Biomarkers Of Breast Cancer Risk Research Articles

Abstract PO4-08-07: Background parenchymal enhancement (BPE) on breast magnetic resonance imaging as a biomarker of breast cancer risk among BRCA1/2 carriers

Abstract Introduction: BRCA1/2 carriers are recommended to undergo dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) screening of the breasts annually. Fibroglandular tissue (FGT) may enhance with MRI contrast agent termed background parenchymal enhancement (BPE). BPE is believed to be a marker of hormonally responsive breast tissue, as BPE is impacted by aromatase inhibition and by oophorectomy. BPE has been shown to be more strongly associated with breast cancer risk than breast density. We hypothesize that BPE may serve as a useful dynamic marker of breast cancer risk among BRCA1/2 mutation carriers, given their frequent MRI screening. Methods: We identified female BRCA1/2 carriers from the University of Pennsylvania Cancer Risk Evaluation Program with no personal history of breast or ovarian cancer at the time of breast MRI. Patients diagnosed within 6 months were excluded to remove prevalent cancer cases at MRI. Among 421 eligible patients, 42 were diagnosed with breast cancer. BPE was quantified from all available breast MRIs using a validated, fully automated method to segment fibroglandular tissue (FGT) and quantify BPE. For preprocessing, the N4ITK algorithm was used for bias-field artifact correction and FGT was segmented using a soft-margin support vector machine classifier using the T1-weighted nonfat-saturated breast MRI. FGT was segmented and quantified in 3D. For BPE quantification, the T1-weighted nonfat-saturated image and the derived FGT mask were registered to the pre-contrast DCE-MRI. The first post contrast DCE-MRI and FGT mask were used to compute the relative enhancement map compared to the pre-contrast image. We calculated BPE metrics in two ways. First, the median BPE enhancement was calculated as the median enhancement of all voxels within the FGT mask. Second the BPE enhancement ratio was calculated as the proportion of voxels enhancing at ≥20%. We performed Cox proportional hazards regression to estimate the hazard ratios for breast cancer per standard deviation unit increase in each BPE metric, with first MRI as the time origin and censoring upon bilateral mastectomy, ovarian cancer diagnosis, death, or loss to follow-up and adjustment for age at MRI, menopause status, volumetric breast density (VBD), body mass index (BMI), and BRCA1 vs. BRCA2 mutation. BPE, VBD, BMI, and menstrual status were included as time varying covariates, enabling inclusion of multiple BPE measurements per woman. Results: The mean age at first MRI was 39 for cases and 41 for non-cases, and the mean number of MRIs was 4. There were no significant differences in median BPE (14% vs. 14%, p=0.965) or BPE ratio (34% vs. 36%, p=0.769) between cases and non-cases. In regression analyses adjusted for age and menstrual status, median BPE was significantly associated with breast cancer risk (HR=1.12, p=0.025). The HR was similar in the fully adjusted model, though did not reach statistical significance (HR=1.11, p=0.055). Higher BPE ratio was significantly associated with increased breast cancer risk in both minimally (HR=1.21, p=0.005) and fully adjusted models (HR=1.21, p=0.012). Conclusions: Our results suggest that BPE is significantly associated with breast cancer diagnosis among BRCA1/2 carriers undergoing breast MRI screening. Both median BPE and BPE ratio metrics were associated with breast cancer risk, even after adjusting for breast density and when accounting for multiple measurements of BPE over time, suggesting that BPE may provide additional risk information for BRCA1/2 carriers. Table 1: Results of Cox proportional hazards regression with time varying covariates Citation Format: Anne Marie McCarthy, Alex Anh-Tu Nguyen, Walter Mankowski, Eric Cohen, Sarah Ehsan, Rhonda Gillette, Lauren Pantalone, Susan Weinstein, Emily Conant, Susan Domchek, Despina Kontos. Background parenchymal enhancement (BPE) on breast magnetic resonance imaging as a biomarker of breast cancer risk among BRCA1/2 carriers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-08-07.

396 Unraveling the Immunological Basis of Lobular Involution in Breast Cancer Development

OBJECTIVES/GOALS: Reveal common immune mechanisms in dysregulated age-related lobular involution (ARLI) and post-partum lobular involution (PPLI) to understand their link to increased breast cancer risk, challenging the traditional view of their distinctiveness. Ultimately, to improve breast cancer risk assessment and personalized prevention METHODS/STUDY POPULATION: The Mayo Clinic Benign Breast Disease (BBD) cohort comprises of ~20,000 women with benign biopsies, including ~1000 women with sequential benign biopsies. Lobular involution (LI) status was assessed by selecting perimenopausal women, ages 45-55, with sequential biopsies, comparing acini number and lobule size between initial and subsequent biopsies. NanoString IO360/ BC360 RNA profiling identified differentially expressed genes associated with dysregulated LI. Using multiplex immunofluorescence (mIF), I'll analyze and spatially map immune biomarkers related to dysregulated ARLI and PPLI in BBD tissue from perimenopausal women who did or did not go on to develop breast cancer, assessing the commonality of ARLI and PPLI markers and exploring their potential as risk biomarkers for breast cancer. RESULTS/ANTICIPATED RESULTS: Preliminary findings link patients who display dysregulated ARLI with an increased breast cancer risk and identify vital PPLI biomarkers in perimenopausal women. I expect the biopsies of women who developed post-menopausal breast cancer (PMBC) and post-partum breast cancer (PPBC) to exhibit elevated levels of dysregulated ARLI immune biomarkers and PPLI biomarkers. Spatially mapping these markers promises to provide a more comprehensive understanding of their interactions, potentially revealing common immunological pathways. These findings could transform our current paradigm of ARLI and PPLI as distinct processes and demonstrate their interconnection in shaping breast cancer risk. DISCUSSION/SIGNIFICANCE: PMBC and PPBC dominate majority of breast cancer cases. Both involve activation of the understudied process of lobular involution, which has been shown to have pro-tumorigenic traits. Elucidating these mechanisms will aid more efficient risk stratification and personalized prevention to reduce incidence and mortality of breast cancer.

