Normal Breast Tissue Research Articles

Abstract P6-10-14: Lysosomal acid lipase (LIPA) as a novel therapeutic vulnerability for treating TNBC

Abstract Background: TNBCs have the highest mortality rate among all BC subtypes. There is thus an urgent and unmet need for effective targeted therapies in TNBC. Recently we, identified a novel agent ERX-41 that showed good efficacy in treating TNBC in preclinical mouse models, however, its molecular action remain unknown. In this study, we identified LIPA as novel molecular target of ERX-41. Methods: We have used CRISPR knockout pooled library and multiple TNBC models for identifying molecular target of ERX-41. Mechanistic studies were performed using LIPA mutants, RNA-seq, Turbo-ID mapping, Mass spectrometry, Immunoprecipitation, and Western blotting. The in vivo efficacy of ERX-41 was examined using four different patient-derived xenograft (PDX) models. We evaluated LIPA protein expression in TNBC using tissue microarray (TMA). Results: To identify the molecular target of ERX-41, we performed an unbiased CRISPR–Cas9 knockout (KO) screen in MDA-MB-231 cells and the results identified LIPA as a top hit. KO of LIPA alone (which encodes lysosomal acid lipase (LAL) abrogated cytotoxic response to ERX-41. Cellular thermal shift assays confirmed that ERX-41 binds to LAL. In silico modelling and mutational studies confirmed that ERX-41 interacts with LAL through residues in its LXXLL domain and that ERX-41 ability to induce ER stress and cell death in TNBC is independent of the lipase activity of LAL. Unbiased RNA-seq studies with and without ERX-41 in parental and LIPA KO SUM-159 cells revealed induction of genes involved in ER stress and UPR response by ERX-41 in parental SUM-159 cells but not in cells with LIPA KO. Ultrastructural studies using live-cell confocal microscopy show that LIPA KO abrogated ER morphological changes at 2 and 4 h after ERX-41 treatment. Further, subcellular localization studies showed LIPA localizes to endoplasmic reticulum (ER). Unbiased proteomic approaches (TurboID and DIA mass spec) identified a core set of proteins that were both LAL binders and affected by ERX-41 treatment. GO analyses of LAL binding proteins confirmed their involvement in protein folding. Tumor micro array (TMA) analyses confirmed that >80% of primary TNBC tumors had significant and detectable LAL protein expression in contrast, normal breast tissue had lower LAL expression. ERX-41 (10 mg/kg body weight) decreased growth of four distinct TNBC patient-derived xenografts (PDXs) in vivo. Conclusions: Our results identified a new molecular target (LAL) for ERX-41 and novel mechanism of action (disruption of protein folding and induction of ER stress) that may have utility in treating patients with TNBC. Citation Format: Suryavathi Viswanadhapalli, Xihui Liu, Uday Pratap, Gangadhara R. Sareddy, Susan T. Weintraub, Ganesh V. Raj, Jung-Mo Ahn, Ratna K. Vadlamudi. Lysosomal acid lipase (LIPA) as a novel therapeutic vulnerability for treating 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 P6-10-14.

Abstract P2-23-15: Histologic, immunohistochemical and genomic comparison between classic Invasive lobular carcinomas and lobular-like invasive ductal carcinomas

Abstract Background: Invasive lobular carcinomas (ILCs) are the most frequent special histologic subtype of breast cancer, accounting for up to 15% of all breast cancer cases. ILCs are characterized by a distinctive discohesive growth pattern, with cells arranged in single cell infiltrative file and dispersed throughout the stroma, which stems from the loss of E-cadherin expression due to bi-allelic inactivation of the CDH1 gene. A subset of breast cancers display a similar single cell infiltrative growth pattern but, in contrast to classic ILC, display diffuse strong membranous E-cadherin reactivity and membranous p120 expression. We refer to such cases as “lobular-like invasive ductal carcinoma” (LLIDC), but it is unclear if this terminology is appropriate and if such cases show biallelic inactivation of CDH1, similarly to ILCs. Here, we sought to define whether LLIDCs would harbor bi-allelic alterations of CDH1 and to perform an exploratory, hypothesis generating analysis of the repertoire of somatic genetic alterations of LLIDCs and classic ILCs. Materials and methods: Representative H&Es, as well as sections subjected to E-cadherin and p120 immunohistochemistry from seven classic ILCs and seven bona fide “lobular-like invasive ductal carcinomas” were retrieved and independently reviewed by two pathologists with experience and expertise in breast pathology. DNA samples were extracted from representative sections from tumor and normal breast tissue from each patient and subjected to an FDA-approved targeted sequencing assay comprising the coding regions and selected regulatory elements of 515 genes. Somatic single nucleotide variants (SNVs) were detected with MuTect, indels with Strelka, Varscan2, Scalpel and Lancet. All mutations were manually inspected using the Integrative Genomics Viewer (IGV). The cancer cell fraction (CCF) of each mutation was inferred, as well as clonal probability, using ABSOLUTE. Copy number alterations and loss of heterozygosity were determined using FACETS. Mutational signatures were inferred using SigMA based on all synonymous and nonsynonymous somatic mutations. Results: Based on the histopathologic evaluation, of the 14 cases analyzed, seven were classified as ILC, and the other seven were classified as LLIDC. Sequencing analysis revealed that the classic ILCs harbored 16q LOH and CDH1 mutations (7/7), of which five were frameshift indel and two were splice site mutations consistently coupled with loss-of-heterozygosity (LOH) of the wild-type allele. Conversely, five of the seven LLIDCs did not harbor CDH1 mutations or genomic rearrangements. CDH1 mutations were identified in 2 LLIDCs: one harbored a subclonal CDH1 in-frame indel mutation coupled with LOH. This case displayed membranous E-cadherin and p120 expression with areas of aberrant expression. The other CDH1-mutated LLIDC harbored a complex in-frame indel with subclonal LOH. This case displayed membranous E-cadherin and p120 expression. The comparative analysis of the repertoire of somatic genetic alterations and mutational signatures present in LLIDCs and classic ILCs did not reveal any significant differences. Conclusion: Despite the histologic similarities, LLIDCs differ from classic lobular carcinomas based on the lack of CDH1 bi-allelic inactivation and the patterns of expression of E-cadherin and p120 catenin. Further whole-genome sequencing analyses are warranted to define the molecular basis of the discohesive cancer cells of LLIDC display. Citation Format: Edaise M. da Silva, Thais Basili, Jing Yu, Juan Blanco-Heredia, Pier Selenica, Qiqi Ye, Arnaud da Cruz Paula, Higinio Dopeso, Antonio Marra, Steffi Oesterreich, Jorge Reis-Filho, Rohit Bhargava. Histologic, immunohistochemical and genomic comparison between classic Invasive lobular carcinomas and lobular-like invasive ductal carcinomas [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-15.

