression in terms of greater age at diagnosis, reduced Nottingham Prognostic Index, lower tumor grade, hormone receptor positivity, HER2-negative status, and luminal subtype p<0.001. In contrast, patients with high MET/ESR2 coexpression had unfavorable tumor features and advanced prognosticators compared to patients with low MET/ESR2 coexpression p<0.001. No significant difference in overall survival was observed based on the MET/ESR coexpression status. However, when data were stratified based on the treatment type (chemotherapy and hormonal therapy), survival was significantly different based on the coexpression status of MET/ESR. Conclusions. Findings from our study add to the growing evidence on the potential crosstalk between MET and estrogen receptors in breast cancer. The expression of the MET/ESR genes could be a novel prognosticator and calls for future studies to evaluate the impact of combinational treatment approaches with MET inhibitors and endocrine drugs in breast cancer.

Enzyme-free and label-free detection of HER2 in human serum based on Ag+-released hybridization chain reaction signal amplification

The overexpression of human epidermal growth factor receptor 2 (HER2) has been linked to aggressive breast cancer and worse clinical outcomes. However, accurately measuring HER2 remains challenging, due to its low serum content. Herein, we developed a novel method for HER2 detection in serum by the combination of aptamer with C-Ag+-C structure, Ag+-released hybridization chain reaction (Ag+-HCR) and coprecipitation of Ag+ and gold nanoparticles (AuNPs) in the solution including Eosin Y (EY) and AgI colloids. Following the aptamer bound to HER2, signal amplification occurred through the release of numerous Ag+ by Ag+-HCR. This process subsequently induced AuNPs aggregation on the surface of AgI colloids, resulting in reduced fluorescence intensity of EY in the supernatant. The reduced value of EY fluorescence intensity in the supernatant was proportional to the level of HER2. Under optimized reaction conditions, a wide detection range was obtained between 0.09 and 9000 pg/mL with a limit of detection of 11.7 fg/mL in diluted human serum, and 96 serum samples in a 96-well plate could be measured simultaneously by a microtiter plate reader within 2 h. Moreover, the quantitative results of HER2 were consistent with those obtained using the enzyme-linked immunosorbent assay kit (r = 0.9995). Therefore, leveraging the delicate design of Ag+-HCR signal amplification, this enzyme-free, label-free, and effective method has demonstrated to be capable of detecting HER2 in serum due to its high sensitivity. Remarkably, this method may be extended to detect other cancer-related proteins and genes by replacing the corresponding target sequences.

Abstract PO4-05-01: Clinical outcomes of breast cancer and its relationship with access to health care in Brazil: prospective study in HER2 negative/hormone receptor positive metastatic disease - BREAST (BRazilian outcomE for metAStatic breasT cancer)

Abstract Background: Cancer survival is one of the key measures of the effectiveness of cancer services and captures whether people have access to effective treatment. The survival rate of breast cancer in low and middle-income countries is notably different from high-income countries; this disparity in survival reflects limited access to first-line systemic therapy for advanced and metastatic tumors. Recent studies have shown benefit of combining cyclin-dependent kinase 4 and 6 inhibitors (CDK 4/6) with endocrine therapies in hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) advanced or metastatic breast cancer. However, in Brazil, the population has limited access to these drugs, mainly in the public health service. The commercialization of CDK 4/6 inhibitors was only released in the national territory in 2018, but until nowadays it is not accessible for most patients. Objective: The aim of this project is to assess whether patients without private health insurance may experience worse outcomes compared to women with private insurance, primarily due to limited access to a specific class of medication during their treatments. The study seeks to gain insights into the clinical outcomes of patients with HR+ HER2- metastatic breast cancer based on their access to health insurance coverage within a real-world setting. Methods: Multicenter, prospective observational study, with patients divided into two groups: one with patients from the public health system and the second with patients treated at the private service. Whereas that the hazard ratio for the primary outcome in the private system compared to the public health system is like that observed in the PALOMA study (0.58), and that event rates after 18 months of follow-up are also similar (approximately 45% and 35%), then a total of 298 patients will provide the study power of 80%, significance level of 5%. This calculation considers a sample size ratio of 1 patient in the private health system for every 2 patients in the public health system. The inclusion criteria specify women with HR+ HER2- metastatic breast cancer who initiated first-line treatment from 2019. Patients will be followed for 24 months and stratified based on the use or non-use of CDK 4/6 inhibitors. The study was approved by the local ethics committee and registered with Clinical Trials (NCT05559528). Results: A total of 300 patients were evaluated, with 199 (66.3%) in the public health system and 101 (33.7%) in the private service. The mean age was 58 years, and 76.2% were postmenopausal. In terms of metastasis site, 84.7% had non-visceral disease, predominantly with bone metastasis (75.7%), which is an expected characteristic of this type of tumor. The use of CDK 4/6 inhibitors at any point in the treatment of metastatic disease was 7.0% in the public health system versus 90.1% in the private service. As a preliminary outcome, with a median time since the diagnosis of metastatic breast cancer of 22.8 months, a mortality rate of 10% was identified in the public health system group compared to 5% in the private service group. Conclusions: The results of our study highlight the disparity in the treatment of metastatic breast cancer among patients in relation to access to healthcare. Patients dependent on the public health system experience higher mortality rates. These findings are crucial for supporting discussions on improving local policies regarding access to medications that have been proven to enhance the prognosis of these patients. Citation Format: Vanessa Sanvido, Lucíola Pontes, Rachel Machado, Marina Nicola, Jackeline Gomes, Letícia Barbante, Nanci Valeis, Alexandre Cavalcanti, Afonso Nazário. Clinical outcomes of breast cancer and its relationship with access to health care in Brazil: prospective study in HER2 negative/hormone receptor positive metastatic disease - BREAST (BRazilian outcomE for metAStatic breasT cancer) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-05-01.

