Sherman, Mark E.

Abstract Purpose Breast cancer risk is elevated in pathogenic germline BRCA 1/2 mutation carriers due to compromised DNA quality control and elevated mutagenic load. We hypothesized that if immunosurveillance promotes tumor suppression, then normal breast lobules from BRCA carriers may demonstrate higher immune cell densities. Methods We assessed immune cell composition in normal breast lobules from age-matched women with progressively increased breast cancer risk, including 1) 19 women who donated normal breast tissue to the Komen Tissue Bank (KTB) at Indiana University Simon Cancer Center, 2) 15 women with biopsy-identified benign breast disease (BBD), and 3) 19 prophylactic mastectomies from women with germline mutations in BRCA1/2 genes. We performed immunohistochemical stains and analysis to quantitate immune cell densities from digital images in up to 10 representative lobules per sample. Median cell counts per mm2 were compared between groups using Wilcoxon rank-sum tests. Results Compared to BBD tissues, normal breast lobules from BRCA carriers had significantly higher densities of CD8 + cells (median 354.4 vs 200.0 cells/mm2, p = 0.01). Compared to KTB normal donor breast tissues, normal breast lobules from BRCA carriers also had significantly higher densities of CD4+ (median 116.3 vs 17.7 cells/mm2), CD8+ (354.4 vs 150.9 cells/mm2), CD11c+ (3.5% vs 0.4% pixels positive), and CD68+ (237.5 vs 57.8 cells/mm2) immune cells (each p < 0.001). Conclusions These preliminary analyses show that normal/benign breast lobules of BRCA mutation carriers contain increased immune cells compared with BBD and normal donated tissues. Future studies will be helpful to define relationships of mutations and immune responses.

Abstract Polycystic ovarian syndrome (PCOS) is a complex health condition associated with metabolic disturbances and infertility. Recent data suggest that the prevalence of PCOS is increasing among women globally, although the etiology of these trends is undefined. Consequently, preclinical models that better reflect the biology of PCOS are urgently needed to facilitate research that can lead to the discovery of prevention strategies or improved management. The existing animal models have several limitations as they do not reflect all the PCOS features metabolically and/or phenotypically. Therefore, there is no clear consensus on the use of appropriate animal model and selection of the most appropriate PCOS-inducing agent. To that end, we have established a Swiss albino mouse model of PCOS based on 3 weeks of daily treatment with letrozole (50 μg/day; intraperitoneal) and dehydroepiandrosterone (DHEA, 6 mg/100 g body weight; subcutaneous) in 5-week-old female mice fed on normal or high-fat diet (HFD). Mice were regularly assessed for body weight, blood glucose, and estrous cycle. Three weeks after drug administration, mice were sacrificed and assessed for blood-based metabolic parameters as well as ovarian function. Our results indicate that DHEA combined with HFD produces changes mimicking those of clinical PCOS, including elevated serum testosterone and luteinizing hormone, dyslipidemia, poor ovarian microenvironment, and development of multiple ovarian cysts, recapitulating cardinal features of PCOS. In comparison, normal diet and/or letrozole produced fewer features of PCOS. The data from the experimental models presented here can improve our understanding of PCOS, a growing concern in women’s health.