Abstract PD4-08: PD4-08 A Novel Single Cell Model of Tamoxifen Response in Primary Human Breast Tumors

Abstract A Novel Single Cell Model of Tamoxifen Response in Primary Human Breast Tumors Austin Whitman, Hyunsoo Kim, Kamila Wisniewska, Rasha Kakati, Susana Garcia Recio, Hector Franco, Charles Perou, Philip Spanheimer Background: Resistance to endocrine therapy is a primary cause of treatment failure and death in patients with estrogen receptor (ER)-positive breast cancer. Intratumor heterogeneity is associated with resistance to therapy across tumors, and specifically in ER+/HER2- breast cancer, heterogeneity in ER and PR expression is associated with a worse response to endocrine therapy. We hypothesize that subpopulations within and across ER+/HER2- human breast tumors have distinct responses to tamoxifen and that discerning heterogeneity in response will improve understanding of inherent and emerging resistance to endocrine therapy. Methods: We developed an operating room-to-laboratory pipeline immediately after surgical resection for studies using alive tissue. Tissue samples were obtained and single cell suspensions created using physical and enzymatic dissociation. Cells were treated with tamoxifen (10 M) or control media for 12 hours in suspension and single cell RNA libraries generated using the 10X Genomics droplet-based kit and sequenced using the Illumina NextSeq2000. Results: We obtained normal breast tissue from 2 women undergoing reduction mammoplasty and tumor tissue from 10 women with ER+/HER2- invasive breast carcinoma. In tamoxifen treated and control matched pairs, a total of 22,195 cells from normal breast and 94,558 cells from tumor samples were sequenced. Computational analysis using consensus clustering was performed and cell types assigned using canonical correlation. Both tumor and normal samples identified clustering by cell type and not by patient revealing significant variability in cell type abundance between samples. In the normal breast samples, we performed differentially expressed genes (DEG) analysis comparing tamoxifen treatment to control for each cell type (Immune cells, fibroblasts, basal epithelial cells, luminal progenitor cells, and mature luminal cells) and enrichment analysis of up- and down-regulated genes performed. Strong depletion of estrogen induced genes was observed in tamoxifen-treated normal luminal progenitor and mature luminal cells, but not in basal epithelial cells or fibroblasts, demonstrating distinct, subpopulation-specific response to tamoxifen. In the 10 tumor matched pairs, 4 had a high epithelial proportion and tumor cells identified using inferred copy number variation. Tumor cells in 3 of these 4 samples showed significant down regulation of estrogen response genes with tamoxifen treatment. Using scBCSubtype to assign PAM50 subtype to individual tumor cells, the 3 responsive tumors were comprised primarily of LumA cells while the unresponsive tumor was predominantly LumB. Finally, we developed a novel score to quantify responsiveness at the single cell level based on downregulation of estrogen response genes with tamoxifen treatment relative to matched cluster-specific untreated expression. This analysis demonstrated heterogeneity in response to tamoxifen in tumor cells and identified distinct subpopulations of responsive and unresponsive tumor cells to tamoxifen treatment. Conclusion: We developed a novel ex vivo model to determine heterogeneity in therapeutic response to tamoxifen in normal human breast tissue and primary human breast tumors. We demonstrate differences in tamoxifen response by cell type and identify distinctly responsive and resistant subpopulations within human tumors. This provides a foundation to define features of responsive and resistant populations on the individual cell and specimen basis, and should allow us to develop precise, single cell-based predictors of response to endocrine therapy, and to identify genes and pathways driving resistance to therapy. Citation Format: Hyunsoo Kim, Austin Whitman, Kamila Wisniewska, Susana Garcia-Recio, Rasha Kakati, Hector L. Franco, Charles M. Perou, Philip Spanheimer. PD4-08 A Novel Single Cell Model of Tamoxifen Response in Primary Human Breast Tumors [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 PD4-08.

Abstract P2-11-22: Expression of milk whey protein genes in human breast carcinoma cells isolated by LCM impacts prediction of clinical outcomes

Abstract Background: Milk whey protein consumption has been associated with breast cancer prevention, therapy and treatment tolerance, and other studies detected these proteins or their mRNAs in breast cancer models or tissues. Our objectives are to ascertain expression of certain whey protein genes in human breast carcinoma cells isolated by Laser Capture Microdissection (LCM) and to determine if gene expression is related to risk of recurrence and overall survival in lesions with various ER/PR status. Genes selected were: LALBA (alpha-lactalbumin), LPO (lactoperoxidase), LTF (lactotransferrin), PAEP (progestogen associated endometrial protein or glycodelin) and SSP1 (secreted phosphoprotein 1 or osteopontin). This retrospective investigation is unique in that highly quantified ER and PR protein levels and ESR1 and PGR relative expression from pure populations of carcinoma cells were employed to stratify cancers into ER/PR subcategories to assess relationships of expression of candidate genes with clinical outcomes. Methods: This investigation of primary breast carcinomas from 170 postmenopausal and 77 premenopausal patients used a matchless deidentified dataset of quantified estrogen receptor (ER) and progestin receptor (PR) protein results with clinical follow-up. ER/PR protein levels, expressed as fmol/mg cytosol protein, were quantified earlier from each breast carcinoma with FDA-approved kits, either Radioligand-Binding Assay (LBA, NEN/DuPont) and/or enzyme immunoassay (EIA, Abbott Labs). Patients were treated with the standard of care at the time of diagnosis. The PixCell IleTM (ThermoFisher, Arcturus) LCM instrument was used to procure only carcinoma cells from tissue sections of de-identified, frozen biopsies, using protocols we employed earlier. Total RNA of each fixed specimen was extracted, purified and amplified to obtain relative expression levels of approximately 22,000 genes by microarray, providing an assessment of mRNA in carcinoma cells only. Relationships of relative gene expression, quantified ER/PR and other features of primary breast cancers were analyzed with clinical results using R software v4.2.0 (Vigorous Calisthenics) by Univariable Cox Regression, Kaplan Meier plots and various tests (i.e., Wilcoxon, Log Ranked, Spearman and/or Chi Square tests). Bidirectional elimination stepwise regression was also used to derive best predictors of patient outcomes. Results: Violin plots of expression distribution of each milk protein gene of 247 biopsies regardless of patient menopausal status revealed that LALBA and SSP1 were highly elevated (p < 0.001) in ER+ (n = 146) or PR+ (n = 146) breast cancer cells while PAEP was quite diminished (p < 0.001). No difference was observed in LPO expression in ER+ or PR+ cells and LTF was only elevated slightly (p < 0.05) in ER+ cells. Regardless of ER/PR status of breast cancer, LTF, LALBA, SSP1, AR and ESR1 gene expression levels were increased significantly in postmenopausal versus premenopausal patients while PAEP was decreased (p < 0.001). No difference was observed in LPO or PGR expression. Correlation matrices examined gene to gene and receptor protein to gene relationships. Using Kaplan Meier analyses of each gene alone with Log Ranked Tests of outcomes, only SSP1 expression was associated with clinical outcome, i.e., overexpression was associated with lower PFS and OS (p = < 0.05). Conclusions: Application of bidirectional elimination stepwise regression of gene expression of the five milk proteins collectively with ER/PR protein status of each carcinoma revealed that LALBA gene expression enhanced prediction of PFS in ER+ cancer while PAEP expression was associated with improved prediction of OS in ER+ carcinomas. We report that expression of LALBA and PAEP genes, largely associated with milk production in normal breast tissue, are also expressed in certain breast carcinomas and influence prediction of patient survival. Citation Format: James L. Wittliff, Michael W. Daniels. Expression of milk whey protein genes in human breast carcinoma cells isolated by LCM impacts prediction of clinical outcomes [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-22.