Abstract 2179: Microbiome-derived metabolites mediate carcinogenic alterations of breast tissue in the context of obesity

Abstract Obesity increases the relative risk for breast cancer incidence. Multiple molecular mechanisms linked to obesity drive breast cancer progression. Obesity shifts the gut microbiome in ways that may increase breast cancer risk. Microbial-associated molecular pattern (MAMP)-proteins and metabolites could directly affect breast epithelial cell signaling. In this study, we focused on lipopolysaccharide (LPS), a structural component of the outer membranes of gram-negative bacteria and a toll-like receptor 4 (TLR4)-agonist. Systemic LPS levels are known to increase in obesity. We hypothesize that chronic low-grade inflammation caused by LPS contributes to breast cancer initiation. To test this hypothesis, we first quantified levels of LPS, along with a panel of adipokines/cytokines, in serum samples from women (n= 33; BMI range= 18.3-45.3 Kg/m2; Age range= 18-51 years). Women with elevated serum LPS had significantly higher BMI, leptin, and leptin/adiponectin ratio, confirming a link between systemic LPS and metabolic markers of obesity. Other biomarkers of breast cancer risk include DNA damage. Analyses of non-cancerous breast tissue sections from matched donors revealed higher densities of DNA double-strand breaks (estimated based on 53BP1 foci counts) in women with high serum LPS. Experiments with HMT-3522 S1 breast acini showed that LPS increases DNA break frequencies and oxidative stress levels in 3D structures of mammary epithelium. Moreover, LPS increases oxidative lesions in the DNA (estimated based on 8-oxoguanine fluorescence in MCF-10A cells) that may lead to increased LPS-mediated DNA damage. Patient obesity status was associated with alterations in oxidative modifications in the breast proteome. We also found that LPS disrupts epithelial polarity, a hallmark of breast tissue homeostasis. In mammary epithelium, LPS activates the nuclear factor-kappa B (NFκB) pathway by binding to the TLR4 receptor, leading to an increased expression of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α). Interestingly, TLR4 blockade prevented the loss of apical polarity and DNA damage induced by LPS. Last, but not least, we show an association between LPS immunogenicity (anti-LPS IgA concentrations) and participant BMI in non-cancerous breast tissue samples. These novel findings show that LPS is a key systemic and local mediator of the obesity-modulated microbiome that increases breast cancer risk. The outcomes of our study underscore the importance of considering the microbiome in the prevention of breast cancer. Citation Format: Mohamed Gaber, Kayla Neary, Julia Holmes, Adam S. Wilson, Adam J. Katz, Pierre-Alexandre Vidi, Katherine L. Cook. Microbiome-derived metabolites mediate carcinogenic alterations of breast tissue in the context of obesity [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 2179.

Abstract 5207: Hypermethylation of SOX17 is an important epigenetic mark in Afro-Brazilian breast cancers

Abstract Afro-Brazilian women suffer from higher excess breast cancer mortality after adjusting for age, level of education and stage at diagnosis. Limited studies have researched disease characteristics in this population, which can benefit from the identification of molecular markers that can provide an insight into who is at greater risk of mortality. Epigenetic biomarkers represent the combined effect of environmental and genetic factors. Particularly DNA methylation biomarkers can be valuable as biomarkers of breast cancer risk and prognosis. While studies in other populations have identified DNA methylation biomarkers associated with breast cancer and its clinicopathological outcomes, research on Brazilian cohorts has been limited in scope and focused on women of European descent. The goal of this work was to determine the epigenetic makeup of breast cancer in Brazilian women of African descent. To identify epigenetic biomarkers in Brazilian women who self-identified as Black or Brown, we conducted a case-series analysis investigating DNA methylation patterns in tumor (T) and adjacent non-tumor (NT) tissue. For this, we used the Illumina EPIC (850k) arrays to carry out DNA methylation profiling in 48 frozen tissue paired T and NT samples of breast cancer patients treated at the National Cancer Institute Breast Cancer Hospital in Rio de Janeiro, Brazil. We identified a total of 7,749 probes with Benjamini-Hochberg adjusted p-value < 0.0001 and differences between T and NT tissue above |0.3|. Of these, 5,976 or 77.12% had lower DNA methylation in T when compared to NT tissue; while 1,773 (22.88%) had higher DNA methylation. These were used to identify 21 differentially methylated regions (DMRs) that contained a minimum of seven cg sites, a mean difference larger than |0.2| and a maximum difference above |0.4|. DMRs were mostly (12) hypermethylated in T, and included PCDHGA, NKAPL, ZKSCAN4, SPAG6, CA3, DPP6, RGS22, NXPH1, ANK3, OTX2, SOX17, and CDO1. We used pyrosequencing to validate the levels of SOX17 in eighty-eight tumor and non-adjacent tumor tissue DNAs. We found SOX17 to be hypermethylated in T (44.5±19.1% vs 10.2±10.6, p<0.0001). Differences in DNA methylation in T and NT tissue we still observed by race/ethnicity categories, 43.9±18.6% vs 11.0.±13.2% (p<0.0001) for Brown, and 45.1±20.7% vs 8.8±3.0% for Black women (p<0.0001), respectively. Similarly to previously reported, SOX17 hypermethylation was found to be associated with Luminal A and B molecular subtypes (p<0.0001). To our knowledge, this is the first study to use a discovery approach to identify epigenetic biomarkers in Black and Brown Brazilian women. Our findings confirming previously reported data on SOX17 hypermethylation in breast cancer are encouraging. We expect to validate other epigenetic markers unique to this population that can ultimately aid in the molecular characterization of Brazilian breast cancers. Citation Format: Lissette Delgado-Cruzata, Diego J. Gomes de Paula, Jennifer G. Vieira, André L. Christianes, Tatiana Simao, Leonor B. Gusmao, Luis Felipe Ribeiro Pinto, Sheila Coelho Soares Lima. Hypermethylation of SOX17 is an important epigenetic mark in Afro-Brazilian breast cancers [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 5207.

