Stallings‐Mann, Melody

Abstract Background The incidence of early onset breast cancers (BCs) has increased, paralleling rising trends in delayed childbearing. We hypothesize that a distinct postpartum BC (PPBC) subtype, identifiable by time since last birth (TSLB) and biomarker expression, contributes to this trend. Methods We applied GeoMx Digital Spatial Profiling (DSP) to measure associations between TSLB and 71 proteins in 640 BCs from women aged ≤40 years included in the Young Women’s Breast Cancer Study. We analyzed data using univariable linear regression and multivariable sliced inverse regression to account for higher order interactions among biomarkers. Results In keratin-rich segments, progesterone receptor (PR) (P = 1.00 × 10−4) and PTEN (P = 1.00 × 10−3) were associated with longer TSLB; multivariable analyses revealed positive associations for GZMB (P = 4.00 × 10−4), SMA (P = 1.00 × 10−4), and NF-1 (P = 1.00 × 10−3). In keratin-poor segments, univariable significant positive associations were found for PR (P = 2.00 × 10−4) and PTEN (P = 1.00 × 10−3), whereas CD20 (P = 3.00 × 10−4) and CTLA4 (P = 4.00 × 10−5) were negatively associated; multivariable significant associations were found for fibronectin (P = 3.00 × 10−5) and pan-Akt (P = 1.00 × 10−3). Associations persisted after adjustment for multiple comparisons and BC molecular subtypes. Associations including for PR, PTEN, and CD20 were strongest among women with the shortest TSLB. OPAL multiplex immunofluorescence assays for PR, PTEN, CD20, SMA, and CTLA4 replicated several DSP findings, particularly when stratified by subtype and with compartment matching. In TCGA, RNA species linked to proteins associated with TSLB correlated strongly with a T-cell exhaustion signature previously linked to poor prognosis among premenopausal women. Conclusion These data support PPBC as a biologically coherent phenotype defined by TSLB and biomarker profile, with potential implications for prevention and therapy.

Background: Discovering risk biomarkers in small benign breast disease (BBD) biopsies is constrained by scarce tissue and microanatomic heterogeneity of terminal duct lobular units (TDLUs). We tested whether tissue-sparing tissue microarray (TMA)–based Digital Spatial Profiling (DSP) can deliver reproducible, biologically coherent protein measurements across benign lobules and breast cancers (BCs), and how well DSP aligns with standard immunoassays. Methods: We performed a pilot using tissues from the Mayo Clinic BBD cohort using TMAs representing four contexts: terminal duct lobular units (TDLUs) from BBD biopsies preceding BC and matched BBD-controls, subsequent BCs, and BC-associated TDLUs. We profiled 79 proteins by DSP (37 retained after QC) and benchmarked against chromogenic IHC and OPAL immunofluorescence. Reproducibility was evaluated using intraclass correlation coefficients (ICCs), cross-platform agreement (weighted kappa), marker correlations, and mixed-effects models with false-discovery-rate (FDR) control. Results: We analyzed 368 BBD-TDLU cores (88 cases; 88 controls), 204 BC cores and 110 BC-associated TDLU cores. ICCs were highest in BC tissues, and lower in BC-associated TDLUs and BBD-TDLUs. Agreement was slight–to-fair in TDLUs but moderate (ER/PR) to substantial (BCL2) in BC. DSP recapitulated expected immunologic correlations (CD45 with T-cell, B-cell, and macrophage markers) and tissue-type gradients (BC > BC-associated TDLUs > BBD-TDLUs). Exploratory case–control differences in BBD-TDLUs did not persist after FDR control. Conclusions: TMA-based DSP is feasible in archival breast tissues and yields biologically coherent, cross-platform-benchmarked profiles that are particularly robust in BC, while conserving scarce TDLUS and clarifying current limits of single-marker risk stratification from benign lobules. These data provide a foundation for refined sampling and expanded panels in future TDLU-focused studies.