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.

The Role of ATG4A Clinical Implication and Potential Function in Breast Cancer

We studied the clinical implication and potential function of ATG4A in breast cancer by using publicly accessible patient data. The mRNA and protein levels of ATG4A were up-regulated in breast cancer tissues compared with normal breast tissues. ATG4A is a potential marker for prognosis, diagnosis and therapeutic target for breast cancer. Autophagy is a conservative catabolic process for cell homeostasis. The mechanism of autophagy in tumors is very complicated, since it can either promote or inhibit the occurrence and development of tumor according to different tumor stages and types. The purpose of this study was to investigate the clinical significance of an autophagy-related gene ATG4A in breast cancer. Transcriptional expression profiles of ATG4A between breast cancer tissues and normal tissues were retrieved from the Cancer Genome Atlas (TCGA). The ATG4A protein level was assessed by the Human Protein Atlas. The co-expression genes with ATG4A expression were analyzed using the LinkedOmics database. Protein–protein interaction (PPI) networks were constructed by the STRING. Paired t-test and Mann-Whitney U-test were used to determine the differences between breast cancer tissues and adjacent normal tissues. Kaplan–Meier curves and log rank tests were conducted for survival analysis. The expression of ATG4A in breast cancer tissues was significantly upregulated than those in adjacent normal tissues. Increased ATG4A mRNA expression was positively associated with lymph node metastases (P <0.05), ER positive (P <0.001), PR positive (P <0.001), HER-2 positive (P <0.05) and over 60 years of age (P <0.005). Compared with breast cancer patients with LumA, LumB and HER-2 positive subtypes, the expression of ATG4A mRNA in breast cancer patients with Basal was significantly lower (P <0.001). Kaplan–Meier survival analysis showed that the OS (P <0.001), DSS (P = 0.017) and PFI (P = 0.020) of breast cancer patients with high ATG4A mRNA expression were significantly shorter than those with low ATG4A mRNA expression. GO and KEGG analysis showed that Endocytosis might be a signal pathway involved in the occurrence and development of breast cancer by ATG4A. Furthermore, we found that RPL4, RPL11, RPL14, RPL29 and RPS9 may be the potential interacting genes of ATG4A in breast cancer by PPI analysis. ATG4A may be a potential diagnostic, prognostic biomarker and therapeutic target gene for breast cancer at the mRNA and protein levels.

Abstract P2-26-08: Influence of genetic ancestry on breast stromal cells provides biologic basis for increased incidence of metaplastic breast cancer in women of African descent

Abstract The biologic basis of genetic ancestry-dependent variability in disease incidence and outcome is just beginning to be explored. We recently reported enrichment of a population of ZEB1-expressing cells located adjacent to the ductal epithelial cells in the normal breast of women of African Ancestry (AA) compared to European Ancestry (EA). By establishing and characterizing cell lines corresponding to these cells and validating in vitro findings with tissue microarrays of healthy breast tissue from AA, EA and Latina Ancestry (LA) women, we demonstrate that these cells have the properties of fibroadipogenic/mesenchymal stromal cells that express PROCR and PDGFR. PROCR+/ZEB1+/PDGFR+ cells, hence renamed as PZP cells, are enriched in the normal breast tissues of AA compared to EA or LA women. In vitro, PZP cells trans-differentiated into adipocytes or osteocytes. In co-culture conditions, PZP:epithelial cell communication resulted in luminal epithelial cells acquiring basal/stem cell characteristics and increased expression of IL-6 suggesting the impact of this communication on the microenvironment and breast epithelial hierarchy. Consistent with this possibility, the level of phospho-STAT3, which is a downstream target of IL-6, was higher in the normal and cancerous breast tissues of AA compared to EA women. PZP cells transformed with HRasG12V ± SV40-T/t antigens generated metaplastic carcinoma in NSG mice suggesting that these cells could be the cell-of-origin of metaplastic breast cancers. Collectively, these results identify a stromal cell component that could influence the biology of breast cancer in AA women. Citation Format: Brijesh Kumar, Katie Batic, Poornima Bhat-Nakshatri, Maggie Granatir, Rebekah Addison, Megan Szymanski, Lee Ann Baldridge, Constance Temm, George Sandusky, Sandra Althouse, Anna Maria Storniolo, Harikrishna Nakshatri. Influence of genetic ancestry on breast stromal cells provides biologic basis for increased incidence of metaplastic breast cancer in women of African descent [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-08.

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.

