Winham, Stacey J.

Abstract Artificial intelligence (AI) models applied to mammography have been shown to improve breast cancer (BC) detection and risk estimation. The Transpara AI detection system has been associated with short and longer-term risk of invasive BC. Whether Transpara can identify changes in the breast associated with tamoxifen (TAM) and aromatase inhibitor (AIs) treatment is unknown. We compare changes in the Transpara malignancy score in the unaffected breast of BC cases using TAM or AIs to those of untreated women without cancer. Cases and controls were sampled from the Mayo Clinic Rochester Breast Screening Practice. Eligible cases were women with breast cancer treated with TAM and/or AIs for at least 8 months who had full field digital (FFDM) mammograms within two years prior to start of treatment (pretreatment mammogram). Controls did not have BC, were not using TAM, AIs, or postmenopausal hormones, and had at least two FFDM screening mammograms. Transpara scores (1-10) were calculated for the contralateral breast in cases and a randomly selected side for controls. Two controls were matched to each case on menopausal status at both mammograms, time between mammograms (< 6 months) and baseline Transpara score (±2 units). Conditional logistic regression was used to assess the association of treatment (case (TAM/AIs) vs. control) with change in Transpara score between the pretreatment mammogram and the mammogram taken closest to 13 months later. Change was classified as having a decrease in Transpara score between the two mammograms vs. no change or an increase. Models were adjusted for age and Transpara score at pretreatment mammogram, and time between the two mammograms. We performed analyses on all women combined and stratified by time on treatment, menopausal status, and treatment type (TAM vs. AIs). A total of 134 cases were identified, with 53 on TAM (21 premenopausal and 32 postmenopausal) and 81 on AIs (postmenopausal); 268 controls were matched to the cases. Characteristics of the study population are summarized (Table). Most case mammograms were obtained within six months prior to treatment start [median 2.63 months (interquartile range (IQR), 1.62, 5.72)] and median time on TAM/AIs was 10.9 months (IQR, 8.03, 16.6) at time of second mammogram. The median absolute change in Transpara score between the two mammograms was similar in cases [0 (IQR, -2, 1)] vs. controls [0 (IQR, -1.25, 1)]; 40.3% of cases and 39.2% of controls had any decrease. Conditional logistic models found no evidence for an association between TAM/AIs and decrease in Transpara score [odds ratio (OR) = 1.07; 95% confidence interval (CI), 0.65, 1.78]. When stratified by median time on treatment (10.9 months), there was suggestion of a stronger association for those on treatment for a longer duration (OR = 1.21; 95% CI, 0.61, 2.40 for >10.9 months vs. OR = 0.96; 95% CI, 0.43, 2.13 for < 10.9 months) but these results were not statistically significant. Similarly, odds ratios were stronger in postmenopausal (OR = 1.18; 95% CI, 0.67, 2.07) compared to premenopausal (OR = 0.91; 95% CI, 0.24, 3.45) women, and for AIs (OR = 1.21, 95% CI, 0.64, 2.29) vs. TAM (OR = 0.93; 95% CI, 0.39, 2.22) but none of these results reached statistical significance. In summary, we found no association between treatment with TAM/AIs and change in Transpara score. However, our study had limited power. Also, longer time on treatment as well as investigation of AI-based scores trained directly to assess BC risk may be necessary to identify relevant changes. Citation Format: Yvonne Wang, Christopher Scott, Matthew Jensen, Dan Hursh, Kathleen Brandt, Aaron Norman, Fang Fang Wu, Stacey Winham, Karla Kerlikowske, Celine Vachon. Changes in an artificial intelligence model score for breast cancer detection (Transpara) with tamoxifen and aromatase inhibitor treatment [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 PO2-09-04.

