Adjacent Normal Breast Tissue Research Articles

Perioperative systemic IL-6 and immune-adipose- metabolism transcription in tumour and tumour adjacent breast cancer.

Surgical resection is the primary treatment approach for patients with breast cancer. Despite optimal multimodal treatment, metastatic recurrence remains a risk. Surgery-mediated systemic inflammation and local tissue inflammation generate an immunosuppressive and wound-healing environment that may accelerate cancer recurrence and metastasis post-operatively. Investigating the impact of surgery on local and systemic inflammation may provide knowledge for improvement of patient prognosis and treatment opportunities. Systemic cytokines were quantified in the blood plasma of patients with breast cancer pre-operatively, early post-operatively, and late post-operatively. Early post-operative levels of IL-6 were significantly elevated in patients who underwent mastectomy compared with wide local excision. Post-operative IL-6 levels correlate with clinicopathological features (age and BMI). The transcriptomes of local matched tumour and normal tumour adjacent (normal) breast tissue, from patients with breast cancer, were analysed by RNA-Seq. Elevated gene expressions of IL6, ADIPOQ, FABP4, LPL, PPARG, and CD36 in normal tissue were associated with worse overall survival of patients with ER-positive breast cancer. In tissue with higher expression of IL6 and ADIPOQ, a higher abundance of M2-like macrophage gene expression was identified. This study revealed perioperative systemic dynamics of inflammatory mediators and identified local immune-adipose-metabolism gene expression in tumour-adjacent tissue associated with pro-tumour function.

Nectin-4 expression in primary breast cancer and associated clinical outcomes.

570 Background: Antibody-drug conjugates (ADC) selectively deliver chemotherapy to tumor cells expressing a target antigen. Currently approved ADCs in breast cancer target HER2 or Trop-2. Enfortumab vedotin, an ADC targeting Nectin-4, a cell adhesion molecule, is approved in advanced/metastatic urothelial cancer. This study evaluated the expression landscape of Nectin-4 in breast cancer and its association with clinical outcomes. Methods: Processed mRNAseq data was assessed for expression of the gene encoding Nectin-4 in primary breast cancer tissue and adjacent normal breast tissue from The Cancer Genome Atlas (TCGA), and normal tissue from the Genotype-Tissue Expression (GTEx) Project. Low gene expression was determined as <100 transcripts per million (TPM). TCGA samples had been annotated for putative copy-number alterations using GISTIC 2.0; genes with a score of 2 were marked as amplified. Multivariable Cox regression analysis was used to analyze the association of low vs medium/high gene expression with disease-free survival (DFS) and overall survival (OS), adjusting for age, stage, and subtype. Results: RNA-Seq data was available for 1086 patients with primary breast cancer, including known hormone receptor positive (HR+)/HER2- (n=530), HER2+ (n=112), and triple negative breast cancer (TNBC) (n=120). NECTIN4was amplified in 15% of cases (n=165): 15% (n=78) in HR+, 15% (n=17) in HER2+ and 21% (n=25) in TNBC. NECTIN4 expression was medium/high in 356 (33%) patients: 149 (28%) HR+/HER2- (median 77.8 TPM, IQR 50.1-116.5), 32 (29%) HER2+ (median 73.7 TPM, IQR 51.9-107.8), and 59 (49%) TNBC (median 99.7 TPM, IQR 57.8-148.3). Median expression in normal breast tissue was 47 TPM (TCGA) & 16.74 TPM (GTEx), and highest in normal skin at 205 TPM. In multivariable analysis, medium/high NECTIN4 expression was associated with worse OS (HR 1.96, 95% CI 1.32-2.99, p<0.001). As a continuous variable, increasing NECTIN4 expression levels were associated with worse OS. After correcting for age, stage and subtype, for each 10-unit increase in NECTIN4TPM, the risk of death increased by 5% (HR 1.0050, 95% CI 1.0028 - 1.0072, p < 0.001). Conclusions: NECTIN4 expression was higher in TNBC and associated with worse overall survival. The high expression level in normal skin possibly contributes to the cutaneous toxicity associated with enfortumab vedotin approval and requires careful consideration as ADCs targeting Nectin-4 are developed to target breast cancer.

Abstract PO3-08-08: Basal-Luminal (BL) Cells in Histologically Normal and Malignant Breast Tissue from BRCA1 and BRCA2 Carriers