Abstract Background: Biopsy diagnoses of benign breast disease (BBD) confer a 1.5- to 4-fold increased risk of developing breast cancer (BC) compared with women without BBD. Previously, we reported that decreased numbers of specific immune cell types in lobules of BBD biopsies predicted increased BC risk, suggesting the promise of future tissue biomarker studies to define BC risk markers among BBD patients. Thus, we applied protein-based GeoMx® Digital Spatial Profiling (DSP) to BBD biopsies preceding BC (cases) and to BBD biopsies from cancer-free patients (controls) to identify possible BC risk markers, which we then evaluated in subsequent BC tissues and in surrounding normal lobules of cases. Methods: Archived pathology slides of BBD biopsies were reviewed and used to guide preparation of TMAs containing 1.0-mm diameter FFPE cores of lobules from an age- and cohort-period-matched set of 91 cases and 88 controls from the Mayo Clinic BBD Cohort. For patients who later developed BC, we prepared TMAs of BC tissue and surrounding mapped normal lobules. We applied GeoMx® DSP (immune and canonical signaling proteins) to both sets of TMAs. Following QC and data normalization, associations of case status with log-transformed biomarker expression in lobules of BBD biopsies were carried out using linear mixed modeling approaches, accounting for multiple ROIs per individual. Biomarkers significantly associated with case status (p&lt;0.05) were further examined in BCs and adjacent normal lobules, using similar approaches. Results: The mean age at BBD biopsy was 52 years, and at BC diagnosis of cases, 61.4 years (mean time from BBD to BC was 10.2 years). A family history of BC was more frequent among cases (70% versus 43%; chi-square p=0.002). Of 72 biomarkers tested, 46 (64.4%) were evaluable after QC and normalization and 5 were associated with BC risk after adjustment for family history of BC: BCL2 (p=0.005), STING (p=0.006), CD44 (p=0.02), S100 protein (p=0.03) and pan-AKT (p=0.05); each showed higher levels in lobules of BBD biopsies of controls than cases. Three unique patterns appeared when examining these biomarkers across tissue type within cases: for BCL2 (p=5 x 10-9) and STING (p=2 x 10-19), levels were high in both BC and lobules surrounding BC but low in preceding BBD; for GAPDH (p=4 x 10-53) and pan-AKT (p=2 x 10-33), levels were high in BC, low in preceding BBD, and moderate in lobules surrounding BC; and for CD44 (p=2 x 10-6) and S100B (p=2 x 10-49), levels were low in BC, high in lobules surrounding BC and moderate in preceding BBD. Conclusions: Using a novel TMA of lobules in combination with DSP, we preliminarily identified immune-based and PI3 kinase-related protein biomarkers in BBD biopsies associated with BC risk. In case-only analyses, these markers demonstrated complex differences between lobules in BBD biopsies, subsequent BCs and adjacent normal lobules. Citation Format: Robert Alan Vierkant, Jodi M. Carter, Stacey J. Winham, Chen Wang, Jennifer M. Kachergus, Ji Shi, Raymond M. Moore, Bryan M. McCauley, Laura M. Pacheco-Spann, E A. Thompson, Derek C. Radisky, Amy C. Degnim, Mark E. Sherman. Towards prediction of breast cancer risk in benign biopsies with high-plex GeoMx spatial protein profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2203.

Abstract Arachidonic acid (AA) metabolizing enzymes, including cyclooxygenases (COX), lipoxygenases, and cytochrome P450s (CYP450), are expressed in breast cancer (BC). Elevated levels of metabolites of COX-derived, pro-inflammatory prostaglandin (PGs) metabolites in urine have been linked to increased risk of developing breast cancer (BC) in two large prospective cohorts. Further, CYP450-derived EETs in BC tumors are associated with metastasis. We hypothesize that measuring levels of AA-derived oxylipins in breast tissues could enable improved BC risk prediction, provide insights into pro-carcinogenic inflammatory mechanisms, and serve as potential surrogate endpoints in chemoprevention trials. Thus, we performed a pilot study to assess the feasibility of measuring these analytes in small breast tissue samples. We analyzed two sample sets, collected under Institutional Review Board (IRB) approved protocols: 50 normal breast tissue cores donated to the Komen Tissue Bank (KTB) via vacuum-assisted biopsy and 10 tissues removed surgically at Mayo Clinic beyond resection margins of lumpectomy samples performed for atypical ductal hyperplasia (ADH). Tissues were frozen and maintained at -80°C. Normal KTB tissue cores were split in half. Tissues for analysis were weighed, extracted in cold methanol containing a cyclooxygenase inhibitor, and assayed for a panel of 42 oxylipins using validated mass spectrometric methods with a limit of detection per analyte of 0.010 ng/mL in supernatant and CVs of approximately &lt;11%. Analyte measurements in paired cores (ng/g tissue) were compared with Spearman correlations and intra-class correlation coefficients. Measurements in surgical samples were assessed descriptively and levels were compared with normal KTB breast tissues.Median weight of the 50 split KTB cores was 0.055 grams. 17 oxylipins were detected in the tissue samples. PGD2 demonstrated the weakest correlation between split cores (r=0.12; p=0.40), whereas 15-HETE demonstrated the strongest correlation (r=0.68; p&lt;0.0001). ICCs ranged from non-significant to nearly perfect, with highest values for 12-HETE (ICC=0.98, 95%CI: 0.97-0.99). Exploratory analyses of the KTB cores demonstrated significant associations between two BC risk factors, alcohol use and family history of breast/ovarian cancer, and several analytes. Levels of most eicosanoids tested were substantially and highly significantly elevated in tissues surrounding ADH versus KTB normal breast tissues. We conclude that eicosanoids can be readily measured in small frozen breast tissue samples obtained via percutaneous biopsy or at surgery. Future studies to assess the associations of eicosanoid levels with BC risk factors and development of BC after a benign biopsy may inform risk prediction and potentially inhibitable inflammatory mechanisms of breast carcinogenesis. Citation Format: Ginger L. Milne, Robert A. Vierkant, Amy C. Degnim, Anna Maria Storniolo, Jill E. Henry, Laura M. Pacheco-Spann, Derek C. Radisky, Mark E. Sherman. Quantification of oxylipins as biomarkers of inflammation and cancer risk in breast tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6333.