Association of Body Mass Index and Weight Gain With Obesity-Related Breast Cancer Risk Biomarkers in Adult Chinese Women

To investigate the associatiojn of body mass index (BMI) at different stages of life and weight gain in adulthood with obesity-related breast cancer risk biomarkers and to provide evidence for formulating policies concerning the prevention and control of breast cancer. A cross-sectional study was designed based on the follow-up cohort of southwest China community-based breast cancer screening of women. Using sequential sampling, eligible participants were enrolled from the cohort as the subjects of the study. Information on the basic risk factors was collected and the height, weight, and plasma biomarker levels were measured. Multiple linear regression model was applied to analyze the associations of early adulthood BMI (defined as the BMI of the participant at age 20), adulthood BMI (defined as the BMI measured at the time of enrollment), and weight gain in adulthood with the biomarkers. The concentrations of the biomarkers were incorporated in the model after log transformation. The average age of the 442 participants was 49 (45, 54) years old, the average early adulthood BMI and adulthood BMI were 21.47 (19.56, 23.11) and 24.10 (22.59, 25.97) kg/m 2, respectively, and the average weight gain in adulthood was 6.60 (2.00, 11.00) kg. Adulthood BMI was negatively associated with adiponectin level ( β=-0.026, 95% CI: -0.045--0.008, P=0.006), and positively associated with C-reactive protein level ( β=0.095, 95% CI: 0.054-0.137, P<0.001) and leptin receptor level ( β=0.090, 95% CI: 0.063-0.117, P<0.001). No association was found between adulthood BMI and resistin levels or between adulthood BMI and insulin-like growth factor-binding protein-3 levels. BMI in early adulthood was found to be negatively associated with only insulin-like growth factor-binding protein-3 levels ( β=-0.039, 95% CI: -0.068--0.010, P=0.009). Further analysis of adulthood weight gain after the age of 20 revealed that average annual weight gain in adulthood was negatively associated with adiponectin levels and positively associated with 4 other biomarkers. Furthermore, compared with those of women whose weight remained stable, the adiponectin level of women whose weight gain in adulthood exceeded 5.00 kg was much lower ( β=-0.185, 95% CI: -0.320--0.049, P=0.008), while their insulin-like growth factor-binding protein-3 ( β=0.389, 95% CI: 0.183-0.594, P<0.001) and leptin receptor ( β=0.245, 95% CI: 0.048-0.442, P=0.015) levels were higher. Weight gain in adulthood is strongly associated with the changes in obesity-related breast cancer risk biomarkers. Women should maintain a stable weight throughout adulthood and it is preferred that their weight gain should not exceed 5.00 kg.

Urinary 2- to 16α-hydroxyestrone ratio did not change with cruciferous vegetable intake in premenopausal women.

The mass ratio of urinary 2-hydroxyestrone to 16-α-hydroxyestrone (2:16) is hypothesized as a biomarker of breast cancer risk in premenopausal women, with higher ratios being theoretically protective. Cruciferous vegetable intake has been associated with higher urinary 2:16 in some studies. We investigated whether a whole-food supplement made from dried Brussels sprouts and kale would increase urinary 2:16 in comparison with placebo or cruciferous vegetables in women. This randomized, parallel arm, placebo-controlled, partly blinded study included 78 healthy premenopausal women (38-50 y) with screening urinary 2:16 ≤3.0. Subjects received either six capsules containing 550 mg dried Brussels sprouts and kale per capsule, 40 g daily alternating broccoli or Brussels sprouts, or placebo for eight weeks. Urinary 2:16 and creatinine were measured at baseline, four, and eight weeks. Intent-to-treat repeated measures-ANOVA with multiple imputation (n=100) for missing values identified no treatment effect (P=0.9) or treatment-by-time interaction (P=0.6); however, a significant time effect was noted (P=0.02). Per-protocol analyses including complete cases found no treatment effect (P=1) or treatment-by-time interaction (P=0.6); however, the significant time effect remained (P=0.03). Restricting analysis to subjects with >80% compliance maintained the time effect (P=0.02). Using Pearson correlations, android-pattern and android:gynoid fat were predictive of change (P≤0.05). In conclusion, neither cruciferous supplements nor an added vegetable serving altered urinary 2:16 in premenopausal women with eight weeks treatment. This ratio did vary with time, which is important for designing future trials.