Abstract Background Single-cell studies of normal breast tissue in BRCA mutation carriers have led to the identification of a candidate cancer precursor cell, the basal-luminal (BL) cell. The localization of BL cells within normal, pre-malignant and malignant breast tissue architecture is not well characterized. Here, using cyclic immunofluorescence (CyCIF), we quantify and localize BL cells within normal and malignant breast tissues from BRCA1 and BRCA2 carriers. Methods Formalin-fixed paraffin-embedded breast tissue sections from 9 BRCA1 and 9 BRCA2 consented patients treated at Dana Farber Cancer Institute (2001-2019) were retrieved from the clinical archives and subjected to CyCIF for luminal and basal markers including CK5, CK14 and CK19. Using one section/case, cells co-expressing basal and luminal cytokeratins (CK14/CK19 and/or CK5/CK19) were visually identified in invasive carcinoma, ductal carcinoma in situ (DCIS) and in adjacent normal breast tissue. In histologically normal tissue, regions of interest (ROIs) with co-expressing cells were classified as terminal duct lobular units (TDLUs) or isolated ducts. Within each ROI, the quantity of BL cells was scored as low (1-5 cells), moderate (6-10 cells), or high ( >10 cells). Results The 9 BRCA1 tumors included 8 invasive ductal carcinomas/no special type and 1 tubular carcinoma. The 9 BRCA2 tumors included 4 invasive ductal carcinomas/no special type and 5 invasive carcinomas with ductal and lobular features. 496 ROIs were evaluated in histologically normal tissue: 297 from BRCA1 cases and 199 from BRCA2 cases. BL cells co-expressing CK14/CK19 and/or CK5/CK19 were identified in normal TDLUs and isolated ducts in both BRCA1 and BRCA2 cases. BL cells were more frequently identified in BRCA2 cases, with at least one BL cell present in 97/297 ROIs (33%) in BRCA1 cases and in 133/199 ROIs (67%) in BRCA2 cases (p< 0.01). 55% of BRCA2 ROIs had >10 BL cells each versus 42% of BRCA1 ROIs; however, this difference was not significant. Four BRCA1 cases showed a diffuse BL cell phenotype within the invasive carcinoma with either a diffuse or scattered BL cell phenotype within the DCIS. The BL cell phenotype was not seen diffusely in invasive carcinoma or DCIS in the other 14 cases; however individual tumor cells or small cell clusters with the BL cell phenotype were occasionally found. Conclusions BL cells are found in histologically normal TDLUs and isolated ducts in breast tissue from BRCA1 and BRCA2 carriers, with greater prevalence in BRCA2 carriers. The BL cell phenotype is present diffusely within tumor cells in almost half of BRCA1-associated cancers and none of the BRCA2-associated cancers in this small cohort. Further study of this interesting cell population is warranted in efforts to understand early changes in epithelium at high risk for the development of cancer. Citation Format: Rodrigo Fonseca Abreu, Tabata Alves Domingos, Roberto Bonfim Pimenta Peixoto, Ashka Patel, Krishan Taneja, McKenna Moore, Alicia Pollaci, Judy Garber, Nomeda Girnius, Deborah Dillon. Basal-Luminal (BL) Cells in Histologically Normal and Malignant Breast Tissue from BRCA1 and BRCA2 Carriers [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 PO3-08-08.

ARHGAP29 Is Involved in Increased Invasiveness of Tamoxifen-resistant Breast Cancer Cells and its Expression Levels Correlate With Clinical Tumor Parameters of Breast Cancer Patients.

Aggressive breast cancer (BC) cells show high expression of Rho GTPase activating protein 29 (ARHGAP29), a negative regulator of RhoA. In breast cancer cells in which mesenchymal transformation was induced, ARHGAP29 was the only one of 32 GTPase-activating enzymes whose expression increased significantly. Therefore, we investigated whether there is a correlation between expression of ARHGAP29 and tumor progression in BC. Since tamoxifen-resistant BC cells exhibit increased mesenchymal properties and invasiveness, we additionally investigated the relationship between ARHGAP29 and increased invasion rate in tamoxifen resistance. The question arises as to whether ARHGAP29 is a suitable prognostic marker for the progression of BC. Tissue microarrays were used to investigate expression of ARHGAP29 in BC and adjacent normal breast tissues. Knockdown experiments using siRNA were performed to investigate the influence of ARHGAP29 and the possible downstream actors RhoC and pAKT1 on invasive growth of tamoxifen-resistant BC spheroids in vitro. Expression of ARHGAP29 was frequently increased in BC tissues compared to adjacent normal breast tissues. In addition, there was evidence of a correlation between high ARHGAP29 expression and advanced clinical tumor stage. Tamoxifen-resistant BC cells show a significantly higher expression of ARHGAP29 compared to their parental wild-type cells. After knockdown of ARHGAP29 in tamoxifen-resistant BC cells, expression of RhoC was significantly reduced. Further, expression of pAKT1 decreased significantly. Invasive growth of three-dimensional tamoxifen-resistant BC spheroids was reduced after knockdown of ARHGAP29. This could be partially reversed by AKT1 activator SC79. Expression of ARHGAP29 correlates with the clinical tumor parameters of BC patients. In addition, ARHGAP29 is involved in increased invasiveness of tamoxifen-resistant BC cells. ARHGAP29 alone or in combination with its downstream partners RhoC and pAKT1 could be suitable prognostic markers for BC progression.

Bioinformatic analysis reveals an association between Metadherin with breast cancer prognosis and tumor immune infiltration

Breast cancer metastasis and invasion are both promoted by the oncoprotein Metadherin (MTDH). However, the the role of Metadherin in breast cancer progression and its role in the immune microenvironment. Are not clear. A bioinformatic analysis was performed to demonstrate the prognostic value of Metadherin in BC. In the present study, we found that Metadherin is overexpressed in BC and is significantly correlated with individual cancer stage, age, subclasses, menopause and nodal metastasis status. Metadherin overexpression was associated with a significant decrease in OS and DSS. Cox multivariate analysis indicated that Metadherin was an independent negative prognostic indicator for OS and DSS. Moreover, Metadherin hypomethylation status was associated with poor prognosis. A negative correlation was also noted between Metadherin overexpression and the number of plasmacytoid dendritic cells, cluster of differentiation 8+ T cells, and natural killer cells. Association patterns varied with different subtypes. Various associations between Metadherin levels and immune cell surface markers were revealed. A total of 40 groups of BC and adjacent normal breast tissue samples were collected. Metadherin mRNA was detected by PCR, and its expression levels in BC tissues were significantly increased compared with those noted in normal tissues. The expression levels of Metadherin were also measured in normal and BC cell lines, respectively, and similar conclusions were obtained. The Metadherin mRNA levels were knocked down in SK-BR3 and MDA-MB-231 cell lines and the cell proliferative and migratory activities were determined using Cell Counting Kit-8 and scratch assays, respectively. The results indicated that the cell proliferative and migratory abilities were reduced following knockdown of Metadherin expression. Therefore, Metadherin may be considered as a novel prognostic biomarker in BC.