e17610 Background: Methylated DNA markers (MDMs) associated with sporadic ovarian cancer (OC) are detected in plasma from patients with treatment naïve tumors, but not cancer-free controls. To assess potential benefits to patients at risk for hereditary OC, the aim of this study was to measure the performance of validated sporadic OC MDMs in OC and benign ovarian tissue (BOT), surgically obtained from germline BRCA1 or BRCA2 ( BRCA1/2) mutation carriers. Methods: BRCA1/2 carriers with OC and BRCA1/2 carriers who had undergone risk-reducing salpingo-oophorectomy (SO) were identified at a comprehensive cancer center. BRCA1/2 carriers in both OC and BOT groups were balanced on age and year of surgery with non- BRCA carriers with sporadic OC or benign indications for SO. OC and BOT formalin fixed paraffin embedded (FFPE) tissues were macrodissected following pathologist selection of representative sites; DNA was extracted, and bisulfite converted. 15 OC MDMs ( GDF6, IFFO1, MAX.chr1.147790358, MAX.chr6.10382190, MAX.chr11.14926602, C2CD4D, PDRM14, NCOR2, SKI, DSCR6, SIM2, PALLD, CDO1, GPRIN1, BCAT1) previously identified in sporadic OC were assayed by quantitative methylation specific PCR, normalized by β-actin, by blinded personnel. Areas under the receiver operating characteristic curve (AUC) were generated for each MDM to assess discrimination of OC from benign tissue and compared between BRCA1/2 and sporadic (non- BRCA) patients, using a z-test. Results: Among BRCA1/2 carriers, there were 48 OC ( BRCA-OC) and 48 BOT ( BRCA-BOT); among non- BRCA, we matched 48 OC (Sporadic-OC) and 48 BOT (non- BRCA-BOT). The median AUC for OC MDM discriminated between OC and BOT with AUCs in BRCA1/2 carriers was 0.87, (IQR, 0.83-0.93). Sporadic-OC vs non- BRCA-BOT median AUC was 0.88 (IQR, 0.83-0.92). AUCs were not significantly different for each MDM when stratified by BRCA or sporadic status. MDMs with an AUC of ≥0.9 in either group are shown (Table). Conclusions: OC MDMs identified and validated in sporadic OC are highly discriminant for OC from BOT in DNA extracted from tissues of people who carry a BRCA1/2 mutation. Testing the performance of these OC MDMs in plasma from BRCA1/2 carriers with and without OC may be of clinical value for high-risk patients and inform multi-cancer early detection testing strategies.[Table: see text]