Abstract P2-26-11: Protein phosphatase 1 catalytic units, PPP1CA, PPP1CB and PPP1CC in breast cancer

Abstract Introduction. Protein phosphatase-1 (PP1) belongs to the Serine/threonine-protein phosphatase family and is known to participate in multiple functions in the cells including glycogen metabolism, cellular proliferation and cell receptor regulations. Protein phosphtase-1 has three catalytic units, namely PPP1CA, PPP1CB and PPP1CC. A PP1 protein complex is composed of one of the catalytic units and at least of one the various phosphatase regulatory units (PPP1Rs). The regulatory units of the phosphatase, abundant in numbers, also influence the functions of the protein phosphatase within cells. PP1 has been indicated to have a role in certain clinical conditions such as the Alzheimer’s and certain viral infections. The value of the PP1 catalytic units in clinical cancers including breast cancer is not well established, therefor this current study aimed to examine the expression profile of the three PP1 catalytic units in human breast cancer and attempted to establish the clinical relevance of the catalytic units in the disease progression of breast cancer. Methods. Fresh frozen normal mammary tissues and breast cancer tissues were investigated for the levels of gene transcripts of the three PP1 catalytic units were quantified and corrected with an epithelial cell marker. The expression and integrated expression profile of PPP1A, PPP1B and PPP1C in the tissues were assessed against the pathological, clinical and outcome of the patients as well as the hormone receptor status. Results. All three catalytic units of PP1 were positively expressed in normal and tumour breast tissues, with PPP1A at significantly higher levels in normal mammary tissues than tumours (p=0.01) and PPP1B and PPP1C showing no significant difference. Expression of PPP1B in breast cancer was significantly lower in high grade breast cancers (p< 0.05 vs low grade), in tumours from patients who died of breast cancer (p=0.047 vs those who survived) and with breast cancer related incidence (p=0.048 vs incidence free). The most significant finding of the present study was a correlation between high levels of all three catalytic units and favourable overall survival of the patients. Those patients with high levels of PPP1A, PPP1B and PPP1C in breast tumours had a mean survival time of 142.6, 141.6 and 140.3 months compared with those with low levels respectively at 111.8, 115.8 and 117.6 months. When the expression profiles of the three units were integrated, the integrated profile formed a highly valuable and independent indicator for both overall survival (p=0.008, HR=0.694) and for disease free survival (p=0.004, HR=0.702). The predictive value of PPP1A/PPP1B/PPP1C for survival was more significant for ER negative tumours than ER positive tumours (p=0.003), but appeared to be indifferent for Her2 status. Finally, our data indicated that the integrated expression of the three catalytic units had a significant predictive value in bone metastasis of the patients (p=0.008, HR=0.546). Discussion. Protein phosphatase-1 catalytic units, PPP1CA, PPP1CB and PPP1CC are expressed in human mammary tissues; raised levels of the catalytic units are a favourable clinical indicator for the survival of patients and for a decreased tendency to develop bone metastasis. Citation Format: Xuefei Dong, Tracey A. Martin, QingPing Dou, Wen G. Jiang, Eleri Davies. Protein phosphatase 1 catalytic units, PPP1CA, PPP1CB and PPP1CC in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-11.

Diagnostic Dilemma of Chest Wall Tuberculosis Masquerading Breast Lump and Sarcoma

Chest wall tuberculosis (TB) is rare and can often masquerade as a tumour. Diagnostic confirmation is made by bacteriological findings of acid-fast bacilli and culture of Mycobacterium tuberculosis or histopathological findings. This is a 37-year-old immunocompetent lady who presented with a 3-month history of gradually increasing right breast lump with suspicious characteristics during clinical examinations. Ultrasound of the breast showed normal breast tissue with a well-defined hypoechoic lesion within the anterior inferior pectoralis muscle. CT scan of the thorax revealed a right anterior chest wall lesion with multiple lung nodules and consolidations. Thus, there was a high suspicion of chest wall tumour and an initial diagnosis of soft tissue sarcoma was made. Biopsy of the lesion showed necrotising granulomatous inflammation but no acid-fast bacilli. A revised diagnosis of chest wall tuberculosis was made. She attended follow-up complaining of shortness of breath and pleuritic chest pain with signs of right pleural effusion. Her symptoms improved after the initiation of anti-TB treatment. This case demonstrated the challenge in making an early diagnosis of chest wall TB and commencement of anti-TB treatment.

Abstract P2-07-01: Hydroxytyrosol, a Component of Olive Oil for Breast Cancer Prevention in Women at High Risk

Abstract Background Breast cancer is the most frequently diagnosed cancer in women in developed countries with increased incidence in women at high risk such as those with strong family history, BRCA mutations, atypical hyperplasia etc. Chemoprevention with drugs like tamoxifen and aromatase inhibitors come with challenges of intolerance and limited efficacy in estrogen receptor negative breast cancers. The purpose of our study was to evaluate the effects of hydroxytyrosol (HT), a component of olive oil on mammographic breast density reduction, which is a validated surrogate biomarker for breast cancer prevention as well as to explore on-target effects on Wingless-related integration site (Wnt) pathway, and alterations in key pathways of proliferation, DNA damage repair and stem cell function. Methods This study was conducted using 25mg/day oral dose of HT for 12 months in both pre- and post-menopausal women who are at increased risk of breast cancer with Gail 5-year risk score ≥ 1.67 and/or > 10% probability of BRCA mutation and had declined standard chemoprevention. These women underwent annual mammograms as well as had the option to have a biopsy of normal breast tissue before and after HT. Maximum volumetric breast density (Max VBD%) was assessed quantitatively using Volpara software (VIS 3.2) and the annualized percent decrease in max VBD% between baseline (BL) and end of treatment (EOT) with HT was analyzed in the study. RNA was extracted from the breast biopsies and RNA sequencing (RNA-Seq) and multiplex analysis of 28 proteins using NanoString nCounter analysis was performed to compare the BL with EOT samples. Results A total of 61 women were screened for the trial, 51 were enrolled and 41 patients completed the study. Median age for the study population was 54 years (range 35-76 years). There were 26 patients with paired BL and EOT mammograms. There were 14 patients with paired BL and EOT breast biopsies and seven patients with only BL and seven with only EOT biopsies. Mammographic density as measured by max VBD% showed a non-significant change of -0.038%, p = 0.49. However, on subgroup analysis in women 60-years or older, the mean decrease in max VBD% was - 3.7% (p = 0.0391), especially in those with high baseline mammographic density (> 10% baseline max VBD%). RNAseq data was generated from 15 BL and 17 EOT samples and pathways were confirmed by NanoString. Our bioinformatics pipeline identified 190 transcripts that were upregulated and 90 that were downregulated in EOT vs. BL by at least 1.5 fold. Reactome pathway R-HSA-195721 (Signaling by WNT) is identified as the top pathway enriched with EOT downregulated genes. On further review of the expression profiles, 82 of 261 member genes had p-value < 0.05 for EOT vs. BL, among which only 4 were upregulated and 78 were downregulated in EOT samples. These 82 genes were highly interactive with each other, with 379 high confidence protein-protein interactions (PPIs) (confidence score > 0.7 from STRING database) involving 81 out of 82 genes. In addition, mitotic telophase/cytokinesis (p = 0.004), ATM signaling (p = 0.007) were some pathways that were upregulated whereas, NOTCH signaling (p = 0.007), oxidative stress induced senescence (p = 0.0001) pathways were downregulated in EOT vs. BL. Conclusions Hydroxytyrosol, a component of olive oil did not show a decrease in breast density in the intention to treat population but showed some reduction in breast density in women 60-years of age and above, especially in those with high baseline breast density. HT showed on target effects by affecting Wnt signaling pathway, as well as decrease in proliferation. This study lays the foundation for future larger studies in exploring a natural compound such as HT for chemoprevention of breast cancer. Citation Format: Akshjot Puri, Zheng Yin, Sergio Granados-Principal, Joe Ensor, Liliana Guzman, Roberto Rosato, Hong Zhao, Stephen Wong, Tejal Patel, Jenny Chang. Hydroxytyrosol, a Component of Olive Oil for Breast Cancer Prevention in Women at High Risk [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-07-01.