Image-only deep learning risk model performance in patients with known germline mutations.

536 Background: Mammograms contain highly predictive biomarkers of future breast cancer risk, which can be identified by deep learning (DL) risk stratification models. DL model performance in discerning risk in patients with known germline mutations has yet to be established. The aim of this study is to assess image-only DL model risk score performance in patients with and without a germline mutation undergoing screening breast MRI. Methods: This retrospective, multisite study included the first screening MRI of consecutive females ≥30 years from 3/1/2011 to 11/30/2021 at four facilities, with a prior bilateral screening mammogram containing a DL risk score and at least one year of follow-up. A mammography-based DL five-year model was used to assess risk, with an increased-risk threshold of ≥2.2 and high-risk of ≥5.8. Patient demographics, germline mutation status, and traditional risk factors were extracted from electronic medical records. Standard screening performance metrics including cancer detection rate (CDR) within one year of MRI were determined. Cancer outcomes were determined via linkage to a regional tumor registry. DL model performance was compared using Pearson’s chi-squared tests. Results: 4025 patients who underwent screening breast MRI met inclusion criteria. 381(9.5%) had a known germline mutation. There was no significant difference in age of those with or without a germline mutation (54.9y ± 10.5 vs 54.8y ± 8.8, p = 0.284) or family history of breast cancer (68.5% vs 65.3%, p = 0.205). Patients with a germline mutation were more likely to be White (95.0 vs 91.4%, p &lt; 0.05), have non-dense breasts (52.8% vs 35.3%, p &lt; 0.001) and less likely to have a personal history of breast cancer (25.7% vs 36.3%, p &lt; 0.001). The CDR in those with a germline mutation was higher than those without a mutation (39.4 vs 20.3, p &lt; 0.05). The median DL risk score in those with a germline mutation was lower than those without a mutation: 1.9 (IQR: 1.4-2.5) vs 2.3 (IQR: 1.7-3.8), p &lt; 0.001. In patients with a germline mutation, the CDR in those with elevated DL scores was higher than those with low DL scores (47.6 vs 34.2, p = 0.512: increased-risk threshold; 60.6 vs 37.4, p = 0.512: high-risk threshold). A similar trend was seen in those without a germline mutation (26.4 vs 12.8, p &lt; 0.01: increased-risk threshold; 27.1 vs 19.0, p = 0.203: high-risk threshold). Trends were demonstrated across all comparisons, but significance was only reached in the largest group. Conclusions: Compared to patients without germline mutations, those with mutations had higher CDRs on MRI but lower DL risk scores. Patients with higher DL risk scores had higher CDRs in those identified as increased or high-risk by DL model, regardless of germline mutation status. Further study to elucidate mechanisms to explain differential performance of DL models in patients with germline mutations is ongoing.

Abstract 1180: Identification of target proteins for breast cancer genetic risk loci and blood risk biomarkers in a large study by integrating genomic and proteomic data

Abstract Genome-wide association studies (GWAS) have identified around 200 loci associated with breast cancer risk. However, protein targets for these loci remain largely unknown. Identifying protein targets and biomarkers can improve the understanding of cancer biology and etiology and identify high-risk individuals for cancer prevention. In this study, we investigated genetically predicted levels of 1,142 circulating proteins with breast cancer risk in 133,384 cases and 113,789 controls of European ancestry included in the Breast Cancer Association Consortium (BCAC). We identified 22 blood protein biomarkers associated with the risk of overall breast cancer at a false discovery rate (FDR) &amp;lt;0.05, including nine proteins encoded by genes located at least 500kb away from previously reported risk variants for breast cancer. Analyses focusing on 124 encoding genes located at GWAS-identified breast cancer risk loci found 20 proteins associated with overall breast cancer risk and one protein associated with triple-negative breast cancer risk at FDR &amp;lt;0.05. Adjustment for the GWAS-identified risk variants significantly attenuated the association for 13 of these proteins, suggesting that these proteins may be the targets of these GWAS-identified risk loci. The identified proteins are involved in various biological processes, including glutathione conjugation, STAT5 signaling, and NF-kB signaling pathways. Our study identified novel protein targets and risk biomarkers for breast cancer risk. Citation Format: Guochong Jia, Yaohua Yang, Jie Ping, Shuai Xu, Lili Liu, Xingyi Guo, Ran Tao, Jirong Long, Wei Zheng. Identification of target proteins for breast cancer genetic risk loci and blood risk biomarkers in a large study by integrating genomic and proteomic data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1180.

Abstract P2-06-02: A randomized controlled trial of soy isoflavone intake on mammographic density among Malaysian women