Abstract Background Mammographic breast density (MBD) decline post-tamoxifen initiation is a favorable prognostic factor in estrogen receptor (ER)–positive breast cancer (BC) and has potential utility as a biomarker of tamoxifen response. However, the prognostic value of MBD decline may vary by molecular characteristics among ER–positive patients. Methods We investigated associations between MBD decline (≥10% vs &lt;10%) and breast cancer–specific mortality (BCSM) among ER–positive breast cancer patients aged 36-87 years at diagnosis treated with tamoxifen at Kaiser Permanente Northwest (1990-2008). Patients who died of BC (case patients; n = 62) were compared with those who did not (control patients; n = 215) overall and by tumor molecular characteristics (immunohistochemistry [IHC]–based subtype [luminal A–like: ER–positive/progesterone receptor [PR]–positive/HER2–negative/low Ki67; luminal B–like: ER–positive and 1 or more of PR–negative, HER2–positive, high Ki67] and modified IHC [mIHC]–based recurrence score of ER/PR/Ki67). Percent MBD was measured in the unaffected breast at baseline mammogram (mean = 6 months before tamoxifen initiation) and follow-up (mean = 12 months post-tamoxifen initiation). Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were computed from logistic regression models. All statistical tests were 2-sided. Results MBD decline was statistically significantly associated with reduced risk of BCSM overall (OR = 0.38, 95% CI = 0.15 to 0.92). This association was, however, stronger among women with aggressive tumor characteristics including luminal B–like (OR = 0.17, 95% CI = 0.04 to 0.73) vs A–like (OR = 0.74, 95% CI = 0.19 to 2.92); large (OR = 0.26, 95% CI = 0.08 to 0.78) vs small (OR = 0.41, 95% CI = 0.04 to 3.79) tumors; PR–negative (OR = 0.02, 95% CI = 0.001 to 0.37) vs PR–positive (OR = 0.50, 95% CI = 0.18 to 1.40) disease; and high (OR = 0.25, 95% CI = 0.07 to 0.93) vs low (OR = 0.44, 95% CI = 0.10 to 2.09) mIHC3 score. Conclusion The findings support MBD decline as a prognostic marker of tamoxifen response among patients with aggressive ER–positive BC phenotypes, for whom understanding treatment effectiveness is critical.

Abstract Breast cancer risk is increased in parous women in the period following childbirth, peaking at five years. After weaning or in the absence of lactation, the breast undergoes postpartum involution (PPI), a process characterized by epithelial cell death and tissue remodeling that returns the mammary gland to a baseline state, and has previously been associated with increased inflammation and breast cancer development and progression in preclinical models. Features of PPI and determinants of related inflammation have been incompletely characterized among women. In this study, we performed visual and AI analysis of 725 digitized H&E images of normal breast tissues donated to the Komen Tissue Bank (KTB) by parous and nulliparous women ≤45 years, and further investigated the immune microenvironment in specimens from 23 women (also from KTB, 13 parous, 10 nulliparous) using NanoString RNA immuno oncology (IO) 360 panels and immune protein biomarkers by NanoString GeoMx Digital Spatial Profiling (DSP). We found that recently parous women (≤5 years of a last birth) had higher numbers of terminal duct lobular units (TDLUs), immune cells, and plasma cells as compared with nulliparous women, and that beyond 5 years of a last birth, TDLU numbers were still higher in parous women, but the immune and plasma cell content of the breast of parous and nulliparous women were not significantly different. Our gene expression analysis revealed differential expression of genes that clustered according to parity status, with interferon stimulated genes; STAT1 and IFITM1, and interferon alpha receptor 1 (IFNAR1), and MHC class II genes (HLA-DMA, HLA-DPA,HLA-DPB1, and HLA-DRA) elevated in parous women. Our DSP analysis revealed that immune biomarkers with immunosuppressive functions, including ARG1, VISTA, CTLA-4, and CD68 were significantly higher in parous women as compared with nulliparous women. Breast tissues of parous women show lobular expansion and alterations in immune cell components compared with nulliparous tissues. Further research is needed to assess how these alterations may impact risk. Citation Format: Joshua Were Ogony, Thomas De Bel, Derek Radisky, Jeroen VanderLaak, Mark Sherman. Pathology AI features and immune biomarkers of postpartum involution: Implications for postpartum breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-11-14.