Nanoindentation study of the viscoelastic properties of human triple negative breast cancer tissues: Implications for mechanical biomarkers.

This paper presents the results of a combined experimental and theoretical study of the structure and viscoelastic properties of human non-tumorigenic mammary breast tissues and triple negative breast cancer (TNBC) tissues of different histological grades. A combination of immunofluorescence and confocal microscopy, and atomic force microscopy is used to study the actin cytoskeletal structures of non-tumorigenic and tumorigenic breast tissues (grade I to grade III). A combination of nanoindentation and statistical techniques is then used to measure viscoelastic properties of non-tumorigenic and human TNBC of different histological grades. A Standard Fluid Model/Anti-Zener Model II is also used to characterize the viscoelastic properties of the non-tumorigenic and tumorigenic TNBC tissues of different grades. The implications of the results are discussed for the potential application of nanoindentation and statistical deconvolution techniques to the development of mechanical biomarkers for TNBC detection/cancer diagnosis. STATEMENT OF SIGNIFICANCE: There is increasing interest in the development of mechanical biomarkers for cancer diagnosis. Here, we show that nanoindentation techniques can be used to characterize the viscoelastic properties of normal breast tissue and TNBC tissues of different histological grades. The Standard Fluid Model (Anti-Zener Model II) is used to classify the viscoelastic properties of breast tissues of different TNBC histological grades. Our results suggest that breast tissue and TNBC tissue viscoelastic properties can be used as mechanical biomarkers for the detection of TNBC at different stages.

Abstract P5-11-04: Differentially expressed genes and their pathways in breast cancer patients with mesenchymal CTC

Abstract Background: Circulating tumor cells (CTC) with phenotype of epithelial-mesenchymal transition (CTC_EMT) represent novel subpopulation of CTC associated with inferior outcome in primary breast cancer (PBC). However, molecular characterization of primary tumors associated with this CTC subpopulation is lacking. The aim of this study was to identify signaling pathways associated with presence of CTC_EMT in PBC patients using a comprehensive genomics approach. Methods: This translational study included 17 patients with PBC and 5 donors of normal breast tissue. CTC_EMT were detected before surgery by quantitative RT-PCR assay for expression of epithelial-mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, ZEB1). Total RNA was extracted, in parallel, from fresh frozen primary tumor and whole-trancriptome profiles were obtained using RNA sequencing and additionally mRNAs profiles by microarray. Genes expressions were further validated by qRT-PCR. Results: Analyzing RNA sequencing and microarray data, we found set of genes differentially expressed in absence or presence of CTC_EMT in PBC. We identified 157 genes differentially expressed in CTC_EMT phenotype compared to patients with non-detectable CTC. Namely, keratin family is represented by genes KRT5, KRT14, KRT17. Gene ontologies related to membrane structure or communication and immunology appears to be involved in CTC-related processes, pathways related to cell junction and various signaling pathways including PI3K and Ras-signaling appear to be significant in processes leading to CTC EMT presence. Conclusions: We suspect multiple genes of having a role in primary tumour processes leading to CTC EMT production in breast cancer patients. Data suggest, that PI3K & Ras-signalling and pathways related to cell junction are the key pathways for changes inside of primary tumour tissue between CTC EMT and CTC- phenotype of breast cancer patients. We propose, additional study with single-cell resolution is needed for better understanding of the processes. Citation Format: Michal Mego, Dominik Hadzega, Gabriel Minarik, Andrea Soltysova, Petra Nemcova, Katarina Kalavska, Marian Karaba, Juraj Benca, Tatiana Sedlackova, Daniel Pindak, Lubos Klucar. Differentially expressed genes and their pathways in breast cancer patients with mesenchymal CTC [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 P5-11-04.

Abstract P5-13-04: Clonal evolution of mammary epithelial cells into breast cancers