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 Breast cancer (BC) remains a leading health concern worldwide and is often undetected until the cancer metastasizes. Early detection can limit the need for aggressive treatments and improve prognosis. We analyzed early immune and molecular markers in benign breast disease (BBD) biopsies prior to BC development to identify women at elevated risk of developing node-positive invasive BC, which might direct surveillance and prevention efforts aimed at reducing incidence of this disease. Utilizing the Mayo Clinic BBD cohort, we compared women who developed node-positive BC following their BBD biopsy (cases; n=42) with those who remained cancer-free (controls; n=37), matched on patient age and biopsy date. We used NanoString nCounter system to identify differentially expressed genes (DEGs) between cases and controls. We also developed a multiplex immunofluorescence (mIF) approach using Opal system by Akoya Biosciences for comprehensive marker detection in FFPE tissue, enabling correlation of cells expressing DEGs with innate and adaptive immune cells. Digitized images were segmented, and cell phenotyping, and spatial relationships were compared between cases and controls. Our findings revealed significantly increased expression levels of IRF8 (interferon regulatory factor 8, a factor involved in immune cell differentiation) in controls as compared to cases and found that increased IRF8 expression in cases is correlated with delayed cancer onset. Additionally, controls exhibited higher frequencies of CD4+, CD8+, CD68+, CD20+ and CD11c+ immune cells, and lower frequency of Ki67+ staining, with a notable increase of CD11c+/CD68+ cells, a marker for M1 type macrophages, indicating a pro-inflammatory, antitumorigenic immune microenvironment polarization. Furthermore, our mIF analyses offer new insights into spatial biomarker localization, enhancing prognostic accuracy. Our findings suggest that decreased numbers of immune cells in BBD biopsies may be associated with increased risk of developing axillary node-positive BC. This research underscores the importance of dissecting molecular and immune interactions in BBD for assessing the risk of this disease. Our results have the potential to improve individualized risk assessment, leading to more targeted surveillance and informed screening, potentially reducing BC incidence and mortality through early intervention. This study not only contributes to our understanding of BC pathogenesis but also opens avenues for developing novel preventive strategies. Citation Format: Matilde Rossi, Nicole Cruz-Reyes, Melody L. Stallings Mann, Bryan M. McCauley, Tanya L. Hoskin, Robert A. Vierkant, Stacey J. Winham, Amy C. Degnim, Mark E. Sherman, Derek C. Radisky. Interferon regulatory factor 8 (IRF8) as a biomarker for early detection and prevention of axillary lymph node-positive breast cancer [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 4773.

Abstract Benign breast disease (BBD) affects over one million women annually in the United States and is diagnosed in nearly half of all women during their lifetime. Pseudoangiomatous stromal hyperplasia (PASH), a common BBD subtype, is characterized by an overaccumulation of tissue fibroblasts. Current research suggests a pivotal role of the immune microenvironment in progression of BBD to breast cancer, suggesting its composition as a determinant of breast cancer risk. Our study evaluates whether fibroblast variations and immune microenvironment differences in BBD biopsies containing PASH lesions are associated with subsequent breast cancer development. We evaluated BBD biopsies from women who subsequently developed breast cancer (cases, N=5) or remained cancer-free (controls, N=5). Using Opal-based multiplex immunofluorescence (mIF) assays, we mapped expression of fibroblast markers linked to breast cancer associated fibroblasts (CAFs), including vimentin (VIM), smooth muscle actin (ACTA2), fibroblast specific protein-1 (S100A4/FSP1), fibroblast associated protein (FAP), integrin beta-1 (CD29/ITGB1), and platelet derived growth factor receptor beta (PDGFRβ) and markers of key immune cells (T cells: CD4, CD8; dendritic cells: CD11c; B cells: CD20; and macrophages: CD68) along with MKI67 as a proliferation marker. Our results revealed marked differences in fibroblast marker expression and immune cell populations between these groups in the normal lobules in the BBD biopsies. PASH cases showed elevated PDGFRβ and CD29/ITGB1 expression in VIM-positive fibroblasts, markers characteristic of CAF subtypes known to accumulate in more aggressive tumor microenvironments. Conversely, a higher percentage of FSP1+/VIM+ cells, similar to that of a different CAF subtype, was noted in the lobules of PASH controls. This difference in biomarker expression, along with corresponding variations in immune cell counts, suggests a complex interplay between fibroblast composition and immune response that ultimately modulate cancer risk in PASH-containing BBD tissues. Additionally, increased MKI67 expression in PASH cases potentially signals the higher likelihood of cancer development. This work advances our understanding of fibroblast heterogeneity in BBD and its implications for breast cancer risk and progression, and paves the way for innovative strategies to improve clinical outcomes for BBD patients to reduce their incidence of breast cancer. Citation Format: Nicole Cruz-Reyes, Matilde Rossi, Melody Stallings Mann, Bryan McCauley, Tanya Hoskin, Robert Vierkant, Stacey Winham, Amy Degnim, Mark E. Sherman, Derek C. Radisky. Fibroblast diversity and immune microenvironment in pseudoangiomatous stromal hyperplasia (PASH): Associations with breast cancer risk [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 283.