Abstract Background: Approximately one million U.S women are diagnosed with benign breast disease (BBD) annually, which increases breast cancer (BC) risk between 1.5- and four-fold, depending on the pathologic characteristics of the lesion and other factors. BBD lesions are associated with inflammation and fibrosis, suggesting that inflammatory pathways may play a role in progression of BBD to BC. Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) are well tolerated, widely used drugs that block inflammation by inhibiting cyclooxygenase enzymes, which lowers prostaglandin synthesis, and by exerting numerous additional anti-inflammatory effects. NSAIDs are inversely associated with several types of cancer, including BC in some, but not all studies, and have been linked to lower BC risk among BBD patients in prior publications. We examined the association between NSAID use and BC risk, overall and by self-reported history of BBD, using data from the American Cancer Society Cancer Prevention Study II (CPS-II). Methods: The Cancer Prevention Study-II (CPS-II) Nutrition Cohort is a study of approximately 189,000 U.S. men and women followed prospectively for cancer incidence and mortality. Study participants filled out a detailed questionnaire at baseline in 1992/1993 and follow-up surveys in 1997 and every two years until 2017. On each follow-up survey, participants reported type, quantity, and duration of NSAID use as well as diagnoses of BBD. Incident BCs were either self-reported and validated via medical records or cancer registry linkage, or identified via National Death Index linkage. Women in the analytic cohort were followed from date of 1997 survey until diagnosis of BC, reported use of tamoxifen, report of cancer other than BC, date of death, loss to follow-up or end of follow-up. Hazard ratios (HRs) and 95% confidence intervals (CIs) assessing associations of BBD and NSAID use with BC risk were estimated using Cox proportional hazards regression analysis. Statistical interactions between BBD and NSAID were evaluated by fitting and testing the corresponding cross-product terms, along with the main effects of each. Both NSAID use and BBD were modeled as time-dependent variables, allowing exposure status to change over the course of the multiple surveys returned. Results: Of the 60,517 women included in the study, 3,899 (6.4%) developed BC. Mean follow-up time was 12.7 years. A total of 23,661 women (39%) reported a diagnosis of BBD at some point during the study period, and 54,656 (90%) reported some NSAID use. Women self-reporting BBD were 46% more likely to develop BC than those without self-reported BBD (HR 1.46, 95% CI 1.36-1.57). Overall, BC risk was not increased among NSAID users versus non-users (HR 0.95, 95% CI 0.88-1.02). In contrast, BC risk was reduced among current NSAID users with a history of BBD (HR 0.87, 95% CI 0.78-0.97), but not among NSAID users who did not report a history of BBD (HR 1.02, 95% CI 0.92-1.13, p-interaction=0.04). Risk did not differ across number of pills per month (RR=0.84 for &lt;15 pills/month, 0.89 for 15-29, 0.88 for 30-44, 0.78 for 45-59, 0.88 for 60+). Conclusion: In our study, NSAID use had a statistically-significant protective effect on BC risk among women with BBD, suggesting its potential use as a chemopreventive agent in this high-risk group of women, but not among those who did not report a history of BBD. Citation Format: Robert A Vierkant, Matthew Masters, Lauren R Teras, Mark E Sherman. Breast cancer (BC) risk among patients with benign breast disease (BBD) by NSAID use [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-11-01.

Abstract Background:Breast terminal duct lobular units (TDLUs), the source of most breast cancer (BC) precursors, are shaped by age-related involution, a gradual process, and postpartum involution (PPI), a dramatic inflammatory process that restores baseline microanatomy after weaning. Dysregulated PPI is implicated in the pathogenesis of postpartum BCs. We propose that assessment of TDLUs in the postpartum period may have value in risk estimation, but characteristics of these tissues in relation to epidemiological factors are incompletely described.Methods: Using validated Artificial Intelligence and morphometric methods, we analyzed digitized images of tissue sections of normal breast tissues stained with hematoxylin and eosin from donors <45 years from the Komen Tissue Bank (180 parous and 545 nulliparous). Metrics assessed by AI, included: TDLU count; adipose tissue fraction; mean acini count/TDLU; mean dilated acini; mean average acini area; mean “capillary” area; mean epithelial area; mean ratio of epithelial area versus intralobular stroma; mean mononuclear cell count (surrogate of immune cells); mean fat area proximate to TDLUs and TDLU area. We compared epidemiologic characteristics collected via questionnaire by parity status and race, using a Wilcoxon rank sum test or Fisher’s exact test. Histologic features were compared between nulliparous and parous women (overall and by time between last birth and donation [ recent birth: ≤5 years versus remote birth: >5 years]) using multivariable regression models.Results:Normal breast tissues of parous women contained significantly higher TDLU counts and acini counts, more frequent dilated acini, higher mononuclear cell counts in TDLUs and smaller acini area per TDLU than nulliparas (all multivariable analyses p<0.001). Differences in TDLU counts and average acini size persisted for >5 years postpartum whereas increases in immune cells were most marked <5 years of a birth. Relationships were suggestively modified by several other factors, including demographic and reproductive characteristics, ethanol consumption and breastfeeding duration. Conclusions:Our study identified sustained expansion of TDLU numbers and reduced average acini area among parous versus nulliparous women and notable increases in immune responses within five years following childbirth. Further, we show that quantitative characteristics of normal breast samples vary with demographic features and BC risk factors.