Abstract Background: Postpartum breast cancer (PPBC) is associated with increased morbidity and mortality among women within 5 to 10 years following childbirth. The postpartum breast undergoes involution, involving tissue remodeling and immune cell infiltration, potentially creating a pro-tumorigenic microenvironment. The molecular mechanisms underlying this elevated risk remain poorly understood. In this study, we compared normal breast tissue samples and human mammary epithelial cells (HMECs) from postpartum women to those from age- and BMI-matched nulliparous women to elucidate potential molecular contributors to PPBC risk. Methods: Transcriptomic profiling was performed on RNA extracted from FFPE normal breast tissues using the NanoString IO 360 and BC 360 platforms to examine gene expression in nulliparous and postpartum women under 45 years of age. HMECs were isolated from organoids derived from breast tissue samples and used for next-generation sequencing and knockdown experiments targeting STAT1 and GBP1. Gene set enrichment analysis (GSEA) was applied to NanoString and RNA-seq datasets to identify differentially enriched genes between postpartum and nulliparous women. Results: Transcriptomic analysis revealed significant upregulation of interferon-stimulated genes, including STAT1 and GBP1, in postpartum breast tissues, consistent with increased immune responses during involution. NanoString data showed that markers of cell proliferation, such as Ki67, STAT1, and GBP1, were significantly higher in samples from parous compared to nulliparous women (p < 0.05). GSEA revealed that cell cycle-promoting genes (GBP1, cyclins, CDKs) were upregulated, while cyclin-dependent kinase inhibitors (CDKNs) were downregulated in postpartum breast tissues. Immunohistochemistry confirmed significantly higher expression of Ki67, STAT1, and GBP1 in postpartum versus nulliparous breast tissues (p < 0.0001). Silencing STAT1 and GBP1 in HMECs resulted in decreased cell proliferation, G1 cell cycle arrest, and increased expression of CDKN1A and CDKN1C. Conclusion: These findings suggest that STAT1 and GBP1 promote proliferation in postpartum breast epithelial cells by downregulating cyclin-dependent kinase inhibitors, potentially contributing to the increased risk of PPBC. Targeting these pathways may offer novel strategies for mitigating postpartum breast cancer risk. Citation Format: Joshua Ogony, Melody Stallings-Mann, Sarah McLaughlin, Mark Sherman, Derek C. Radisky. STAT1 and GBP1 promote proliferation in postpartum breast epithelium by downregulating cyclin-dependent kinase inhibitors: implications for increased postpartum breast cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 187.

Abstract Background: Breast cancer (BC) remains a leading health concern worldwide and is often undetected until it metastasizes, limiting treatment options and worsening prognosis. Early detection of molecular and immune markers in benign breast disease (BBD) could identify women at elevated risk of developing invasive, node-positive BC, enabling targeted surveillance and prevention efforts. Building on this premise, we investigated the immune and molecular landscape of BBD biopsies preceding BC development to enhance risk stratification and inform preventive strategies. Methods: Utilizing the Mayo Clinic BBD cohort, we conducted a case-control study comparing women who developed node-positive BC following their BBD biopsy (cases; n=8) to those who remained cancer-free (controls; n=11), matched on patient age and biopsy date. Differential gene expression was assessed using the NanoString nCounter system to identify differentially expressed genes between cases and controls. We developed a multiplex immunofluorescence (MxIF) approach using the Opal system by Akoya Biosciences for comprehensive marker detection in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Digitized images were segmented for cell phenotyping, and spatial relationships of immune cells were analyzed and compared between cases and controls. Results: Our findings revealed significantly increased expression levels of immunosuppressive genes in the case group, including CD48, CD84, IL-32, and LAIR1. Immunohistochemical analyses showed that controls exhibited higher frequencies of CD68+ (macrophages), CD11c+ (M1 macrophages), and CD206+ (M2 macrophages) cells compared to cases. Notably, controls had a significant increase in CD68+CD11c+ cells, indicative of a pro-inflammatory, antitumorigenic M1 macrophage polarization. Furthermore, our MxIF analyses provided novel insights into spatial biomarker localization, enhancing prognostic accuracy. Conclusions: The decreased numbers of immune cells and increased expression of immunosuppressive genes in BBD biopsies may be associated with an elevated risk of developing axillary node-positive BC. These findings emphasize the importance of dissecting molecular and immune interactions in BBD for risk assessment. Our results have the potential to improve individualized risk stratification, leading to more targeted surveillance and informed screening. This research not only contributes to our understanding of BC pathogenesis but also opens avenues for developing novel preventive strategies, potentially reducing BC incidence and mortality through early intervention. Citation Format: Matilde Rossi, Nicole Cruz-Reyes, Trista Navarro, Melody L. Stallings-Mann, Bryan M. McCauley, Robert A. Vierkant, Stacey J. Winham, Mark E. Sherman, Amy C. Degnim, Derek C. Radisky. Immune and molecular markers in benign breast disease predict risk of node-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1912.