Abstract PO3-25-01: CTLA4 expression profiles and their association with clinical outcomes of breast cancer

Abstract The cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is involved in the progression of various cancers, but its biological roles in breast cancer (BRCA) remain unclear. Therefore, we performed a systematic multiomic analysis to expound on the prognostic value and underlying mechanism of CTLA4 in BRCA. First, the expression of CTLA4 in pan-cancer was evaluated using the platforms of Oncomine. Afterward, the web-based bioinformatics platforms of GEPIA and UCLAN were used to compare the expression of CTLA4 in breast cancer and normal tissues. Next, we assessed the relationship between CTLA4 mRNA expression and prognosis in breast cancer patients using the PrognoScan Database, Kaplan-Meier Plotter, and R2: Kaplan-Meier Scanner. Additionally, the correlation between CTLA4 expression and immune cell infiltration in breast cancer and functional enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed to elucidate the potential biological functions of CTLA4. Finally, we used the STRINGdb and cytoHubba plugin Cytoscape to create a PPI network and identify known and predicted structural essentials for CTLA4 functions and the top five hub genes. CTLA4 was highly expressed in BRCA tumor tissue compared to normal tissue (p< 0.01). The CTLA4 mRNA levels in BRCA based on breast cancer subtypes of luminal, Human epidermal growth factor receptor 2 (HER-2), and Triple-negative breast cancer (TNBC) were considerably higher than in normal tissues (p< 0.001). However, the overexpression of CTLA4 was associated with a better prognosis in BRCA (P< 0.001), and was correlated with clinicopathological characteristics including age, T-stage, estrogen receptors (ER), progesterone receptors (PR), and PAM50 (P< 0.01). The infiltration of multiple immune cells was associated with increased CTLA4 expression in BRCA (P< 0.001). CTLA4 was highly enriched in antigen binding, immunoglobulin complexes, lymphocyte-mediated immunity, and cytokine-cytokine receptor interaction. This study provides suggestive evidence of the prognostic role of CTLA4 in breast cancer, which may be a therapeutic target for breast cancer. Furthermore, CTLA4 may influence breast cancer prognosis through antigen binding, immunoglobulin complexes, lymphocyte-mediated immunity, and cytokine-cytokine receptor interaction. These findings help us understand how CTLA4 plays a role in breast cancer and set the stage for more research. Citation Format: Woo Chul Noh, Young Bum Yoo, Kyoung Sik Park, Sang Eun Nam, Seung Hwan Lim. CTLA4 expression profiles and their association with clinical outcomes of breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-01.

Abstract PO3-24-09: May ESR1 mutant breast cancer cells influence fibroblast phenotype?