Abstract Introduction: Soy intake is associated with lower breast cancer risk in observational studies of Asian women, but clinical trials of soy isoflavone (ISF) supplements report no effect on biomarkers of breast cancer risk among Caucasian women. To date, there are no such trials among Asian women living in Asia. We conducted a three-armed, randomized controlled trial (RCT) to assess the effects of one-year intervention of soy isoflavone supplements (100mg/day) or soy isoflavones through diet (50mg/day) on mammographic density (MD) change among peri- and postmenopausal Malaysian women. Methodology: Healthy women between 45-65 years old were enrolled between November 2018 and December 2019 at a private tertiary hospital in Malaysia. Women were randomly assigned into the 100mg/day ISF Supplement arm, 50mg/day ISF Diet arm, or Control arm. Dense area was assessed from digital mammograms conducted at enrolment and after 12 months. We compared absolute and relative change in dense area over the study period by study arm using Kruskal Wallis tests. Results: Of the 118 women who received the intervention, 91 women completed the study whilst 27 women (23%) were lost to follow up. After 12 months of intervention, women in the ISF Supplement arm observed a marginally larger decline in dense area (-1.3cm2), compared to women in the ISF Diet arm (-0.5cm2) and Control arm (-0.8cm2), but this difference was not statistically significant (p=0.479). Notably, these effects appear to be stronger and limited to women who enrolled within 5 years of menopause, where up to 6cm2 decline in MD was observed in the ISF Supplement arm, compared to &amp;lt; 1.0cm in the Control arm (p=0.131). Conclusion: This RCT demonstrates a possible causal association between soy ISF intake and lower breast cancer risk among Asian women, specifically around the time of menopause, but these findings will require confirmation in a larger trial. Citation Format: Nadia Rajaram, Beverley Yap, Mikael Eriksson, Shivaani Mariapun, Lee Mei Tan, Hamizah Saat, Evelyn Ho, Nur Aishah Mohd Taib, Geok Lin Khor, Cheng Har Yip, Weang Kee Ho, Per Hall, Soo Hwang Teo. A randomized controlled trial of soy isoflavone intake on mammographic density among Malaysian women [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 P2-06-02.

Menstrual Phase and Menopausal Status Classification of Benign Breast Tissue Using Hormone-Regulated Gene Expression and Histomorphology: A Validation Study.

The validation of breast cancer risk biomarkers in benign breast samples (BBS) is a long-sought goal, hampered by the fluctuation of gene and protein expression with menstrual phase (MP) and menopausal status (MS). Previously, we identified hormone-related gene expression and histomorphology parameters to classify BBS by MS/MP. We now evaluate both together, to validate our prior results. BBS were obtained from consenting women (86 premenopausal, 55 postmenopausal) undergoing reduction mammoplasty (RM) or contralateral unaffected breast (CUB) mastectomy. MP/MS was defined using classical criteria for menstrual dates and hormone levels on the day of surgery. BBS gene expression was measured with reverse transcription quantitative polymerase chain reaction (RT-qPCR) for three luteal phase (LP) genes (TNFSF11, DIO2, MYBPC1) and four menopausal genes (PGR, GREB1, TIFF1, CCND1). Premenopausal samples were classified into LP or non-LP, using published histomorphology parameters. Logistic regression and receiver-operator curve analysis was performed to assess area under the curve (AUC) for prediction of MP/MS. In all 131 women, menopausal genes plus age>50 years predicted true MS [AUC 0.93, 95% confidence interval (CI) 0.89, 0.97]. Among premenopausal women, high TNFSF11 expression distinguished non-LP from LP samples (AUC 0.80, 95% CI 0.70, 0.91); the addition of histomorphology improved the prediction nonsignificantly (AUC 0.87, 95% CI 0.78, 0.96). In premenopausal subsets, addition of histomorphology improved LP prediction in RM (AUC 0.95, 95% CI 0.87, 1.0), but not in CUB (0.84, 95% CI 0.72, 0.96). Expression of five-gene set accurately predicts menopausal status and menstrual phase in BBS, facilitating the development of breast cancer risk biomarkers using large, archived sample repositories.

Identification of target proteins for breast cancer genetic risk loci and blood risk biomarkers in a large study by integrating genomic and proteomic data.

Genome-wide association studies (GWAS) have identified around 200 loci associated with breast cancer risk. However, protein targets for these loci remain largely unknown. Identifying protein targets and biomarkers can improve the understanding of cancer biology and etiology and identify high-risk individuals for cancer prevention. In this study, we investigated genetically predicted levels of 1142 circulating proteins with breast cancer risk in 133 384 cases and 113 789 controls of European ancestry included in the Breast Cancer Association Consortium (BCAC). We identified 22 blood protein biomarkers associated with the risk of overall breast cancer at a false discovery rate (FDR) <0.05, including nine proteins encoded by genes located at least 500 kb away from previously reported risk variants for breast cancer. Analyses focusing on 124 encoding genes located at GWAS-identified breast cancer risk loci found 20 proteins associated with overall breast cancer risk and one protein associated with triple-negative breast cancer risk at FDR <0.05. Adjustment for the GWAS-identified risk variants significantly attenuated the association for 13 of these proteins, suggesting that these proteins may be the targets of these GWAS-identified risk loci. The identified proteins are involved in various biological processes, including glutathione conjugation, STAT5 signaling and NF-κB signaling pathways. Our study identified novel protein targets and risk biomarkers for breast cancer risk.

Application of Novel Breast Biospecimen Cell-Type Adjustment Identifies Shared DNA Methylation Alterations in Breast Tissue and Milk with Breast Cancer-Risk Factors.