Calreticulin, a potential coregulator of immune checkpoints and biomarker associated with tumor microenvironment and clinical prognostic significance in breast invasive carcinoma.

As a promising immune checkpoint of immunogenic cell death (ICD) and multifunctional calcium-binding molecular chaperone, calreticulin (CALR) has been attracting increasing attention. CALR mainly locates in cellular endoplasmic reticulum and significantly affects cell proliferation, invasion, induction of apoptosis, and angiogenesis in breast invasive carcinoma (BRCA). CALR overexpression might be correlated with a worse outcome. Nonetheless, it remains obscure how CALR correlates with immune infiltration and survival prognosis of BRCA. In this study, we investigated CALR expression utilizing RNAseq data from the cancer genome atlas (TCGA) and genotype-tissue expression (GTEx) database. The prognostic value of CALR was analyzed using clinical survival data. Enrichment analysis was conducted using the R package "clusterProfiler." We downloaded the immune cell infiltration score of TCGA samples from published articles and online databases and performed a correlation analysis between immune cell infiltration levels and CALR expression. We further assessed the association between CALR and immunomodulators. Moreover, we also evaluated the expression of CALR in 100 formalin-fixed and paraffin-embedded breast cancer and adjacent normal breast tissue specimens. Our results found that CALR was highly expressed in BRCA, and CALR expression levels differed in pathological stages, T stages, and N stages. Besides, these results suggested that CALR overexpression may have adverse effects on the progression-free interval (PFI) and disease-free interval (DFI), which may be related to tumor proliferation, invasion, and metastasis, leading to tumor deterioration. Meanwhile, immune cell infiltration analysis revealed a correlation between the expression of CALR and the number of neutrophils and dendritic cells, suggesting that CALR was highly correlated with many immunomodulators in BRCA. Our results provide potential biomarkers of CALR in BRCA. CALR may interact synergistically with other immunomodulators to regulate the immune microenvironment, which could be utilized to develop new immunotherapy drugs.

HTRA1 expression is associated with immune-cell infiltration and survival in breast cancer.

High temperature requirement A1 (HTRA1), a member of the HTRA family, is a serine peptidase involved in many crucial bioprocesses such as proliferation, mitochondrial homeostasis, apoptosis, and protein quality control. It also plays an important role in the development of various tumors. However, the potential role and mechanisms of action of HTRA1 in breast cancer (BRCA) remain unclear. We conducted a bioinformatics-based study to investigate HTRA1 expression in BRCA alongside its associations with immune-cell infiltrates and survival outcomes. The expression of HTRA1 in BRCA samples was analyzed using RNAseq datasets from The Cancer Genome Atlas and Gene Expression Omnibus. R software was employed to assess the relationship between HTRA1 expression and clinicopathological characteristics, tumor-infiltrating immune cells, and immunity-associated biomarkers in BRCA. MethSurv and cBioPortal database were utilized to evaluate DNA methylation and genovariation within the HTRA1 DNA. Receiver operating characteristic curves, Kaplan-Meier analysis, and Cox regression were performed to estimate the impact of HTRA1 on diagnosis, prognosis, and response to chemotherapy in BRCA. HTRA1 expression was significantly downregulated in BRCA tissues compared to adjacent normal breast tissue controls. Differentially expressed genes associated with HTRA1 expression primarily enriched in cell proliferation pathways. Furthermore, altered HTRA1 expression significantly correlated with patient age, tumor histological type, T stage, progesterone receptor/estrogen receptor status, and PAM50 subtype of BRCA. Both positive and negative associations were observed between HTRA1 levels and the abundance of different types of immune cells, as well as immune biomarkers, including resting mast cells, follicular helper T cells, PD-L1, p53, and Ki67. Low HTRA1 expression was related with pathological complete response in luminal B BRCA patients undergoing chemotherapy. Additionally, lower HTRA1 expression in BRCA was associated with inferior overall survival and relapse-free survival. HTRA1 expression is associated with immune-cell infiltration, response to chemotherapy, and survival outcomes in BRCA. HTRA1 has the potential to serve as a promising biomarker and therapeutic target moving forward.

Breast tissue analysis using a clinically compatible combined time-resolved fluorescence and diffuse reflectance (TRF-DR) system.