Abstract For almost 50 years, it has been postulated that carcinogenesis relies on the intrinsic genetic abnormalities of tumor cells, but in the last decades, it has been reported that their functional interactions with the tumor microenvironment (TME) might affect therapy response and contribute to the disease progression. Based on these observations, the aim of the present study was to investigate the functional interactions between one of the essential components of TME, the fibroblasts, and breast cancer (BC) cells, expressing mutations in the hormone-binding domain (HBD) of the gene encoding for the estrogen receptor alpha (ESR1). Thus, we employed estrogen receptor-positive MCF-7 BC cells CRISPR engineered to express the Y537S-HBD mutation. Y537S is the most commonly detected mutation harbored in HBD-ESR1 and is a major contributor to endocrine therapy resistance and poor clinical outcome in BC. We collected conditioned media (CM) from BC cells and fibroblasts isolated from primary human tissues (normal fibroblasts, NFs, and cancer-associated fibroblasts, CAFs) in order to assess in vitro co-culture systems that can mimic the complex in vivo TME. It was interesting to observe that CAFs displayed a significant increase in their proliferation and migration when exposed to CM of mutant BC cells with respect to the parental ones. These latter findings fit with the immunofluorescence analysis showing a more evident formation of actin stress fibers. However, proteomic analysis demonstrated, in the same circumstances, more significant changes in NFs than in CAFs. Among the up-regulated proteins revealed by NFs upon Y537S-CM exposure, most of them overlap the up-regulated proteins featuring the intrinsic phenotype of CAFs, mainly involved in the cellular ultrastructural organization (i.e. cytoskeleton, focal adhesion, vesicle, and organelle) as evidenced by Metacore functional analysis. On the other side, we demonstrated that mutant clones showed more aggressive behavior in terms of proliferation, growth, migration, and invasion, compared to the parental counterpart upon exposure to the CM-derived from NFs and CAFs. This led us to further investigate the intrinsic properties of the mutated clones through their genomic and proteomic profiles that evidenced, by using Metacore software, an enrichment of insulin-like growth factor 1 receptor (IGF1R) pathway. The relevance of the latter datum was confirmed by the higher content of the IGF-1 ligand found in the secretome of mutant cells. Thus, the short autocrine loop maintaining IGF1/IGF1R signaling activation in mutant clones might reasonably play an important role in mediating the autocrine and paracrine effects between mutant cells and fibroblasts. For instance, the pharmacological blockade of IGF1R signaling interfered with the reciprocal interactions between fibroblasts and BC cells that sustained tumor growth and progression as evidenced in both “in vitro” and “in vivo” experiments. In conclusion, our study addressed the IGF1R pathway as a potential druggable target helpful to disconnect mutant breast cancer cell-fibroblast crosstalk that potentiates breast tumor growth and progression. Citation Format: Luca Gelsomino, Amanda Caruso, Rocco Malivindi, Adel Elisabetta Leonetti, Emine Tasan, Salvatore Panza, Giuseppina Daniela Naimo, Ines Barone, Cinzia Giordano, Daniela Bonofiglio, Loredana Mauro, Guowei Gu, Suzanne Fuqua, Stefania Catalano, Sebastiano Andò. May ESR1 mutant breast cancer cells influence fibroblast phenotype? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-09.

Abstract PO1-17-05: Real-world effectiveness of palbociclib plus aromatase inhibitors (AI) in metastatic breast cancer patients with cardiovascular diseases

Abstract Background Breast cancer survivors are at risk for mortality from cardiovascular diseases (CVD). CVD and their treatments can impact breast cancer treatment selection and clinical outcomes. A cyclin dependent kinase 4/6 inhibitor (CDK4/6i) combined with endocrine therapy (ET) is more effective than ET alone for hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2) metastatic breast cancer (MBC). CDK4/6i plus ET is now the standard of care in the first-line setting for HR+/HER2 MBC. However, data on the effectiveness of CDK4/6i in MBC patients with CVD are limited. We compared overall survival (OS) and real-world progression-free survival (rwPFS) of palbociclib plus AI (PAL+AI) vs AI alone in HR+/HER2 MBC patients with CVD in routine US clinical practices. Methods The Flatiron Health longitudinal database contains electronic health records from >280 cancer clinics, representing >3 million actively treated cancer patients in the US. Using the Flatiron database, we conducted a retrospective analysis of 469 patients with HR+/HER2 MBC and CVD who started PAL+AI or AI as first-line therapy between February 2015 and March 2020. CVD prior to the initiation of PAL+AI or AI were identified based on their definitions within the National Cancer Institute-Comorbidity Index (NCI-CI), including myocardial infarction, congestive heart failure, peripheral vascular diseases, and cerebrovascular diseases. Patients were assessed from start of PAL+AI or AI to September 30, 2020 (data cutoff date), death, or last visit, whichever came first. OS was defined as months from start of PAL+AI or AI to death. rwPFS was defined as months from start of PAL+AI or AI to death or disease progression, evaluated based on clinical assessment or radiographic scan/tissue biopsy. Stabilized inverse probability treatment weighting (sIPTW) as primary analysis was used to balance baseline demographics and clinical characteristics. Cox proportional-hazards models were used to estimate the relative effectiveness of PAL+AI vs AI alone. Results Of the 469 eligible patients, 160 (34.1%) were treated with PAL+AI and 309 (65.9%) were treated with AI alone. Compared with AI-alone patients, those treated with PAL+AI were younger and were more likely to have de novo MBC, ≥2 metastatic sites, and lung/liver involvement. After sIPTW, patient characteristics were generally balanced. After sIPTW, median OS (95% confidence interval [CI]) was 40.7 months (30.956.0) in PAL+AI patients and 26.5 months (23.337.3) in AI patients (hazard ratio [HR]=0.732, 95% CI=0.5370.997, p=0.0476). Median rwPFS (95% CI) was 20.0 months (11.7–27.5) in PAL+AI patients and 12.5 months (9.718.3) in AI patients (HR=0.679, 95% CI=0.5120.900, p=0.0070). Consistent results were observed with multivariate Cox proportional-hazards models as sensitivity analysis. See Table for baseline patient characteristics and outcome results. Conclusions First-line PAL in combination with AI is associated with prolonged OS and rwPFS in patients with HR+/HER2 MBC and CVD in a real-world setting compared with AI alone. Further studies with larger cohorts and comprehensive assessments of comorbidities are needed to provide additional evidence of outcomes and safety of CDK4/6i plus AI for MBC patients with various comorbid conditions in routine clinical practice. Citation Format: Adam Brufsky, Xianchen Liu, Benjamin Li, Lynn McRoy, Connie Chen, Doris Makari, Rachel Layman, Hope Rugo. Real-world effectiveness of palbociclib plus aromatase inhibitors (AI) in metastatic breast cancer patients with cardiovascular diseases [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-17-05.