DNA methylation patterning is cell-type-specific and altered DNA methylation is well established to occur early in breast carcinogenesis, affecting non-cancerous, histopathologically normal breast tissue. Previous work assessing risk factor-associated alterations to DNA methylation in breast tissue has been limited, with even less published research in breast milk, a noninvasively obtained biospecimen containing sloughed mammary epithelial cells that may identify early alterations indicative of cancer risk. Here, we present a novel library for the estimation of the cellular composition of breast tissue and milk and subsequent assessment of cell-type-independent alterations to DNA methylation associated with established breast cancer-risk factors in solid breast tissue (n = 95) and breast milk (n = 48) samples using genome-scale DNA methylation measures from the Illumina HumanMethylation450 array. We identified 772 hypermethylated CpGs (P < 0.01) associated with age consistent between breast tissue and breast milk samples. Age-associated hypermethylated CpG loci were significantly enriched for CpG island shore regions known to be important for regulating gene expression. Among the overlapping hypermethylated loci mapping to genes, a differentially methylated region was identified in the promoter region of SFRP2, a gene observed to undergo promoter hypermethylation in breast cancer. Our findings suggest the potential to identify epigenetic biomarkers of breast cancer risk in noninvasively obtained, tissue-specific breast milk specimens. This work demonstrates the potential of using breast milk as a noninvasive biomarker of breast cancer risk, improving our ability to detect early-stage disease and lowering the overall disease burden.

Association of preoperative serum adipokines, insulin, and sex steroid hormones with breast cancer risk in the Indian women.

Obesity-related factors such as adipokines, insulin, insulin-like growth factors, and sex steroid hormones have been reported to be involved in breast carcinogenesis. Studies across the world suggest an important, but still controversial, role of obesity in breast cancer risk. This study aims to evaluate the association of obesity-related factors such as adipokines, insulin, insulin resistance, and sex steroid hormones with breast cancer risk in the Indian population. Anthropometric and biochemical measurements were taken in 60 newly diagnosed and histologically confirmed breast cancer patients and 50 healthy controls. Preoperative serum levels of adiponectin, leptin, insulin, estrogen, and testosterone were measured using ELISA (enzyme-linked immunosorbent assay). The data were analyzed and compared. The mean serum total cholesterol (T.CHOL) and leptin levels were significantly higher ( P = 0.047), whereas testosterone levels were significantly lower in patients than in controls. Waist circumference (WC) and leptin levels showed a significant positive association with breast cancer risk. Association of serum leptin levels with breast cancer risk persisted after adjusting for age, body mass index (BMI), and WC parameters (odds ratio [ OR ] = 1.042, P = 0.03). Leptin levels positively correlated with WC and triglycerides (TG), whereas insulin and insulin resistance positively correlated with BMI, WC, TG, and T.CHOL ( P < 0.05). Among the tumor characteristics, serum adiponectin showed a positive correlation with lymph node involvement, whereas serum estradiol levels were positively correlated with ER (estrogen receptor) and PR (progesterone receptor) status. Together, our study supports the association of obesity (WC) with breast cancer risk and also suggests the potential role of leptin as a biomarker for breast cancer risk, independent of obesity.

Abstract P037: Association between circulating osteoprotegerin levels and estimated risk of BRCA1-associated breast cancer

Abstract Purpose: Dysregulation of the receptor activator of nuclear factor κB (RANK) pathway may contribute to the pathogenesis of BRCA1-associated breast cancer. Specifically, lower levels of osteoprotegerin (OPG), an endogenous inhibitor of RANK-ligand (RANKL) that competes with RANK for RANKL binding, have been reported among women with pathogenic or likely pathogenic variant (mutation) in the BRCA1 gene. It has been suggested that lower OPG levels may be associated with an increased risk of breast cancer, proposing that OPG may serve as a biomarker for breast cancer risk. Whether OPG is associated with estimated breast cancer risk remains unknown. The goal of this study was to evaluate the relationship between OPG and the estimated risk of breast cancer in women with a BRCA1 mutation. Methods: Women were eligible for inclusion if they had a BRCA1 mutation, were between ages 18 and 79, had a pedigree, had no prior history of cancer, had not undergone prophylactic bilateral mastectomy and provided a blood sample available for OPG analysis. Detailed information on various risk factors (i.e., reproductive, hormonal, family history) was collected using biennial questionnaires. An enzyme-linked immunosorbent assay was used to quantify serum OPG levels (pg/mL). Risk of breast cancer was estimated using Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm version 6 incorporated in the CanRisk tool. The relationship between OPG and estimated lifetime (up to age 80) and ten-year (age 40–50) risks of breast cancer was evaluated using quantile regression analyses. Analyses were further stratified by age (≤50 vs &amp;gt;50 years). Results: A total of 539 women were included in the analysis. The median age at blood collection was 39 years (18–77 years). For lifetime breast cancer risk, a 1 standard deviation (SD; 38.40 pg/ml) increase in OPG level was associated with a 5.4% lower risk at the 25th percentile (P ≤ 0.001), a 4.2% lower risk at the 50th percentile (P ≤ 0.001), and a 1.5% lower risk at the 75th percentile (P ≤ 0.001). For ten-year breast cancer risk, a 1 SD increase in OPG level was associated with a 1.2% lower risk at the 25th percentile (P ≤ 0.001), a 0.8% lower risk at the 50th percentile (P ≤ 0.001), and a 0.8% lower risk at the 75th percentile (P = 0.005). The association between OPG and both estimated risks did not vary in the analysis stratified by age (≤50 vs &amp;gt;50 years). Conclusion: Higher circulating OPG levels were associated with significantly lower estimated lifetime and ten-year risks of BRCA1-associated breast cancer. These associations were stronger for women at a lower risk of developing lifetime breast cancer. Further research is needed to determine whether integrating circulating OPG levels can improve existing risk prediction models. These data contribute to ongoing interest in the role of RANK signaling in mammary tumorigenesis. Inhibiting RANK signaling may therefore be a potential target for breast cancer prevention in women with a BRCA1 mutation and other high-risk populations. Citation Format: Sarah S. Park, Tasnim Zaman, Shana J. Kim, Jennifer D. Brooks, Andy K. O. Wong, Jan Lubiński, Steven A. Narod, Leonardo Salmena, Joanne Kotsopoulos. Association between circulating osteoprotegerin levels and estimated risk of BRCA1-associated breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P037.