This work aims to develop a clinically compatible system that can perform breast tissue analysis in a more time efficient process than conventional histopathological assessment. The potential for such a system to be used in vivo in the operating room or surgical suite to improve patient outcome is investigated. In this work, 80 matched pairs of invasive ductal carcinoma and adjacent normal breast tissue were measured in a combined time-resolved fluorescence and diffuse reflectance (DA) system. Following measurement, the fluorescence intensity of collagen and flavin adenine dinucleotide (FAD); the fluorescence lifetime of collagen, nicotinamide adenine dinucleotide (NADH), and FAD; the DA; absorption coefficient; and reduced scattering coefficient were extracted. Samples then underwent histological processing and H&E staining to classify composition as tumor, fibroglandular, and/or adipose tissue. Statistically significant differences in the collagen and FAD fluorescence intensity, collagen and FAD fluorescence lifetime, DA, and scattering coefficient were found between each tissue group. The NADH fluorescence lifetime and absorption coefficient were statistically different between the tumor and fibroglandular groups, and the tumor and adipose groups. While many breast tissue analysis studies label fibroglandular and adipose together as "normal" breast tissue, this work indicates that some differences between tumor and fibroglandular tissue are not the same as differences between tumor and adipose tissue. Observations of the reduced scatter coefficient may also indicate further classification to include fibro-adipose may be necessary. Future work would benefit from the additional tissue classification. With observable differences in optical parameters between the three tissue types, this system shows promise as a breast analysis tool in a clinical setting. With further work involving samples of mixed composition, this combined system could potentially be used intraoperatively for rapid margin assessment.

Exploring the presence of bovine leukemia virus among breast cancer tumors in a rural state.

The bovine leukemia virus (BLV) is a deltaretrovirus that causes malignant lymphoma and lymphosarcomas in cattle globally and has high prevalence among large scale U.S. dairy herds. Associations between presence of BLV DNA in human mammary tissue and human breast cancer incidence have been reported. We sought to estimate the prevalence of BLV DNA in breast cancer tissue samples in a rural state with an active dairy industry. We purified genomic DNA from 56 fresh-frozen breast cancer tissue samples (51 tumor samples, 5 samples representing adjacent normal breast tissue) banked between 2016 and 2019. Using nested PCR assays, multiple BLV tax sequence primers and primers for the long terminal repeat (LTR) were used to detect BLV DNA in tissue samples and known positive control samples, including the permanently infected fetal lamb kidney cell line (FLK-BLV) and blood from BLV positive cattle. The median age of patients from which samples were obtained at the time of treatment was 60 (40-93) and all were female. Ninety percent of patients had invasive ductal carcinoma. The majority were poorly differentiated (60%). On PCR assay, none of the tumor samples tested positive for BLV DNA, despite having consistent signals in positive controls. We did not find BLV DNA in fresh-frozen breast cancer tumors from patients presenting to a hospital in Vermont. Our findings suggest a low prevalence of BLV in our patient population and a need to reevaluate the association between BLV and human breast cancer.

Survivin Expression in Luminal Breast Cancer and Adjacent Normal Tissue for Immuno-Oncology Applications.

Survivin (BIRC5) is a tumor-associated antigen (TAA) overexpressed in various tumors but present at low to undetectable levels in normal tissue. Survivin is known to have a high expression in breast cancer (e.g., Ductal Carcinoma in situ (DCIS) and triple negative breast cancer). Previous studies have not compared survivin expression levels in DCIS tumor samples to levels in adjacent, normal breast tissue from the same patient. To ensure the effective use of survivin as a target for T cell immunotherapy of breast cancer, it is essential to ascertain the varying levels of survivin expression between DCIS tumor tissue samples and the adjacent normal breast tissue taken from the same patient simultaneously. Next-generation sequencing of RNA (RNA-seq) in normal breast tissue and tumor breast tissue from five women presenting with DCIS for lumpectomy was used to identify sequence variation and expression levels of survivin. The identity of both tumor and adjacent normal tissue samples were corroborated by histopathology. Survivin was overexpressed in human breast tissue tumor samples relative to the corresponding adjacent human normal breast tissue. Wild-type survivin transcripts were the predominant species identified in all tumor tissue sequenced. This study demonstrates upregulated expression of wild type survivin in DCIS tumor tissue versus normal breast tissue taken from the same patient at the same time, and provides evidence that developing selective cytotoxic T lymphocyte (CTL) immunotherapy for DCIS targeting survivin warrants further study.

Mass Spectrometry Imaging of Lumpectomy Specimens Deciphers Diacylglycerols as Potent Biomarkers for the Diagnosis of Breast Cancer.

Detecting breast tumor markers with a fast turnaround time from frozen sections should foster intraoperative histopathology in breast-conserving surgery, reducing the need for a second operation. Hence, rapid label-free discrimination of the spatially resolved molecular makeup between cancer and adjacent normal breast tissue is of growing importance. We performed desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of fresh-frozen excision specimens, including cancer and paired adjacent normal sections, obtained from the lumpectomy of 73 breast cancer patients. The results demonstrate that breast cancer tissue posits sharp metabolic upregulation of diacylglycerol, a lipid second messenger that activates protein kinase C for promoting tumor growth. We identified four specific sn-1,2-diacylglycerols that outperformed all other lipids simultaneously mapped by the positive ion mode DESI-MSI for distinguishing cancers from adjacent normal specimens. This result contrasts with several previous DESI-MSI studies that probed metabolic dysregulation of glycerophospholipids, sphingolipids, and free fatty acids for cancer diagnoses. A random forest-based supervised machine learning considering all detected ion signals also deciphered the highest diagnostic potential of these four diacylglycerols with the top four importance scores. This led us to construct a classifier with 100% overall prediction accuracy of breast cancer by using the parsimonious set of four diacylglycerol biomarkers only. The metabolic pathway analysis suggested that increased catabolism of phosphatidylcholine in breast cancer contributes to diacylglycerol overexpression. These results open up opportunities for mapping diacylglycerol signaling in breast cancer in the context of novel therapeutic and diagnostic developments, including the intraoperative assessment of breast cancer margin status.