Transcriptomic analysis revealed potential regulatory biomarkers and repurposable drugs for breast cancer treatment.

Breast cancer (BC) is the most widespread cancer worldwide. Over 2 million new cases of BC were identified in 2020 alone. Despite previous studies, the lack of specific biomarkers and signaling pathways implicated in BC impedes the development of potential therapeutic strategies. We employed several RNAseq datasets to extract differentially expressed genes (DEGs) based on the intersection of all datasets, followed by protein-protein interaction network construction. Using the shared DEGs, we also identified significant gene ontology (GO) and KEGG pathways to understand the signaling pathways involved in BC development. A molecular docking simulation was performed to explore potential interactions between proteins and drugs. The intersection of the four datasets resulted in 146 DEGs common, including AURKB, PLK1, TTK, UBE2C, CDCA8, KIF15, and CDC45 that are significant hub-proteins associated with breastcancer development. These genes are crucial in complement activation, mitotic cytokinesis, aging, and cancer development. We identified key microRNAs (i.e., hsa-miR-16-5p, hsa-miR-1-3p, hsa-miR-147a, hsa-miR-195-5p, and hsa-miR-155-5p) that are associated with aggressive tumor behavior and poor clinical outcomes in BC. Notable transcription factors (TFs) were FOXC1, GATA2, FOXL1, ZNF24 and NR2F6. These biomarkers are involved in regulating cancer cell proliferation, invasion, and migration. Finally, molecular docking suggested Hesperidin, 2-amino-isoxazolopyridines, and NMS-P715 as potential lead compounds against BC progression. We believe that these findings will provide important insight into the BC progression as well as potential biomarkers and drug candidates for therapeutic development.

Assessment of body composition in breast cancer patients: concordance between transverse computed tomography analysis at the fourth thoracic and third lumbar vertebrae.

Specific body composition markers derived from L3 axial computed tomography (CT) images predict clinical cancer outcomes, including chemotherapy toxicity and survival. However, this method is only applicable to those undergoing lumbar (L3) CT scanning, which is not universally conducted in early breast cancer cases. This study aimed to evaluate CT analysis at T4 as a feasible alternative marker of body composition in breast cancer. All patients participated in the Investigating Outcomes from Breast Cancer: Correlating Genetic, Immunological, and Nutritional (BeGIN) Predictors observational cohort study (REC reference number: 14/EE/1297). Staging chest-abdomen-pelvic CT scan images from 24 women diagnosed with early breast cancer at University Hospital Southampton were analysed. Adipose tissue, skeletal muscle, and muscle attenuation were measured from the transverse CT slices' cross-sectional area (CSA) at T4 and L3. Adipose tissue and skeletal muscle area measurements were adjusted for height. Spearman's rank correlation coefficient analysis was used to determine concordance between body composition measurements using CT analysis at L3 and T4 regions. Derived estimates for total adipose tissue, subcutaneous adipose tissue, and intramuscular adipose tissue mass following adjustment for height were highly concordant when determined from CSAs of CT slices at T4 and L3 (Rs = 0.821, p < 0.001; Rs = 0.816, p < 0.001; and Rs = 0.830, p < 0.001). In this cohort, visceral adipose tissue (VAT) and skeletal muscle estimates following height adjustment were less concordant when measured by CT at T4 and L3 (Rs = 0.477, p = 0.039 and Rs = 0.578, p = 0.003). The assessment of muscle attenuation was also highly concordant when measured by CT at T4 and L3 (Rs = 0.840, p < 0.001). These results suggest that the CT analysis at T4 and L3 provides highly concordant markers for total adipose, subcutaneous adipose, and intramuscular adipose estimation, but not VAT, in this breast cancer population. High concordance between T4 and L3 was also found when assessing skeletal muscle attenuation. Lower concordance was observed for the estimates of skeletal muscle area, potentially explained by differences in the quantity and proportions of axial and appendicular muscle between the thorax and abdomen. Future studies will determine the value of T4 metrics as predictive tools for clinical outcomes in breast cancer.