Objectives/Goals: This project will enrich our understanding of basal breast carcinogenesis, highlighting the distinct biological differences in breast cancer risk between non-Hispanic Black (NHB) or non-Hispanic White (NHW) women, but also has the potential to provide real-world solutions thereby contributing to reducing health disparities in breast cancer outcomes Methods/Study Population: In the biological investigation, I am utilizing a unique cohort of normal breast tissues from Mayo Clinic patients who underwent breast reduction surgery that have self-identified as NHB or NHW premenopausal women. From these tissues, we are able to subculture human mammary epithelial cells, which we will use for our consequent experiments. For the qualitative study, I aim to recruit up to 40–50 eligible NHB women identified at increased risk for breast cancer and conduct semi-structured qualitative interviews informed by the National Institute on Minority Health and Health Disparities Research Framework. Results/Anticipated Results: Gene expression profiling on our cohort of 15 NHB and 40 NHW premenopausal women identified a gene expression signature in NHB women indicative of elevated Hedgehog signaling, a key factor in triple-negative breast cancer (TNBC) development and progression. I hypothesize that persistent activation of Hedgehog signaling within NHB women instigates the proliferation and transformation of basal stem cells within the breast, thus fueling TNBC development. Understanding the perceptions and experiences of NHB women that are identified as being at an increased risk for BC will aid in the identification of barriers and facilitators to their risk reduction care and in turn provide implementation strategies that could contribute to alleviating the racial disparity seen in TNBC morbidity, mortality, and preventative care. Discussion/Significance of Impact: The significance of this project lies in its dual approach, uncovering basal breast cancer mechanisms in a high-risk group using normal breast tissue and evaluating the perspectives from NHB women to enhance the practical relevance of our findings.

Abstract Transcriptome profiling using RNA sequencing (RNA-seq) of bulk formalin-fixed paraffin-embedded (FFPE) tissue blocks is a standard method in biomedical research. However, when used on tissues with diverse cell type compositions, it yields averaged gene expression profiles, complicating biomarker identification due to variations in cell proportions. To address the need for optimized strategies for defining individual cell type compositions from bulk FFPE samples, we constructed single-cell RNA-seq reference data for breast tissue and tested cell type deconvolution methods. Initial simulation experiments showed similar performances across multiple commonly used deconvolution methods. However, the introduction of FFPE artifacts significantly impacted their performances, with a root mean squared error (RMSE) ranging between 0.04 and 0.17. Scaden, a deep learning-based method, consistently outperformed the others, demonstrating robustness against FFPE artifacts. Testing these methods on our 62-sample RNA-seq benign breast disease cohort in which cell type composition was estimated using digital pathology approaches, we found that pre-filtering of the reference data enhanced the accuracy of most methods, realizing up to a 32% reduction in RMSE. To support further research efforts in this domain, we introduce SCdeconR, an R package designed for streamlined cell type deconvolution assessments and downstream analyses.