A Randomized, Double-Blind, Placebo-Controlled Investigation of Selenium Supplementation in Women at Elevated Risk for Breast Cancer: Lessons for Re-Emergent Interest in Selenium and Cancer.

Damage to cellular macromolecules such as DNA and lipid, induced via reactive oxygen species, and indicators of cell proliferation potential such as insulin-like growth factor (IGF) metabolic status are intermediate biomarkers of breast cancer risk. Based on reports that selenium status can affect these markers, a randomized, placebo-controlled, double-blind experiment was conducted to investigate the potential of selenium supplementation to modulate breast cancer risk. Using a placebo tablet or a tablet containing 200 μg selenium provided as high-selenium yeast daily for one year, concentrations of the biomarkers in blood or urine were assessed at baseline and after 6 and 12 months of intervention. The selenium intervention used in this study is presumed to mediate its effect via the induction of glutathione peroxidase activity and the consequential impact of the active form of this protein on oxidative damage. We found no evidence to support this hypothesis or to indicate that systemic IGF metabolic status was affected. Critical knowledge gaps must be addressed for the resurgence of interest in selenium and cancer to garner clinical relevance. Those knowledge gaps include the identification of a specific, high-affinity selenium metabolite and the cellular target(s) to which it binds, and the demonstration that the cellular determinant that the selenium metabolite binds plays a critical role in the initiation, promotion, or progression of a specific type of cancer.

Association between vitamin D and calcium intakes, breast microcalcifications, breast tissue age-related lobular involution and breast density.

To demystify the potential role of vitamin D and calcium intakes in breast carcinogenesis, we explored the association between these two nutrients and three biomarkers of breast cancer risk: the presence of microcalcifications, age-related lobular involution and breast density. A total of 82 premenopausal and 79 postmenopausal women diagnosed with breast cancer completed a food frequency questionnaire to assess their total vitamin D and calcium intakes. Presence of microcalcifications was determined by reviewing pathology reports. Age-related lobular involution was assessed in nontumoral breast tissue on hematoxylin-eosin-stained slides and percent breast density was assessed by a computer-assisted method. Multivariate generalized linear models were used to evaluate associations between quartiles of vitamin D and calcium intakes and the biomarkers of breast cancer risk. Increasing quartiles of vitamin D intake were inversely associated with the presence of microcalcifications (fourth quartile [Q4] prevalence ratio [PR] = 0.55; Ptrend = 0.021) and breast density (Q4-Q1 = -7.7%; Ptrend = 0.023) in postmenopausal women, and positively associated with age-related lobular involution in women with microcalcifications (Q4 PR = 1.62; Ptrend = 0.036). Increasing quartiles of calcium intake were inversely associated with microcalcifications among all (Q4 PR = 0.44), premenopausal (Q4 PR = 0.37) and postmenopausal women (Q4 PR = 0.38; Ptrend < 0.014 for all). It was also inversely associated with breast density in women without microcalcification (Q4-Q1 = -8.3%; Ptrend = 0.047), but positively associated with breast density in women with microcalcifications (Q4-Q1 = 10.0%; Ptrend = 0.032). Results suggest that the association between vitamin D and calcium intakes and breast cancer risk factors could be influenced by the presence of microcalcifications.

New Moms Wellness Study: the randomized controlled trial study protocol for an intervention study to increase fruit and vegetable intake and lower breast cancer risk through weekly counseling and supplemental fruit and vegetable box delivery in breastfeeding women

BackgroundLaboratory studies indicate that chemicals in fruits and vegetables have anti-carcinogenic and anti-inflammatory activities that can lower breast cancer risk. However, epidemiologic studies of the association between fruit and vegetable intake and breast cancer risk have produced mixed results. Measurement error, confounding, and an emphasis on diet in later adulthood may contribute to weak associations. This paper describes a randomized controlled diet intervention trial in breastfeeding women to examine the effect of high fruit and vegetable intake on breast cancer risk factors, including weight, DNA methylation and inflammatory markers.MethodsEligible breastfeeding women who reside within a 35-mile radius of Amherst, MA are enrolled at five to six weeks postpartum and randomly assigned to a Fruit and Vegetable Intervention Arm (target n = 200) or to a USDA MyPlate Control Arm (target n = 200). The Fruit and Vegetable Intervention group receives weekly telephone or video-based counseling to encourage intake of at least eight to ten daily servings of fruits and vegetables and a weekly delivery of a supplemental box of fruits and vegetables for 20 weeks, and less intensive counseling for up to one year. Breastmilk and infant fecal specimens are collected at baseline, 10 and 20 weeks. Anthropometric measurements are obtained at these timepoints and at the 1-year follow-up. The primary outcomes are change in DNA methylation in breast epithelial cells and change in inflammatory markers in breastmilk from randomization to 20 weeks; and change in weight, waist circumference, and fruit and vegetable intake for the period from randomization to 20 weeks and 1 year.DiscussionThis 1-year randomized diet intervention trial in breastfeeding women will assess whether intake of at least eight to ten daily servings of fruits and vegetables per day improves biomarkers of breast cancer risk directly in the breast (i.e., DNA methylation and inflammatory markers) and helps women maintain a healthy weight.Trial registration: ClinicalTrials.gov Identifier: NCT04374747. Registered May 5, 2020. https://www.clinicaltrials.gov/ct2/show/NCT04374747.