A Novel Disulfidptosis-related lncRNAs Prognostic Signature for Prognosis Predicting and Immune Microenvironment Characterization in Breast Cancer.

Breast cancer (BRCA) is one of the leading causes of cancer-related death in women. The improvement of the BRCA risk assessment method is of positive clinical significance. Although many clues showed the potential role of disulfidptosis in BRCA as a novel type of programmed cell death, whether disulfidptosis is involved in BRCA tumorigenesis remains unclear. We used LASSO-univariate Cox analysis and multivariate Cox analysis to identify six disulfidptosis-related lncRNAs (DPLs) that correlated with BRCA clinical outcome and confirmed that these DPLs were independent prognostic factors for BRCA (YTHDF3-AS1, AC002398.1, AL451085.2, AC092718.4, AC097662.1 and AC053503.5). The BRCA risk prognosis model was subsequently established based on these DPLs. After verifying the model reliability in predicting prognosis, immune infiltration and somatic mutation analysis showed significant differences in the immune microenvironment and mutation of DPLs by risk stratification. Immunotherapy response and drug resistance analysis suggest the reference value of DPLs in clinical individualized therapy. The abnormal expressions of selected DPLs were further validated by the BRCA cell line experiment. Our results shed new light on the role of DPLs in BRCA.

The relationship between cancer associated fibroblasts biomarkers and prognosis of breast cancer: a systematic review and meta-analysis.

To elucidate the relationship between cancer-associated fibroblast (CAFs) biomarkers and the prognosis of breast cancer patients for individualized CAFs-targeting treatment. PubMed, Web of Science, Cochrane, and Embase databases were searched for CAFs-related studies of breast cancer patients from their inception to September, 2023. Meta-analysis was performed using R 4.2.2 software. Sensitivity analyses were performed to explore the sources of heterogeneity. Funnel plot and Egger's test were used to assess the publication bias. Twenty-seven studies including 6,830 patients were selected. Univariate analysis showed that high expression of platelet-derived growth factor receptor-β (PDGFR-β) (P=0.0055), tissue inhibitor of metalloproteinase-2 (TIMP-2) (P<0.0001), matrix metalloproteinase (MMP) 9 (P<0.0001), MMP 11 (P<0.0001) and MMP 13 (P=0.0009) in CAFs were correlated with reduced recurrence-free survival (RFS)/disease-free survival (DFS)/metastasis-free survival (MFS)/event-free survival (EFS) respectively. Multivariate analysis showed that high expression of α-smooth muscle actin (α-SMA) (P=0.0002), podoplanin (PDPN) (P=0.0008), and PDGFR-β (P=0.0470) in CAFs was associated with reduced RFS/DFS/MFS/EFS respectively. Furthermore, PDPN and PDGFR-β expression in CAFs of poorly differentiated breast cancer patients were higher than that of patients with relatively better differentiated breast cancer. In addition, there is a positive correlation between the expression of PDPN and human epidermal growth factor receptor-2 (HER-2). The high expression of α-SMA, PDPN, PDGFR-β in CAFs leads to worse clinical outcomes in breast cancer, indicating their roles as prognostic biomarkers and potential therapeutic targets.

Abstract A071: Identification and characterization of functionally distinct isoforms of estrogen receptor alpha in breast cancer