Abstract Background Alcohol consumption behaviors and alcohol use disorder risk and presentation differ by sex, and these complex traits are associated with blood concentrations of the steroid sex hormones, testosterone and estradiol, and their regulatory binding proteins, sex hormone binding globulin (SHBG) and albumin. Genetic variation is associated with alcohol consumption and alcohol use disorder, as well as levels of steroid sex hormones and their binding proteins. Methods To assess the contribution of genetic factors to previously described phenotypic associations between alcohol-use traits and sex-hormone levels, we estimated genetic correlations (rg) using summary statistics from prior published, large sample size genome-wide association studies (GWAS) of alcohol consumption, alcohol dependence, testosterone, estradiol, SHBG, and albumin. Results For alcohol consumption, we observed positive genetic correlation (i.e. genetic effects in the same direction) with total testosterone in males (rg = 0.084, p = 0.007) and trends toward positive genetic correlation with bioavailable testosterone (rg = 0.060, p = 0.084) and SHBG in males (rg = 0.056, p = 0.086) and with albumin in a sex-combined cohort (rg = 0.082, p = 0.015); however in females, we observed positive genetic correlation with SHBG (rg = 0.089, p = 0.004) and a trend toward negative genetic correlation (i.e. genetic effects in opposite directions) with bioavailable testosterone (rg = -0.064, p = 0.032). For alcohol dependence, we observed a trend toward negative genetic correlation with total testosterone in females (rg = -0.106, p = 0.024) and positive genetic correlation with BMI-adjusted SHBG in males (rg = 0.119, p = 0.017). Several of these genetic correlations differed between females and males and were not in the same direction as the corresponding phenotypic associations. Conclusions Findings suggest that shared genetic effects may contribute to positive associations of alcohol consumption with albumin in both sexes, as well as positive associations between alcohol consumption and bioavailable testosterone and between alcohol dependence and SHBG in males. However, relative contributions of heritable and environmental factors to associations between alcohol-use traits and sex-hormone levels may differ by sex, with genetic factors contributing more in males and environmental factors contributing more in females.

Abstract Background: Women with an inherited pathogenic variant in BRCA1 or BRCA2 have a greatly increased risk of developing ovarian cancer, but the importance of behavioral factors is less clear. We used a case-only design to compare the magnitude of associations with established reproductive, hormonal, and lifestyle risk factors between BRCA mutation carriers and noncarriers. Methods: We pooled data from five studies from the Ovarian Cancer Association Consortium including 637 BRCA carriers and 4,289 noncarriers. Covariate-adjusted generalized linear mixed models were used to estimate interaction risk ratios (IRR) and 95% confidence intervals (CI), with BRCA (carrier vs. noncarrier) as the response variable. Results: IRRs were above 1.0 for known protective factors including ever being pregnant (IRR = 1.29, 95% CI; 1.00–1.67) and ever using the oral contraceptive pill (1.30, 95% CI; 1.07–1.60), suggesting the protective effects of these factors may be reduced in carriers compared with noncarriers. Conversely, the IRRs for risk factors including endometriosis and menopausal hormone therapy were below 1.0, suggesting weaker positive associations among BRCA carriers. In contrast, associations with lifestyle factors including smoking, physical inactivity, body mass index, and aspirin use did not appear to differ by BRCA status. Conclusions: Our results suggest that associations with hormonal and reproductive factors are generally weaker for those with a pathogenic BRCA variant than those without, while associations with modifiable lifestyle factors are similar for carriers and noncarriers. Impact: Advice to maintain a healthy weight, be physically active, and refrain from smoking will therefore benefit BRCA carriers as well as noncarriers.