Abstract Over 1 million women in the U.S. undergo radiologically guided percutaneous biopsies annually. Of these, 75% reveal benign breast disease (BBD), typically surrounded by normal breast lobules, which are proposed to reflect the biology of the breast parenchyma diffusely. With aging, normal lobules undergo involution, and delays in this process are linked to increased breast cancer (BC) risk. Because BBD patients are at increased risk of developing BC, and pathologic classification only stratifies risks among groups of patients, novel methods for individual BC risk prediction are needed to guide precision management. Prior literature has shown that most proliferating epithelial cells in normal lobules do not express estrogen receptor (i.e., ER-/Ki67+), whereas co-expression is frequent in BC precursors and in BCs (i.e., ER+/Ki67+). Thus we developed and applied a novel quantitative dual ER/Ki67 stain to BBD biopsies from the well-characterized contemporary Mayo Clinic BBD cohort (2002-2013) to assess differences by levels of lobular involution and tissue type (BBD vs. normal), as well as associations with BC risk. We identified 100 BBD biopsies preceding BC (cases) and 100 biopsies from cancer-free women (controls), frequency-matched on age and length of follow-up. A multiplex immunofluorescence assay was developed to detect ER/Ki67 coexpression in cytokeratin positive epithelial cells in normal lobules or BBD lesions in FFPE tissue sections. An inverse normal transformation was applied to cell counts and proportions and associations were assessed for levels of lobular involution, tissue type (normal lobule vs. BBD), and case status using t-tests and linear regression models. The final data set included 1152 normal lobules or BBD lesions in biopsies of 177 subjects (88 cases and 89 controls). In univariate analyses, normal lobules showing delayed age related involution included higher counts of ER-/Ki67+, ER+/Ki67- and ER+/Ki67+ cells, and the lowest proportion of ER+/Ki67- cells (p<0.03; all). In separate analyses comparing presence vs. absence, detection of any ER+/Ki67+ cells was suggestively more common in non-involuted vs. involuted lobules. Compared with normal lobules, BBD showed higher counts and proportions of ER+/Ki67- and ER+/Ki67+ cells and lower values for ER-/Ki67+ (all p<0.001). In these preliminary results, marker expression in normal lobules and in BBD did not differ by case-control status. Preliminarily, we find that ER+/Ki67+ cells are increased in normal lobules with delayed age-related involution and in BBD lesions, consistent with their associations with increased BC risk. These findings are consistent with the risk reducing effects of endocrine chemoprevention and may serve as useful biomarkers in prevention therapy. In this analysis, limited to only a few lobules and BBD lesions per biopsy, ER+/Ki67+ cell counts and proportions did not vary significantly by case-control status. Citation Format: Melody L. Stallings-Mann, Robert A. Vierkant, Stacey J. Winham, Bryan McCauley, Matt R. Jensen, Sophie Noel, Laura Pacheco-Spann, Celine M. Vachon, Amy C. Degnim, Derek C. Radisky, Mark E. Sherman. Measurement of ER and Ki67 coexpression in cytokeratin positive epithelial cells in benign breast disease (BBD) percutaneous biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB328.