Abstract We used long- and short-read RNA sequencing of breast tumor tissue, breast cancer cell lines, and healthy tissues to create a comprehensive annotation of alternative mRNA isoforms of estrogen receptor alpha (ER, gene symbol ESR1) and combined this with functional studies of alternative protein isoforms. The ER is frequently overexpressed in breast cancer and is used in the clinic as a prognostic and treatment-predictive factor. It is also a drug target. The ER is a transcription factor and regulates gene expression in response to estrogen, a hormone that can stimulate breast cell proliferation and contribute to tumor growth. The gene has multiple promoters, and alternative mRNA isoforms have been linked to more aggressive breast tumors, suggesting that the splicing pattern of ER transcripts may influence breast cancer biology and clinical outcome. We cloned and characterized six alternative protein isoforms of ER. They were selected based on expression level in sequencing data, splice junction support, and only contained one alternative splicing event each compared to the full-length ER. Experimental characterization was done in cell lines and included transcription factor activity, alone and upon co-expression with full-length ER, response to the ER-targeting drugs tamoxifen and fulvestrant, as well as subcellular localization. Two alternative isoforms exhibited transcription factor activity, although at lower levels than the full-length ER, while isoforms with larger deletions in the DNA-binding domain or lacking parts of the ligand-binding domain were inactive. One isoform had a dominant negative effect on the activity of the full-length protein. Furthermore, full-length ER and three alternative isoforms were found in both cytoplasm and nucleus, while the remaining isoforms were only present in the cytoplasm. Isoforms capable of entering the nucleus showed increased nuclear localization upon estradiol treatment, with one isoform accumulating significantly higher and one lower than the full-length ER. Co-transfection of full-length ER and isoforms lacking transcription factor activity did not alter their distribution, suggesting a possible role of heterodimerization with full-length ER for the dominant negative isoform in the nucleus. In ongoing experiments, we are studying the regulation, expression, and function of alternative isoforms of ER in cell line models of drug resistance. These findings highlight the complexity and diversity of ER isoforms in breast cancer and suggest that specific isoforms may have distinct functional roles in tumor progression and response to therapy. Our work provides insights into the role of alternative mRNA splicing in breast cancer development and progression. Citation Format: Juliane Albrecht, Carlos Enrique Balcazar, Helena Persson, Völundur Hafstað, Cornelia Börjesson Freitag, Johan Vallon-Christersson, Jari Häkkinen. Identification and characterization of functionally distinct isoforms of estrogen receptor alpha in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A071.

Expression of transglutaminase-2 (TGM2) in the prognosis of female invasive breast cancer

BackgroundTransglutaminase 2 (TGM2) is a protein expressed in several isoforms in both intra- and extra-cellular tissue compartments. It has multiple functions that are important in cancer biology and several small studies have suggested expression of TGM2 in breast cancers is associated with a poorer prognosis. The aim of this study was to evaluate the role of intra-cellular and extra-cellular TGM2 expression in breast cancer and to determine whether there were any differences by hormone receptor status.MethodsWe carried out TGM2 immunostaining of tissue micro-arrays comprising 2169 tumour cores and scored these for both intra- and extra-cellular and expression.ResultsIntra-cellular (tumour cell) TGM2 positivity was associated with a better prognosis (HR = 0.74, 95% CI 0.59–0.92) with a larger effect stronger in hormone-receptor-negative cases (HR = 0.56, 95% CI 0.37–0.85). Extra-cellular (stromal) TGM2 expression was associated with a poorer prognosis (HR = 1.47, 95% CI 1.06–2.03) with a stronger association in hormone-receptor-positive cases (HR = 1.60, 95% CI 1.09–2.34).ConclusionTissue compartment and hormone receptor status differences in the effect of TGM2 expression on clinical outcomes of breast cancer may reflect the different functions of TGM2.

Waist Circumference as a Tool for Identifying Visceral Fat in Women with Non-Metastatic Breast Cancer

Abdominal adiposity is associated with tumor development and poor clinical outcomes in breast cancer (BC) and can be identified by the measurement of waist circumference (WC) and visceral adipose tissue (VAT). This study aimed to evaluate the association between waist circumference (WC) and imaging measurement of central adiposity according to age group in women with BC. Abdominal adiposity was assessed by WC and VAT, obtained by dual-energy X-ray absorptiometry (DXA). Body mass index (BMI) was assessed. The presence of inflammation was investigated by measuring C-Reactive Protein (CRP) levels. Multivariate linear regression models were applied to verify the association between WC and VAT. The significance level adopted for all tests was 5%. This study included 112 women with a mean age of 55.5 ± 11.4 years. After adjusted models, WC remained associated with VAT and for every centimeter increase in WC, there was an increase of 3.12 cm2 (CI: 2.40 − 3.85; p < 0.001) in VAT. WC was associated with VAT in women with breast cancer, proving to be a simple, fast, and noninvasive approach that can be used as a proxy to identify visceral fat.

Contrastive learning-based histopathological features infer molecular subtypes and clinical outcomes of breast cancer from unannotated whole slide images

The artificial intelligence-powered computational pathology has led to significant improvements in the speed and precision of tumor diagnosis, while also exhibiting substantial potential to infer genetic mutations and gene expression levels. However, current studies remain limited in predicting molecular subtypes and clinical outcomes in breast cancer. In this paper, we proposed a weakly supervised contrastive learning framework to address this challenge. Our framework first performed contrastive learning pretraining on a large number of unlabeled patches tiled from whole slide images (WSIs) to extract patch-level features. The gated attention mechanism was leveraged to aggregate patch-level features to produce slide feature that was then applied to various downstream tasks. To confirm the effectiveness of the proposed method, three public cohorts and one external independent cohort of breast cancer have been used to conducted evaluation experiments. The predictive powers of our model to infer gene expression, molecular subtypes, recurrence events and drug responses were validated across cohorts. In addition, the learned patch-level attention scores enabled us to generate heatmaps that were highly consistent with pathologist annotations and spatial transcriptomic data. These findings demonstrated that our model effectively established the high-order genotype-phenotype associations, thereby potentially extend the application of digital pathology in clinical practice.