SDC4-rs1981429 and ATM-rs228590 may provide early biomarkers of breast cancer risk

In Australia, 13% of women are diagnosed with breast cancer (BC) in their lifetime with approximately 20,000 women diagnosed with the disease in 2021. BC is characterised by complex histological and genomic influences with recent advances in cancer biology improving early diagnosis and personalised treatment interventions. The Phosphatidyl-inositol-3-kinase/Protein kinase B (PI3K/AKT) pathway is essential in apoptosis resistance, cell survival, activation of cellular responses to DNA damage and DNA repair. Heparan sulfate proteoglycans (HSPGs) are ubiquitous molecules found on the cell surface and in the extracellular matrix with essential functions in regulating cell survival, growth, adhesion and as mediators of cell differentiation and migration. HSPGs, particularly the syndecans (SDCs), have been linked to cancers, making them an exciting target for anticancer treatments. In the PI3K/AKT pathway, syndecan-4 (SDC4) has been shown to downregulate AKT Serine/Threonine Kinase (AKT1) gene expression, while the ATM Serine/Threonine Kinase (ATM) gene has been found to inhibit this pathway upstream of AKT. We investigated single-nucleotide polymorphisms (SNPs) in HSPG and related genes SDC4, AKT1 and ATM and their influence on the prevalence of BC. SNPs were genotyped in the Australian Caucasian Genomics Research Centre Breast Cancer (GRC-BC) population and in the Griffith University–Cancer Council Queensland Breast Cancer Biobank (GU-CCQ BB) population. We identified that SDC4-rs1981429 and ATM-rs228590 may influence the development and progression of BC, having the potential to become biomarkers in early BC diagnosis and personalised treatment.

Effect of Dietary Energy Restriction on the Expression of Genes Encoding the Enzymes Involved in Steroidogenesis in Normal Breast Epithelium and Abdominal Adipose Tissue of Women with Excessive Adiposity

Background: Weight gain and excessive adiposity are associated with an increased risk of breast cancer, especially in postmenopausal women with a high circulating level of testosterone. In this in silico study, the effect of the dietary energy restriction (DER) on the expression of a panel of genes coding for the main enzymes involved in androgens and estrogens production was investigated in breast epithelium and abdominal adipose tissue of healthy women with excessive adiposity and randomly allocated to DER for one menstrual cycle or asked to continue their usual diet. Methods: Wilcoxon-Signed-Rank test was used to assess the difference between paired samples before and after DER in the breast epithelium or adipose tissue, and Spearman’s rank correlation was used to investigate the correlation between genes. Results: In response to DER, the expression of HSD17B12, the gene encoding the enzyme that catalyzes the conversion of inactive estrone into bioactive estradiol, was significantly (P=0.0059) downregulated in breast epithelium and adipose tissue. Furthermore, the expression of HSD17B12 negatively correlated with that of the LRP1 gene (r= -0.91, P&lt;0.0001), which codes for a multitasking protein recently proposed as a tumor suppressor in breast epithelium. Conclusion: Present results showed that, in addition to the recognized systemic effects (reduction of the fatty mass and decrease of circulating biomarkers of breast cancer risk), DER could act locally by down-regulating the expression of some genes pivotally involved in the production of biologically active estrogens, thus contributing to prevent the estrogen-dependent initiation of breast cancer.

Effect of 10-hours Time Restricted Diet on Breast Cancer Risk in Pre and Postmenopausal Women with Overweight or Obesity: TRED Pilot Study

ObjectivesThis study examined the effect of 10-h time restricted diet on breast cancer risk, assessing changes in body weight, insulin, IGF-1, and IGF binding protein 1, 2, and 3 among pre and postmenopausal women with overweight and obesity.MethodsWomen with overweight or obesity (n = 25) were enrolled into a time-restricted diet for 6 months. Participants were instructed to eat only from 10am until 8pm and to record consumption on the TRED smartphone app. At weeks 1, 2, 12, and 24, participants were asked to complete food diaries. During the other weeks, participants simply recorded the category of food consumed (meal or snack). Compliance was calculated as percentage of days when food consumption was reported.Results25 women with a BMI between 25 and 40 (32.3 ± 4.2) and age between 18–65 years old (46.5 ± 11.0) were enrolled. Average weight loss was 0.8 ± 4.6 kg (−0.6%). Fat percentage, muscle mass remained unchanged. Insulin and IGFBP 2 significantly increased from baseline to post intervention and to follow up. However, the change is not clinically significant. IGF-1 and IGFBP1 significantly decreased from baseline to follow up. Average adherence to the diet protocol was high: 78% of participants recorded dietary intake on at least 77% of intervention days.ConclusionsThe TRED 10-hour eating window did not lead to weight loss, body fat loss, or decreasing waist circumference among pre and postmenopausal women with overweight or obesity. However, the intervention significantly improved some biomarkers of breast cancer risk (IGF-1, IGFBP 2 and IGFBP 3).Funding SourcesThis study was supported by NIH/NCI training grant T32 CA193193.