Abstract Background: Age-related lobular involution (ARLI), characterized by regression of breast epithelial tissue during the peri-and post-menopausal period, plays a crucial role in breast cancer risk. Delayed or incomplete ARLI is linked with increased postmenopausal breast cancer susceptibility.This study focuses on unraveling the molecular mechanisms of ARLI, drawing parallels with the well-characterized postpartum involution (PPI). Our aim was to discover biomarkers differentiating women who progressed through ARLI from those with stagnant ARLI. Methods: We utilized the Mayo Clinic Benign Breast Disease (BBD) cohort, analyzing 108 perimenopausal women aged 45-55 who had a subsequent benign biopsy between 2-15 years later. We assessed Lobular Involution (Ll) status by comparing the number and size of lobules in initial and subsequent biopsies. RNA profiling of initial biopsies was performed using NanoString 10360 and BC360, identifying differentially expressed genes between ARLI progression and stagnation. Immunohistochemistry (IHC) mapped the expression of these biomarkers in epithelial lobules. Results: We identified 37 genes with significant differential expression, highlighting Leukemia Inhibitory Factor Receptor (LIFR) as upregulated in ARLI stagnant samples. LIFR, part of the IL-6 cytokine signaling pathway, is known for its role in the induction of apoptosis during PPI via pSTAT3 activation. Our IHC analyses of initial biopsies of women who differentially progress through ARLI confirmed differential expression of LIFR that is coordinately regulated with pSTAT3. Conclusion: Our findings reveal significant parallels between ARLI and PPI. Future research will assess how LIFR/pSTAT3 signaling is linked to induction of apoptotic markers in perimenopausal women and will investigate how anomalies in the LIFR/pSTAT3 pathway might contribute to heightened breast cancer risk in women with delayed or incomplete ARLI. This study not only sheds light on the complex biological processes of aging in breast tissue but also opens new avenues for breast cancer risk assessment and prevention strategies. Citation Format: Jaida C. Lue, Derek Radisky, Melody Stallings-Mann, Mark Sherman, Amy Degnim, Bryan McCauley, Tanya Hoskin. Deciphering the link between aging, lobular involution, and breast cancer risk: The role of LIFR/pSTAT3 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 849.

Abstract Triple negative breast cancer (TNBC) represents an aggressive and therapeutically challenging breast cancer subtype with higher incidence and mortality rates in non-Hispanic Black (NHB) women. This disparity persists even after adjusting for socioeconomic factors, tumor biology, and quality of care. The study aims to unravel specific molecular mechanisms underlying this disparity, with a focus on Hedgehog signaling pathways in premenopausal NHB women. Understanding these mechanisms is vital for developing targeted therapies and personalized medicine approaches to address these health disparities. Our research utilized a unique cohort of NHB and non-Hispanic White (NHW) women who underwent breast reduction surgery at the Mayo Clinic and donated breast tissue samples for our study. We isolated RNA from whole tissue sections from a cohort of 12 NHB and 40 NHW women who were parous and premenopausal at time of surgery. We performed gene expression profiling using NanoString BC360 and IO360 assays. This allowed us to identify a distinct gene expression signature characteristic of basal-like/TNBC and indicative of heightened activity in the Hedgehog signaling pathway. We also employed advanced cell culture techniques to generate tissue organoids and subculture human mammary epithelial cells (HMECs) that preserve the intrinsic characteristics of the original tissue samples. We found that significantly differential expression of IL20RA and SOX10, principal factors in the gene expression signature, were preserved in both the derived organoids and the HMECs. These results suggest a potential intrinsic difference in the activation of the Hedgehog pathway in the breast epithelial cells of NHB women. This activation may contribute to increased proliferation and transformation of basal stem cells within the breast, possibly fueling the development and progression of TNBC. The implication of activated Hedgehog signaling in NHB women opens new avenues for understanding TNBC disparities. IL20RA and SOX10 represent potential biomarkers for TNBC risk that may serve as a stratification tool for NHB women. The results of this study have the potential to improve assessment and management of TNBC risk among NHB women, thereby contributing significantly to reducing health disparities in breast cancer outcomes. Citation Format: Jennifer Vanessa Cabezas, Savanna Touré, Matthew Wilson, Melody L. Stallings Mann, Laura M. Pacheco-Spann, Joshua W. Ogony, Mark E. Sherman, Derek C. Radisky. Identifying molecular mechanisms in triple negative breast cancer disparities: Unveiling the role of Hedgehog signaling in non-Hispanic Black women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2139.