AbstractDeveloping novel cancer prevention medication strategies is important for reducing mortality. Identification of common genetic variants associated with cancer risk suggests the potential to leverage these discoveries to define causal targets for cancer interception. Although each risk variant confers small increases in risk, researchers propose that blocking those that produce causal carcinogenic effects might have large impacts on cancer prevention. While a promising concept, we describe potential hurdles that may need to be scaled to reach this goal, including: (i) understanding the complexity of risk; (ii) achieving statistical power in studies with binary outcomes (cancer development: yes or no); (iii) characterization of cancer precursors; (iv) heterogeneity of cancer subtypes and the populations in which these diseases occur; (v) impact of static genetic markers across complex events of the life course; (vi) defining gene–gene and gene–environment interactions and (vii) demonstrating functional effects of markers in human populations. We assess short-term prospects for this research against the backdrop of these challenges and the potential to prevent cancer through other means.See related commentary by Peters and Tomlinson, p. 7

Importance Benign breast disease (BBD) comprises approximately 75% of breast biopsy diagnoses. Surgical biopsy specimens diagnosed as nonproliferative (NP), proliferative disease without atypia (PDWA), or atypical hyperplasia (AH) are associated with increasing breast cancer (BC) risk; however, knowledge is limited on risk associated with percutaneously diagnosed BBD. Objectives To estimate BC risk associated with BBD in the percutaneous biopsy era irrespective of surgical biopsy. Design, Setting, and Participants In this retrospective cohort study, BBD biopsy specimens collected from January 1, 2002, to December 31, 2013, from patients with BBD at Mayo Clinic in Rochester, Minnesota, were reviewed by 2 pathologists masked to outcomes. Women were followed up from 6 months after biopsy until censoring, BC diagnosis, or December 31, 2021. Exposure Benign breast disease classification and multiplicity by pathology panel review. Main Outcomes The main outcome was diagnosis of BC overall and stratified as ductal carcinoma in situ (DCIS) or invasive BC. Risk for presence vs absence of BBD lesions was assessed by Cox proportional hazards regression. Risk in patients with BBD compared with female breast cancer incidence rates from the Iowa Surveillance, Epidemiology, and End Results (SEER) program were estimated. Results Among 4819 female participants, median age was 51 years (IQR, 43-62 years). Median follow-up was 10.9 years (IQR, 7.7-14.2 years) for control individuals without BC vs 6.6 years (IQR, 3.7-10.1 years) for patients with BC. Risk was higher in the cohort with BBD than in SEER data: BC overall (standard incidence ratio [SIR], 1.95; 95% CI, 1.76-2.17), invasive BC (SIR, 1.56; 95% CI, 1.37-1.78), and DCIS (SIR, 3.10; 95% CI, 2.54-3.77). The SIRs increased with increasing BBD severity (1.42 [95% CI, 1.19-1.71] for NP, 2.19 [95% CI, 1.88-2.54] for PDWA, and 3.91 [95% CI, 2.97-5.14] for AH), comparable to surgical cohorts with BBD. Risk also increased with increasing lesion multiplicity (SIR: 2.40 [95% CI, 2.06-2.79] for ≥3 foci of NP, 3.72 [95% CI, 2.31-5.99] for ≥3 foci of PDWA, and 5.29 [95% CI, 3.37-8.29] for ≥3 foci of AH). Ten-year BC cumulative incidence was 4.3% for NP, 6.6% for PDWA, and 14.6% for AH vs an expected population cumulative incidence of 2.9%. Conclusions and Relevance In this contemporary cohort study of women diagnosed with BBD in the percutaneous biopsy era, overall risk of BC was increased vs the general population (DCIS and invasive cancer combined), similar to that in historical BBD cohorts. Development and validation of pathologic classifications including both BBD severity and multiplicity may enable improved BC risk stratification.

Mendelian randomization has become a popular tool to assess causal relationships using existing observational data. While randomized controlled trials are considered the gold standard for establishing causality between exposures and outcomes, it is not always feasible to conduct a trial. Mendelian randomization is a causal inference method that uses observational data to infer causal relationships by using genetic variation as a surrogate for the exposure of interest. Publications using the approach have increased dramatically in recent years, including in the field of hepatology. In this concise review, we describe the concepts, assumptions, and interpretation of Mendelian randomization as related to studies in hepatology. We focus on the strengths and weaknesses of the approach for a non-statistical audience, using an illustrative example to assess the causal relationship between body mass index and NAFLD.