Abstract [Introduction] Proliferative lesions in the breast have been implicated in the development of breast cancer. Previous studies showed that some proliferative lesions and adjacent breast cancers shared common genetic alterations, suggesting that these originated from the same ancestral cell. However, the clonal structure of normal epithelia and their clonal history during evolution to cancer are poorly understood. In this study, we analyzed genetic profiles of normal epithelia and proliferative lesions in the cancer-borne breast to illustrate the clonal evolution of cancer from a normal epithelial cell. [Methods] Single cell-derived organoids (n=47) were established from breast milk of 4 healthy women aged 22–36 and normal breast tissue of 15 breast cancer patients aged 29–83 to evaluate somatic mutation rate in normal epithelial cells. Multiple normal lobules and proliferative lesions together with cancer lesions were collected using laser-capture micro-dissection (LCM) from fresh frozen (n=3) or formalin-fixed paraffin-embedded (n=5) surgical specimens in 9 premenopausal breast cancer patients. Somatic mutations and copy number alterations were evaluated using whole-genome sequencing. [Results] The mutation profile of single cell-derived organoids suggests that somatic mutations accumulate in normal mammary epithelial cells at a constant rate of 19.4/genome/year before menopause, and the mutation rate decreases to 6.9/genome/year after menopause. Parity was negatively associated with mutation number (-49.3 per life birth). In total, we analyzed 143 LCM samples, including those from 72 normal lobules, 43 proliferative lesions, and 19 non-invasive and 9 invasive cancer samples. Five cases showed a large expansion of proliferative lesions sharing a substantial number of somatic mutations with cancer. These lesions expanded over a distance of 35-90 mm, sharing tens to hundreds of mutations including those in breast cancer-related driver genes, such as PIK3CA, AKT1, GATA3, CBFB and PTEN, while harboring private mutations or copy number alterations of their own. Of interest, the cancers in 4 out of these 5 cases was luminal-A type invasive ducal carcinoma or ER-positive HER2-negative ductal carcinoma in situ, and characterized in common by the presence of der(1;16), concurrent whole-arm 1q gain and 16q loss, in both cancer and proliferative lesions. Phylogenetic analysis adapted with the mutation rate in normal cells predicted that der(1;16) had been acquired between puberty and early 20’s, and the common ancestors of non-cancerous and cancerous lesions emerged by early 30’s, >10 years earlier than at the time of cancer diagnosis. By contrast, analysis of non-cancerous lobules unrelated to cancer showed that der(1;16)-negative non-cancer clones that had emerged after puberty stayed within a single lobule or spatially confined to adjacent lobules and rarely expanded to a large area as observed for those carrying der(1;16), even if the clones had acquired mutations in driver genes such as PIK3CA and PIK3R1, which highlighted the role of der(1;16) in wide clonal expansion. [Conclusions] Our results suggest that in some breast cancer cases, particularly in those with der(1;16), a highly recurrent translocation accounting for the major subset of Luminal A breast cancer, the clones with the funder driver alterations expanded macroscopically long before the onset of cancer, in which further clonal evolutions recursively occur multi-focally, giving rise to multiple proliferative lesions and ultimately, invasive cancers. Our findings provide new insight into the early development of breast cancer. Citation Format: Tomomi Nishimura, Nobuyuki Kakiuchi, Kenichi Yoshida, Takaki Sakurai, Tatsuki R. Kataoka, Eiji Kondoh, Yoshitsugu Chigusa, Masahiko Kawai, Morio Sawada, Takuya Inoue, Yasuhide Takeuchi, Hirona Maeda, Satoko Baba, Yusuke Shiozawa, Ryunosuke Saiki, Masahiro M. Nakagawa, Yasuhito Nannya, Yotaro Ochi, Tomonori Hirano, Yukiko Inagaki-Kawata, Kosuke Aoki, Masahiro Hirata, Eiji Suzuki, Masahiro Takada, Masahiro Kawashima, Kosuke Kawaguchi, Kenichi Chiba, Yuichi Shiraishi, Junko Takita, Satoru Miyano, Masaki Mandai, Kengo Takeuchi, Hironori Haga, Masakazu Toi, Seishi Ogawa. Clonal evolution of mammary epithelial cells into breast cancers [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 P5-13-04.

K-Ras4A Plays a More Significant Role than K-Ras4B in Ductal Carcinoma of Breast.

K-Ras mutations rarely occur in breast cancer. However, studies have supported that K-Ras upregulation is involved in breast cancer pathogenesis. Two main K-Ras transcript variants; K-Ras4A and K-Ras4B, originate from the alternative splicing of exon 4. In this study, we aimed to evaluate variations in the expression of K-Ras4A and K-Ras4B and their role in breast ductal carcinoma. Total RNA was extracted from breast tumors, and the NATs obtained via mastectomy. Patients were selected from new cases of breast cancer with no prior history of chemotherapy. Relative mRNA expression was calculated based on a pairwise comparison between the tumors and the NATs following normalization to the internal control gene. Predictive values of the transcript variants were examined by ROC curve analysis. A statistically significant increase was found in the K-Ras4A and K-Ras4B expression with the mean fold changes of 7.58 (p = 0.01) and 2.47 (p = 0.001), respectively. The K-Ras4A/K-Ras4B ratio was lower in the tumors than that of the normal tissues. ROC curve analysis revealed the potential of K-Ras4A (AUC: 0.769) and K-Ras4B (AUC: 0.688) in breast cancer prediction. There was also a significant association between K-Ras4B expression and HER2 statues (p = 0.04). Furthermore, a significant link was detected between K-Ras4A expression and pathological prognostic stages (p = 0.04). Our findings revealed that expression levels of K-Ras4A and K-Ras4B is higher in the tumor compared to the normal breast tissues. Increase in K-Ras4A expression was more significant than that of K-Ras4B.

Abstract GS4-01: Impact of Breast Conservation Therapy on Local Recurrence in Patients with Multiple Ipsilateral Breast Cancer – Results from ACOSOG Z11102 (Alliance)

Abstract Background Breast conserving therapy (BCT) is accepted as a preferred option for unifocal breast cancer. However, the oncologic safety of BCT for multiple ipsilateral breast cancer (MIBC), has not been demonstrated in a prospective study. The ACOSOG (Alliance) Z11102 phase II single arm prospective trial was designed to evaluate outcomes with BCT for MIBC. Methods Women age 40+ with 2 or 3 foci of biopsy proven breast cancer (BC) (each site < 5cm in size with at least 1 site invasive) separated by >2-3 cm of normal breast tissue and disease limited to two quadrants of the breast with cN0 or cN1 disease were eligible. All patients had pre-operative mammogram and breast MRI was initially required and subsequently made optional. Neoadjuvant therapy was not allowed. Patients were treated with lumpectomy resected to negative margins followed by whole breast radiation with boost to all lumpectomy beds. The primary endpoint of Z11102 is the cumulative incidence of local recurrence (LR, defined as histologic evidence of ductal carcinoma in situ or invasive BC in the ipsilateral breast or chest wall) at 5 years (treating death and distant and nodal/regional recurrence as competing risks) to assess whether the rate is greater than 8%. Data were frozen 5/25/2022. Results From 11/2012-8/2016, 270 women were enrolled. Of these, 33 were ineligible, 14 converted to mastectomy, 11 were unable to meet protocol-specific radiation endpoints, 1 had no definable tumor, 1 had 4 sites of cancer and 16 withdrew before completing surgery and radiation, leaving 194 patients [median age 61 (range 40-87)] eligible who completed breast conserving surgery and radiation therapy. With median follow-up of alive patients of 66.6 months (range: 4.1, 90.6), 6 patients have developed LR (5 ipsilateral breast and 1 chest wall), corresponding to an estimated cumulative incidence of local recurrence of 3.2% (95% CI: 1.3, 6.4) at 5 years. No patients have developed regional recurrence, 5 patients developed distant recurrence, 0 patients developed local and distant recurrence, 5 patients developed contralateral BC, 3 new non-BC primaries and 8 patients have died (1 related to BC). The rate of local recurrence in patients without a breast pre-op MRI (n=14) was 22.6% at 5 years compared to 1.7% among the 180 patients with a preop MRI (p=0.002). Patient age, number of sites of preoperative biopsy proven BC, HER2 status, pathologic T and N category were not statistically significantly associated with risk of LR. Conclusion The Z11102 clinical trial demonstrates that for women with MIBC breast conserving surgery with adjuvant radiation with lumpectomy site boosts has an acceptably low LR rate (3.2% at 5 years), making this a reasonable consideration for women with 2-3 ipsilateral foci. The LR rate was significantly higher in the small cohort of patients without preoperative breast MRI. Support: U10CA180821, U10CA180882; https://acknowledgments.alliancefound.org. ClinicalTrials.gov Identifier: NCT01556243 Table 1: Factors associated with LR after BCT for MIBC Citation Format: Judy C. Boughey, Kari M. Rosenkranz, Karla V. Ballman, Linda McCall, Bruce G. Haffty, Laurie W. Cuttino, Charlotte D. Kubicky, H. T. Carisa Le-Petross, Kimberly Van Zee, Armando E. Giuliano, Olwen M. Hahn, Kelly K. Hunt, Lisa Carey, Ann Partridge. Impact of Breast Conservation Therapy on Local Recurrence in Patients with Multiple Ipsilateral Breast Cancer – Results from ACOSOG Z11102 (Alliance) [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 GS4-01.