CTLA4 expression profiles and their association with clinical outcomes of breast cancer: a systemic review.

The cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is involved in the progression of various cancers, but its biological roles in breast cancer (BRCA) remain unclear. Therefore, we performed a systematic multiomic analysis to expound on the prognostic value and underlying mechanism of CTLA4 in BRCA. We assessed the effect of CTLA4 expression on BRCA using a variety of bioinformatics platforms, including Oncomine, GEPIA, UALCAN, PrognoScan database, Kaplan-Meier plotter, and R2: Kaplan-Meier scanner. CTLA4 was highly expressed in BRCA tumor tissue compared to normal tissue (P < 0.01). The CTLA4 messenger RNA levels in BRCA based on BRCA subtypes of Luminal, human epidermal growth factor receptor 2, and triple-negative BRCA were considerably higher than in normal tissues (P < 0.001). However, the overexpression of CTLA4 was associated with a better prognosis in BRCA (P < 0.001) and was correlated with clinicopathological characteristics including age, T stage, estrogen receptors, progesterone receptors, and prediction analysis of microarray 50 (P < 0.01). The infiltration of multiple immune cells was associated with increased CTLA4 expression in BRCA (P < 0.001). CTLA4 was highly enriched in antigen binding, immunoglobulin complexes, lymphocyte-mediated immunity, and cytokine-cytokine receptor interaction. This study provides suggestive evidence of the prognostic role of CTLA4 in BRCA, which may be a therapeutic target for BRCA. Furthermore, CTLA4 may influence BRCA prognosis through antigen binding, immunoglobulin complexes, lymphocyte-mediated immunity, and cytokine-cytokine receptor interaction. These findings help us understand how CTLA4 plays a role in BRCA and set the stage for more research.

GREB1L overexpression is associated with good clinical outcomes in breast cancer

BackgroundBreast cancer is the most common malignant tumor among women worldwide. GREB1L is a protein-coding gene. Previous studies have shown that GREB1L plays a vital role in lung and gastric adenocarcinoma. Currently, there is no relevant report about its role in breast cancer.MethodsThe Cancer Genome Atlas database was used to compare the expression level of GREB1L between tumor and normal tissues. The TISIDB website was used for prognosis analysis. The LinkedOmics database was used to predict the potential biological mechanism of GREB1L in breast cancer. Immunohistochemistry was used to detect the GREB1L expression level in breast tissue. Western blotting was used to detect the GREB1L expression level in cell lines. Transwell assays, CCK-8 cell proliferation assays, and colony formation assays were used to detect the migration, invasion, proliferation, and colony formation abilities of cells. Subcutaneous xenograft models were used to detect the in vivo tumor formation abilities of cells.ResultsGREB1L is highly expressed in breast cancer tissues and breast cancer cells. KEGG enrichment analysis suggested that GREB1L participates in the regulation of the Hedgehog signaling pathway; changes in GREB1L expression affected the migration and invasion abilities of MCF7 and MDA-MB-231 cells. Although changes in GREB1L expression did not affect their proliferation and colony formation abilities in vitro and in vivo, they affected the expression of tumor metastasis-related genes in vivo. The overexpression of GREB1L in breast cancer predicted a favorable prognosis.ConclusionThese results showed that GREB1L is involved in the development of breast cancer, and it may be a potential molecular marker for predicting the prognosis of breast cancer.

Prognostic value analysis of cholesterol and cholesterol homeostasis related genes in breast cancer by Mendelian randomization and multi-omics machine learning.