Disequilibrium between BRCA1 and BRCA2 Circular and Messenger RNAs Plays a Role in Breast Cancer.

Breast cancer is a frequent disease for which the discovery of markers that enable early detection or prognostic assessment remains challenging. Circular RNAs (circRNAs) are single-stranded structures in closed loops that are produced by backsplicing. CircRNA and messenger RNA (mRNA) are generated co-transcriptionally, and backsplicing and linear splicing compete against each other. As mRNAs are key players in tumorigenesis, we hypothesize that a disruption of the balance between circRNAs and mRNAs could promote breast cancer. Hence, we developed an assay for a simultaneous study of circRNAs and mRNAs, which we have called splice and expression analyses by exon ligation and high-throughput sequencing (SEALigHTS). Following SEALigHTS validation for BRCA1 and BRCA2, our hypothesis was tested using an independent research set of 95 pairs from tumor and adjacent normal breast tissues. In this research set, ratios of BRCA1 and BRCA2 circRNAs/mRNAs were significantly lower in the tumor breast tissue compared to normal tissue (p = 1.6 × 10-9 and p = 4.4 × 10-5 for BRCA1 and BRCA2, respectively). Overall, we developed an innovative method to study linear splicing and backsplicing, described the repertoire of BRCA1 and BRCA2 circRNAs, including 15 novel ones, and showed for the first time that a disequilibrium between BRCA1 and BRCA2 circRNAs and mRNAs plays a role in breast cancer.

Abstract 6521: Subtype-specific molecular signatures of field cancerization in patients with sporadic breast cancer

Abstract Background: We have previously shown the presence of molecular field cancerization in patients with sporadic breast cancer. Given the heterogeneity of breast cancer, we hypothesized that such field cancerization is likely to be subtype-specific. Methods: RNA exome sequencing was performed on 360 samples obtained from 75 breast cancer patients undergoing mastectomy; 25 cases were selected from each subtype: hormone receptor (HR) positive, Her2 positive, triple negative (TN) breast cancer. Four sites were sampled from each patient: primary tumor (A), adjacent normal parenchyma within 2cm of the tumor (B), 2 separate, histologically normal sites at least 2cm away from the tumor (C &D) or, if available, tissue from the unaffected contralateral breast (E). Normal breast tissue (N) from cancer-free controls was obtained from reduction mammoplasty. Tumor-associated genes (TAGs) were identified by comparison of primary tumor to tissue from cancer free controls. We estimated tumor content that was shared across samples B-E by applying deconvolutional analysis to the tumor associated genes, to calculate indices that ranged from 0, indicating normal, to 1, indicating tumor. Molecular field-associated genes and pathways were identified by Spearman’s correlation coefficient analysis between the differential genes/pathways and ISTC. Results: Across all subtypes, the proportion of tumor content present in the histologically normal breast tissue samples (samples B, C, D, E) obtained from cancer patients ranged from 80% in tissue adjacent to the tumor to approximately 50% in tissue obtained from the contralateral breast. We found over 600 deregulated genes to constitute the molecular field across all of the 3 major subtypes tested (Her2+, HR, and TNBC), of which approximately 20% were shared in the molecular field of all 3 subtypes of breast cancer. Among the 664 genes noted to be part of the molecular field in TNBC, 23.8% were unique to this subtype. In contrast, only 13% of the genes that constituted the field cancerization in the Her2 and HR+ subtypes were subtype-specific. PIP3-AKT, TCR signaling, and DNA synthesis were among the top pathways specifically activated in the molecular field of the Her2+ subtype of breast cancer. The molecular field of HR+ breast cancer was uniquely characterized by upregulation in the mitotic cell cycle, G1- S transition, and RB pathway. The field of cancerization in the TNBC subtype showed an upregulation in MEK-MAPK, mTOR, and JNK-JUN-TAK1 pathways. Conclusions: Our study suggests the presence of a breast cancer molecular field effect that extends beyond the adjacent normal breast tissue and includes the entire mammary gland. A substantial proportion of this field cancerization is subtype specific with uniquely deregulated pathways within each subtype. These findings provide new opportunities for developing subtype-specific chemoprevention strategies. Citation Format: Anjana Bhardwaj, Zhenlin Ju, Constance Albarracin, Celestine Trinidad, Preethi Gunaratne, Jing Wang, Randa El-Zein, Isabelle Bedrosian. Subtype-specific molecular signatures of field cancerization in patients with sporadic 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 6521.