Abstract P3-03-27: Assessment of the proliferative activity of breast tissue after the administration of combined oral hormonal contraceptive, associated or not with estriol

Abstract The molecular mechanisms related to the breast cancer development are poorly understood, particularly those associated with estrogen. Evidence is mounting that developmental differences may influence the risk of cancer. However, understanding breast development, morphology and the biochemical factors that influence them is pertinent to study pre-malignant and malignant conditions that affect the breasts. Objective: To evaluate the proliferative activity of normal breast epithelium, through the c-myc expression, after the administration of combined oral hormonal contraceptive, associated or not with estriol. Retrospective double-blind study, in which the effect of combined oral contraceptives on the normal breast epithelium in women with fibroadenoma was evaluated. We analyzed 33 women In a cohort of 70 selected (37 excluded). The control group (1) received a compound containing levonorgestrel 0.15mg (LNG) and ethinylestradiol 0.03mg (EE), associated with 2mg of placebo (PLC) manufactured in the same capsule ingested daily for 21 days with an interval of 7 days, during four cycles. The study group (2) received a compound containing levonorgestrel 0.15 mg (LNG) and ethinylestradiol 0.03 mg (EE), associated with estriol 2 mg (E3) manufactured in the same capsule ingested daily for 21 days with an interval of 7 days, up to four cycles, when was proceeded the lumpectomy plus normal breast tissue sample. The UNIFESP-EPM Department of Pathology received the samples collected and fixed in 10% buffered formalin. We used an automation device to perform the Immunohistochemistry reactions, using c-myc antibodies, added DAB developer (diaminobenzidine) that provides a bright brown color that provides good contrast. We used conventional optical microscopy to read TMA slides. We considered positive the nuclei stained with dark brown in contrast to the blue negative to the c-myc reactions. We evaluated the epithelial areas at least five acinar units were present in the sample at 40X magnification. In the statistical analysis, the initial stages of consolidation as well as the analysis were performed in Software R (2020) and Rstudio (version 4.1), using the packages Tidyverse (2016), Psyco, SummaryTools, Janitor and DataExplorer. After verifying the assumptions of normality, independence and linearity, Student’s T test was performed for independent samples. An alpha of 0.05 was chosen for rejection of the null hypothesis, that is, a confidence interval of the test results of 95%. An effect size measure of the difference between the groups was also performed, called Cohen’s D; it ranges from -1 to 1, and the closer to 0, the smaller the effect size of the difference between the groups. Regarding the control group (30.30%) and the case group (69.70%). The table 2 presents the results for group 1 (COC + Placebo), while table 3 presents the results for group 2 (COC + Estriol). The T test was performed to verify a possible significant difference in the levels of cell proliferation in patients with or without Estriol. The normality was verified by the Shapiro-Wilk test, with data following a normal distribution (W = 0.901 and 0.96, p value > 0.3). The equitable variance was verified by the Levine test, which verified the equitable variance in both groups (F (1) = 0.24, p value = 0.62). A mean positive nucleus of 14 (SD = 10.65) was observed in the group of patients with contraceptive plus placebo and a mean of positive nuclei of 20.81 (SD = 9.88) in the group of patients with contraceptive plus Estriol. The ratio of positive nuclei to total was higher in the estriol group than in the control group. However, it was not possible to verify a significant difference in the two groups (T (14.57) = 1.3, p value = 0.215, d Cohen = 0.53). Cohen’s d pointed to a moderate difference effect (table 4). Table 2. Number of positive, negative and total normal epithelial cell nuclei in immunohistochemical investigation of c-myc antigens in Group 1 (COC + Placebo) (N = 10) Table 3. Number of positive, negative and total normal epithelial cell nuclei in immunohistochemical investigation of c-myc antigens in Group 2 (COC + Estriol) (N = 23) Table 4. Inferential results of the ratio of positive normal epithelial cell nuclei as a function of the control and case groups Citation Format: Osmar Pellegrini, Jr., Afonso Nazário, Angela Flavia L. Waitzberg. Assessment of the proliferative activity of breast tissue after the administration of combined oral hormonal contraceptive, associated or not with estriol [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 P3-03-27.