The high incidence of breast cancer (BC) prompted us to explore more factors that might affect its occurrence, development, treatment, and also recurrence. Dysregulation of cholesterol metabolism has been widely observed in BC; however, the detailed role of how cholesterol metabolism affects chemo-sensitivity, and immune response, as well as the clinical outcome of BC is unknown. With Mendelian randomization (MR) analysis, the potential causal relationship between genetic variants of cholesterol and BC risk was assessed first. Then we analyzed 73 cholesterol homeostasis-related genes (CHGs) in BC samples and their expression patterns in the TCGA cohort with consensus clustering analysis, aiming to figure out the relationship between cholesterol homeostasis and BC prognosis. Based on the CHG analysis, we established a CAG_score used for predicting therapeutic response and overall survival (OS) of BC patients. Furthermore, a machine learning method was adopted to accurately predict the prognosis of BC patients by comparing multi-omics differences of different risk groups. We observed that the alterations in plasma cholesterol appear to be correlative with the venture of BC (MR Egger, OR: 0.54, 95% CI: 0.35-0.84, p<0.006). The expression patterns of CHGs were classified into two distinct groups(C1 and C2). Notably, the C1 group exhibited a favorable prognosis characterized by a suppressed immune response and enhanced cholesterol metabolism in comparison to the C2 group. In addition, high CHG score were accompanied by high performance of tumor angiogenesis genes. Interestingly, the expression of vascular genes (CDH5, CLDN5, TIE1, JAM2, TEK) is lower in patients with high expression of CHGs, which means that these patients have poorer vascular stability. The CAG_score exhibits robust predictive capability for the immune microenvironment characteristics and prognosis of patients(AUC=0.79). It can also optimize the administration of various first-line drugs, including AKT inhibitors VIII Imatinib, Crizotinib, Saracatinib, Erlotinib, Dasatinib, Rapamycin, Roscovitine and Shikonin in BC patients. Finally, we employed machine learning techniques to construct a multi-omics prediction model(Risklight),with an area under the feature curve (AUC) of up to 0.89. With the help of CAG_score and Risklight, we reveal the signature of cholesterol homeostasis-related genes for angiogenesis, immune responses, and the therapeutic response in breast cancer, which contributes to precision medicine and improved prognosis of BC.

Occupational exposure to pesticides dysregulates systemic Th1/Th2/Th17 cytokines and correlates with poor clinical outcomes in breast cancer patients

Pesticides are compounds known to cause immunetoxicity in exposed individuals, which have a potential to substantially modify the prognosis of pathologies dependent on an efficient immune response, such as breast cancer. In this context, we examined the circulating cytokine profile of Th1/Th2/Th17 patterns in women occupationally exposed to pesticides and their correlation with worse prognostic outcomes. Peripheral blood samples were collected from 187 rural working women with breast cancer, occupationally exposed or not to pesticides, to quantify the levels of cytokines IL-1β, IL-12, IL-4, IL-17-A, and TNF -α. Data on the disease profile and clinical outcomes were collected through medical follow-up. IL-12 was reduced in exposed women with tumors larger than 2 cm and in those with lymph node metastases. Significantly reduced levels of IL-17A were observed in exposed patients with Luminal B subtype tumors, with high ki67 proliferation rates, high histological grade, and positive for the progesterone receptor. Reduced IL-4 was also seen in exposed women with lymph node invasion. Our data show that occupational exposure to pesticides induces significant changes in the levels of cytokines necessary for tumor control and correlates with poor prognosis clinical outcomes in breast cancer.

The heterogeneity of tumour immune microenvironment revealing the CRABP2/CD69 signature discriminates distinct clinical outcomes in breast cancer.

It has been acknowledged that the tumour immune microenvironment (TIME) plays a critical role in determining therapeutic responses and clinical outcomes in breast cancer (BrCa). Thus, the identification of the TIME features is essential for guiding therapy and prognostic assessment for BrCa. The heterogeneous cellular composition of the TIME in BrCa by single-cell RNA sequencing (scRNA-seq). Two subtype-special genes upregulated in the tumour-rich subtype and the immune-infiltrating subtype were extracted, respectively. The CRABP2/CD69 signature was established based on CRABP2 and CD69 expression, and its predictive values for the clinical outcome and the neoadjuvant chemotherapy (NAT) responses were validated in multiple cohorts. Moreover, the oncogenic role of CRABP2 was explored in BrCa cells. Based on the heterogeneous cellular composition of the TIME in BrCa, the BrCa samples could be divided into the tumour-rich subtype and the immune-infiltrating subtype, which exhibited distinct prognosis and chemotherapeutic responses. Next, we extracted CRABP2 as the biomarker for the tumour-rich subtype and CD69 as the biomarker for the immune-infiltrating subtype. Based on the CRABP2/CD69 signature, BrCa samples were re-divided into three subtypes, and the CRABP2highCD69low subtype exhibited the worst prognosis and the lowest chemotherapeutic response, while the CRABP2lowCD69high subtype showed the opposite results. Furthermore, CARBP2 functioned as a novel oncogene in BrCa, which promoted tumour cell proliferation, migration, and invasion, and CRABP2 inhibition triggered the activation of cytotoxic T lymphocytes (CTLs). The CRABP2/CD69 signature is significantly associated with the TIME features and could effectively predict the clinical outcome. Also, CRABP2 is determined to be a novel oncogene, which could be a therapeutic target in BrCa.