DNA methylation age in paired tumor and adjacent normal breast tissue in Chinese women with breast cancer

BackgroundFew studies have examined epigenetic age acceleration (AA), the difference between DNA methylation (DNAm) predicted age and chronological age, in relation to somatic genomic features in paired cancer and normal tissue, with less work done in non-European populations. In this study, we aimed to examine DNAm age and its associations with breast cancer risk factors, subtypes, somatic genomic profiles including mutation and copy number alterations and other aging markers in breast tissue of Chinese breast cancer (BC) patients from Hong Kong.MethodsWe performed genome-wide DNA methylation profiling of 196 tumor and 188 paired adjacent normal tissue collected from Chinese BC patients in Hong Kong (HKBC) using Illumina MethylationEPIC array. The DNAm age was calculated using Horvath’s pan-tissue clock model. Somatic genomic features were based on data from RNA sequencing (RNASeq), whole-exome sequencing (WES), and whole-genome sequencing (WGS). Pearson’s correlation (r), Kruskal–Wallis test, and regression models were used to estimate associations of DNAm AA with somatic features and breast cancer risk factors.ResultsDNAm age showed a stronger correlation with chronological age in normal (Pearson r = 0.78, P < 2.2e−16) than in tumor tissue (Pearson r = 0.31, P = 7.8e−06). Although overall DNAm age or AA did not vary significantly by tissue within the same individual, luminal A tumors exhibited increased DNAm AA (P = 0.004) while HER2-enriched/basal-like tumors exhibited markedly lower DNAm AA (P = < .0001) compared with paired normal tissue. Consistent with the subtype association, tumor DNAm AA was positively correlated with ESR1 (Pearson r = 0.39, P = 6.3e−06) and PGR (Pearson r = 0.36, P = 2.4e−05) gene expression. In line with this, we found that increasing DNAm AA was associated with higher body mass index (P = 0.039) and earlier age at menarche (P = 0.035), factors that are related to cumulative exposure to estrogen. In contrast, variables indicating extensive genomic instability, such as TP53 somatic mutations, high tumor mutation/copy number alteration burden, and homologous repair deficiency were associated with lower DNAm AA.ConclusionsOur findings provide additional insights into the complexity of breast tissue aging that is associated with the interaction of hormonal, genomic, and epigenetic mechanisms in an East Asian population.

Abstract P4-08-03: Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC

Abstract Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC Background and Premise: Triple-negative breast cancer (TNBC) is an aggressive, metastatic disease with high mortality. The standard-of-care neoadjuvant chemotherapy (NACT) in TNBC shows an initial response but followed by an increase in tumor relapse and distant metastases. Furthermore, metastatic TNBC (mTNBC) patients often develop resistance to NACT and targeted therapies. Immunotherapy with therapeutic antibodies such as anti-programmed death ligand-1 (PD-L1) has shown efficacy in treating a subset of TNBC patients, but currently only 20% of patients qualify for that treatment. Hence, there is an unmet need for developing a precision approach against novel actionable targets along with immunotherapy to treat mTNBC. Eukaryotic initiation factor 4A1 (eIF4A1) is an integral part of the translational machinery for many oncogenic mRNAs including survivin, c-MYC, Rho kinase1, and Cyclins D1 and D3, which all require the helicase activity of eIF4A1 for their translation. Our lab previously established that the eIF4A1 is a vulnerable target in breast cancer stem-like cells (BCSCs) and bulk tumor cells. We further demonstrated a key role for eIF4A1 in cancer stemness and drug resistance. Interestingly, we also observed a reduction in levels of PD-L1 when eIF4A1 is pharmacologically inhibited or genetically ablated. Many downstream effectors of eIF4A1 such as c-MYC, STAT1 and ARF6 (involved in recycling of PD-L1 back to the plasma membrane) are indeed upstream regulators of PD-L1 gene expression and cell surface expression of PD-L1. In this study, we examine human TNBC biospecimens for total eIF4A1 level, levels of STAT1, c-MYC, ARF6 and PD-L1. This will help establish the differential operation of the eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant primary and mTNBC. Approach: Human biospecimens were obtained from CHTN NIH repositories and now we have a collection of more than 100 TNBC tumor samples as well as some lymph node, lung and brain metastases and one sample of male breast primary tumor. These specimens are analyzed for role of eIF4A1 in translation of oncogenic mRNAs that contribute to the gene expression of immune checkpoint PD-L1 and other emerging immune checkpoint markers such as VISTA, TIGIT, and LAG3. To mimic the inclusion criteria for current PD-L1 monoclonal antibody treatment, TNBC tumors are examined for PD-L1 expression via immunohistochemistry after subjecting the samples to deglycosylation for effective detection and also by immunoblotting. Results: The levels of total eIF4A1 from 16 matched, therapy naive TNBC tissue were compared to adjacent normal breast tissue from the same patient via immunoblotting. Total eIF4A1 levels were found to be significantly elevated in TNBC compared to adjacent normal tissue. The downstream effectors of eIF4A1 such as STAT1 and ARF6 were also significantly elevated indicating a robust helicase activity of eIF4A1 in tumor samples. Importantly, PD-L1 levels was found to be elevated. Additionally, the total eIF4A1 level from tumor lysates from TNBC patients that had received a variety of NACT (i.e., patients with demonstrated drug resistance) were compared against normal untreated breast tissue from volunteers who underwent reduction mammoplasty by immunoblotting. The total eIF4A1 was found to be elevated in drug-resistant TNBC specimens compared to control, therapy-naïve TNBC samples. Conclusion: The eIF4A1-STAT1-PD-L1 axis is highly active in TNBC especially in drug-resistant situations. Currently, we are evaluating emerging immune checkpoints such as VISTA, TIGIT, and LAG3. Citation Format: Andrew Boring, Dharmindra Dulal, Dayanidhi Raman. Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-03.