Abstract P2-14-01: MOLECULAR FLUORESCENCE-GUIDED SURGERY USING BEVA800 FOR THE ASSESSMENT OF TUMOR MARGINS DURING BREAST CONSERVING SURGERY OF PATIENTS WITH PRIMARY BREAST CANCER (MARGIN-II)

Abstract Introduction: The goal of breast conserving surgery (BCS) for early breast cancer (EBC) is to remove the tumor in toto and preserving as much of the normal breast tissue as possible. In 20-50% of cases a re-excision is necessary because of involved margins. Repeat surgeries are not only a burden to patients physically but also psychologically and can delay recommended adjuvant therapies. Accurate determination of tumor margins during surgery is therefore a critical need. Breast cancer tissue produces significantly higher amounts of VEGF-A than healthy tissue. VEGF-A stimulates tumor angiogenesis and is therefore a target for molecular imaging techniques. The fluorescence imaging agent bevacizumab-IRDye800CW (Beva800) is a conjugate of bevacizumab and IRDye800CW and binds specifically to VEGF-A. Beva800 provides a potentially efficacious approach to imaging specimen and cavity margins during BCS. We are presenting a phase II study that combined Beva800 with the SurgVision Explorer Air camera for intraoperative margin assessment during BCS for EBC. Methods: MARGIN II is a multicenter open-label single arm prospective clinical trial aimed at evaluating Beva800 for assessment of tumor margins in women with EBC scheduled for BCS. The study was a within-patient comparison of positive tumor margin rates using BCS standard of care margin assessment compared to intraoperative assessment with 4.5 mg Beva800 and fluorescence imaging with the SurgVision Explorer Air camera. All patients received an i.-v. bolus injection of 4.5 mg of Beva800 three days before surgery. The fluorescent signal was visualized during surgery using NIR fluorescence imaging (700–1000 nm). Standard of care margin assessment was defined as visual inspection, palpation and, in cases of pre-operative wire marking, specimen sonography or mammography. Beva800 efficacy was determined as the number of patients in which a pathology-confirmed positive margin was identified by fluorescence-guided surgery using Beva800 but not by standard of care BCS. Results: 49 patients were included in 5 centers. 4 training cases were only included in the safety analysis, 45 patients were evaluable for the efficacy analysis. 8 patients (17.8%) had involved margins after standard of care BCS, 4 of which were detected by molecular fluorescence intraoperatively resulting in the reduction of patients with positive margins by 50% (95% CI: 15.7%, 84.3%). 4 patients (8.9%; 95% CI: 2.5%, 21.1%) needed a re-excision because of involved margins. In 27 patients (60.0%) the additional molecular fluorescence guided cavity shaving did not change the resection status from positive to negative (false positive). Adverse events were reported by 16 of 49 patients (32.7%), but only 3 (6.1%) were related to Beva800 (syncope, hot flush, hypertensive crisis). One patient experienced a treatment related SAE (hypertensive crisis). No anti-Beva800 antibodies were detected. Conclusion: In our analysis the rate of necessary second operations was reduced by 50% using Beva800 and the SurgVision Explorer Air camera. The safety analysis confirmed the positive safety profile of Beva800 found in previous studies. Molecular fluorescence-guided surgery may have the potential to change the practice of breast conserving surgery by reducing unnecessary re-excisions. Future studies will have to address the high false positive rates. Citation Format: Hans-Christian Kolberg, Carmen Röhm, Angrit Stachs, Florian Schütz, Jens-Uwe Blohmer, Sarah Wetzig, Steffi Hartmann, Jörg Heil, Markus Hahn. MOLECULAR FLUORESCENCE-GUIDED SURGERY USING BEVA800 FOR THE ASSESSMENT OF TUMOR MARGINS DURING BREAST CONSERVING SURGERY OF PATIENTS WITH PRIMARY BREAST CANCER (MARGIN-II) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-14-01.

Transcriptomic profiles-based approach to decode the role of miR-122 in triple negative breast cancer.

miR-122 has been considered both as tumor suppressor miRNA and oncomiR in breast tumor phenotypes. However, the role of miR-122 in triple-negative breast cancer (TNBC) is still unknown. In this study, the clinical value of miR-122 was used to describe the transcriptomic landscape of TNBC tumors obtained from The Cancer Genome Atlas database. Low expression levels of miR-122 were associated with poor overall survival (OS) of TNBC patients than those with higher expression levels of miR-122. We identified gene expression profiles in TNBC tumors expressed lower or higher miR-122. Gene coexpression networks analysis revealed gene modules and hub genes specific to TNBC tumors with low or high miR-122 levels. Gene ontology and KEGG pathways analysis revealed that gene modules in TNBC with gain of miR-122 were related to cell cycle and DNA repair, while in TNBC with loss of miR-122 were enriched in cell cycle, proliferation, apoptosis and activation of cell migration and invasion. The expression of hub genes distinguished TNBC tumors with gain or loss of miR-122 from normal breast tissues. Furthermore, high levels of hub genes were associated with better OS in TNBC patients. Interestingly, the gene coexpression network related to loss of miR-122 were enriched with target genes of miR-122, but this did not observed in those with gain of miR-122. Target genes of miR-122 are oncogenes mainly associated with cell differentiation-related processes. Finally, 75 genes were identified exclusively associated to loss of miR-122, which are also implicated in cell differentiation. In conclusion, miR-122 could act as tumor suppressor by controlling oncogenes in TNBC.

A Ribosome-Related Prognostic Signature of Breast Cancer Subtypes Based on Changes in Breast Cancer Patients’ Immunological Activity

Background and Objectives. The prognostic role of adjacent nontumor tissue in patients with breast cancer (BC) is still unclear. The activity changes in immunologic and hallmark gene sets in normal tissues adjacent to BC may play a crucial role in predicting the prognosis of BC patients. The aim of this study was to identify BC subtypes and ribosome-associated prognostic genes based on activity changes of immunologic and hallmark gene sets in tumor and adjacent nontumor tissues to improve patient prognosis. Materials and Methods. Gene set variation analysis (GSVA) was applied to assess immunoreactivity changes in the overall sample and three immune-related BC subtypes were identified by non-negative matrix factorization (NMF). KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) analyses were after determining the prognostic gene set using the least absolute shrinkage and selection operator (LASSO) method. Ribosome-related genes were identified by PPI (protein-protein interaction) analysis, and finally a prognostic risk model was constructed based on the expression of five ribosomal genes (RPS18, RPL11, PRLP1, RPL27A, and RPL38). Results. A comprehensive analysis of immune and marker genomic activity changes in normal breast tissue and BC tissue identified three immune-related BC subtypes. BC subtype 1 has the best prognosis, and subtype 3 has the worst overall survival rate. We identified a prognostic gene set in nontumor tissue by the least absolute shrinkage and selection operator (LASSO) method. We found that the results of both KEGG and GO analyses were indistinguishable from those of ribosome-associated genes. Finally, we determined that genes associated with ribosomes exhibit potential as a reliable predictor of overall survival in breast cancer patients. Conclusions. Our research provides an important guidance for the treatment of BC. After a mastectomy, the changes in gene set activity of both BC tissues and the nontumor tissues adjacent to it should be thoroughly evaluated, with special attention to changes in ribosome-related genes in the nontumor tissues.