Abstract P1-05-33: RNA in Extracellular vesicles used for the prediction of distant metastasis of breast cancer and the function of TGM2 in promoting breast cancer development

Abstract Background: Extracellular vesicles (EVs) can be released by living cells during the process of tumor metastasis. Since EVs could contain various of molecular with high stability, a growing number of evidences indicated that those molecular can be utilized as biomarkers of liquid biopsy for metastatic prediction. Therefore, we aimed to identify genes and build a model that can predict the risk of metastasis in breast cancer patients with acceptable sensitivity and accuracy. Methods: Pretreatment plasma EV-RNAs of patients diagnosed at Fudan University Shanghai Cancer Center from 2017 to 2018 were extracted and analyzed by next generation sequencing (NGS) and bioinformatics, including breast cancer patients (n=114) and benign cases (n=18). Weighted correlation network analysis (WGCNA) was utilized to determine the relationship between clinical features and genes. The Kaplan-Meier was used for survival analysis in public database. By 100 times of 5 folds cross-validation, we used logistic regression analysis to set up the model. The receiver operating characteristic curve (ROC) and area under curve (AUC) were used to assess the predicted capacity of the model. QRT-PCR was conducted to further confirm the expression of genes selected by the predicting model in 175 breast cancer patients with and without metastasis, along with 5 EV-RNAs of tumor samples and paired normal adjacent breast tissues. The functions of candidate genes on cell proliferation, metastasis, and invasion were determined by a series of in vitro experiments in cancer lines. RNA sequencing and bioinformatic analysis were performed to explore the mechanism of the candidate genes. Moreover, co-immunoprecipitation (Co-IP) was used to reveal interacting proteins. Results: WGCNA screened 40 hub genes which were significantly associated with the distant metastasis in patients with breast cancer. A total of 207 upregulated genes were identified in patients with distant metastasis. After intersection, 6 genes were selected. Survival analysis suggested that high expression of IGFBP5, BCL6B, TGM2, and SH3PXD2A were correlated with poor distant metastasis-free survival (DMFS). The metastasis predicting model built based on those genes showed an AUC value of 0.923 with diagnostic accuracy of 91.2%. In the validation cohort, data showed the AUC value of each single gene were 0.835, 0.818, 0.845, 0.834, for TGM2, IGFBP5, BCL6B, and SH3PXD2A respectively. Among those four genes, TGM2 was significantly upregulated with clinical stages and correlated with poor prognosis. Moreover, TGM2 showed the highest abundance in tumor tissue EVs. Functional experiments revealed that TGM2 promoted breast cancer proliferation, metastasis, and invasion in vitro. The initially upregulated expression of TGM2 in EVs of cell lines was significantly decreased by adding the exosome release inhibitor GW4869. In addition, EVs from TGM2 overexpressed cells promoted cell migration and invasion of wild-type cells. Mechanistically, TGM2 was positively correlated with EMT, Hedgehog and IL6-JAK-STAT3 pathways. Co-IP assay found that TGM2 interacted with TMF1, which was an effector for degradation of STAT3 through the ubiquitin-proteasome pathway. Interfering the expression of TMF1 remarkably promoted the migration ability of cells and reversed by TGM2 overexpression. Conclusion: Based on the plasma EV-RNAs, we constructed a model with promising predicted capacity of distant metastasis in patients with breast cancer. TGM2, as the most effective predictor, can promote the progression and metastasis of breast cancer by targeting TMF1/STAT3. Key words Breast Cancer; Extracellular vesicles; Metastasis; Prediction model; TGM2 Citation Format: Jiong Wu, Yayun Chi. RNA in Extracellular vesicles used for the prediction of distant metastasis of breast cancer and the function of TGM2 in promoting breast cancer development [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-05-33.

Comprehensive Analysis of the Expression and Prognosis of chromobox Family Members in Breast Cancer

Background: Chromobox proteins are canonical components of the Polycomb group family and play pivotal roles in several cancers. However, little is known about the function, prognostic value and drug sensitivity of CBX family members in breast cancer.Methods: In this study we investigated the expression, prognosis value and drug sensitivity of CBX family in breast cancer using the ONCOMINE, GEPIA, Human Protein Atlas and Kaplan-Meier Plotter databases, etc. and preliminary verified the expression of CBX family in breast cancer cell lines by RT-qPCR.Results: We found that the expression levels of CBX1/2/3/4/8 members were elevated in breast cancer tissues compared to adjacent normal breast tissues, while the expression levels of CBX6/7 genes were reduced in breast cancer tissue. In vitro qRT-PCR validated the expression differences of CBX1/2/3/4/8 in breast cancer cell lines. Further analysis showed expression of CBX family members was remarkably correlated with cancer subgroups. As nodal metastasis status increased, the mRNA expression of CBX1/2/3/4/8 members tended to be higher, while CBX6/7 tended to be lower. The expression of CBX1/2/3 was higher in patients with TP53 mutation and CBX6/7 expression tended to be lower in patients with TP53 mutation groups. High transcription levels of CBX2/3 were significantly associated with shorter overall survival in breast cancer patients, while lower expression of CBX4/5/6/7 members was associated with unfavorable overall survival. Moreover, a high mutation rate of CBX gene members (43%) was observed in breast cancer patients, and genetic alterations in CBX genes was associated with poor prognosis.Conclusion: Taken together, our results indicated that CBX2/3/6/7/8 could be considered prognostic and therapeutic biomarkers of breast cancer and are worthy of further study.

The expression analysis of long non-coding RNAs and p53 as a possible genetic signature in luminal: An invasive breast ductal carcinoma

Breast cancer is the second most common cause of cancer-related death among females. The long non-coding RNAs (lncRNAs) are a significant population of non-coding RNAs with well-defined functions in both adjacent normal cells and tumorigenesis. Miss expression of them has been associated with the development of different kinds of cancers. The aim of this study was to investigate the alterations in 3 lncRNAs and p53 in tissue samples of a group of women with luminal type A breast adenocarcinoma. In this case-control study, the expression levels of p53 and three lncRNAs were evaluated in association with luminal A breast cancer in 80 ductal carcinoma tumors and adjacent normal breast tissues. Quantitative real-time PCR was used to measure the expression of the mentioned genes. The data were analyzed using t-tests. The expression levels of IGFBP7-AS1, and RHPN1-AS1, showed a significant increase (P> 0.05) while P53 and LINC00861 had a significant decrease in expression level in tumor tissues compared to adjacent normal tissues (P> 0.05). Receiver Operating Characteristic (ROC) curve indicated the diagnostic power of LINC00861 in differentiating tumor tissues from adjacent normal tissue with 90% sensitivity and 96% specificity, which can also interact with P53. P53 and the LncRNAs including Linc00861, RHPN1-AS1, and IGFBP7-AS1 were dysregulated in invasive breast ductal carcinoma samples. Based on the area under the curve (AUC) value, expression of Linc00861, p53, RHPN1-AS1, and IGFBP-AS1 genes can help to differentiate patients from healthy individuals with diagnostic power of 0.855, 0.824, 0.778, and 0.659, respectively. The key roles of the latest genes in the control of cancer-related pathways and the dysregulation of their expression in studied malignancies imply that they can be exploited as biomarkers for cancer diagnosis, prognosis, and possible therapeutic targets. Here, the roles of lncRNAs in invasive breast ductal carcinoma type luminal A and their importance in prognosis and patient treatment are discussed.

Species-Level Characterization of the Microbiome in Breast Tissues with Different Malignancy and Hormone-Receptor Statuses Using Nanopore Sequencing

Unambiguous evidence indicates that microbes are closely linked to various human diseases, including cancer. Most prior work investigating the microbiome of breast tissue describes an association between compositional differences of microbial species in benign and malignant tissues, but few studies have examined the relative abundance of microbial communities within human breast tissue at the species level. In this work, a total of 44 breast tissue samples including benign and malignant tissues with adjacent normal breast tissue pairs were collected, and Oxford Nanopore long-read sequencing was employed to assess breast tissue microbial signatures. Nearly 900 bacterial species were detected from the four dominant phyla: Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. The bacteria with the highest abundance in all breast tissues was Ralstonia pickettii, and its relative abundance increased with decreasing malignancy. We further examined the breast-tissue microbiome composition with different hormone-receptor statuses, and the relative abundance of the genus Pseudomonas increased most significantly in breast tissues. Our study provides a rationale for exploring microbiomes associated with breast carcinogenesis and cancer development. Further large-cohort investigation of the breast microbiome is necessary to characterize a microbial risk signature and develop potential microbial-based prevention therapies.

Association of body mass index and inflammatory dietary pattern with breast cancer pathologic and genomic immunophenotype in the nurses’ health study

BackgroundBreast tumor immune infiltration is clearly associated with improved treatment response and outcomes in breast cancer. However, modifiable patient factors associated with breast cancer immune infiltrates are poorly understood. The Nurses’ Health Study (NHS) offers a unique cohort to study immune gene expression in tumor and adjacent normal breast tissue, immune cell-specific immunohistochemistry (IHC), and patient exposures. We evaluated the association of body mass index (BMI) change since age 18, physical activity, and the empirical dietary inflammatory pattern (EDIP) score, all implicated in systemic inflammation, with immune cell-specific expression scores.MethodsThis population-based, prospective observational study evaluated 882 NHS and NHSII participants diagnosed with invasive breast cancer with detailed exposure and gene expression data. Of these, 262 women (training cohort) had breast tumor IHC for four classic immune cell markers (CD8, CD4, CD20, and CD163). Four immune cell-specific scores were derived via lasso regression using 105 published immune expression signatures’ association with IHC. In the remaining 620 patient evaluation cohort, we evaluated association of each immune cell-specific score as outcomes, with BMI change since age 18, physical activity, and EDIP score as predictors, using multivariable-adjusted linear regression.ResultsAmong women with paired expression/IHC data from breast tumor tissue, we identified robust correlation between novel immune cell-specific expression scores and IHC. BMI change since age 18 was positively associated with CD4+ (β = 0.16; p = 0.009), and CD163 novel immune scores (β = 0.14; p = 0.04) in multivariable analyses. In other words, for each 10 unit (kg/m2) increase in BMI, the percentage of cells positive for CD4 and CD163 increased 1.6% and 1.4%, respectively. Neither physical activity nor EDIP was significantly associated with any immune cell-specific expression score in multivariable analyses.ConclusionsBMI change since age 18 was positively associated with novel CD4+ and CD163+ cell scores in breast cancer, supporting further study of the effect of modifiable factors like weight gain on the immune microenvironment.