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Perspective on This Article from p16INK4a Expression and Breast Cancer Risk in Women with Atypical Hyperplasia

ERβ staining in atypical ductal hyperplasia (ADH) versus atypical lobular hyperplasia (ALH).

Over one million women in the United States receive biopsy diagnoses of benign breast disease (BBD) each year, which confer a 1.5–4.0-fold increase in breast cancer risk. Studies in the general population suggest that nonsteroidal anti-inflammatory agents (NSAID) lower breast cancer risk; however, associations among women with BBD are unknown. We assessed whether NSAID use among women diagnosed with BBD is associated with lower breast cancer risk. Participants included 3,080 women (mean age = 50.3 ± 13.5 years) in the Mayo BBD surgical biopsy cohort diagnosed between January 1, 1992 and December 31, 2001 who completed breast cancer risk factor questionnaires that assessed NSAID use, and whose biopsies underwent detailed pathology review, masked to outcome. Women were followed from date of BBD biopsy to breast cancer diagnosis (main outcome) or censoring (death, prophylactic mastectomy, reduction mammoplasty, lobular carcinoma in situ or last contact). Median follow-up time was 16.4 ± 6.0 years. Incident breast cancer was diagnosed among 312 women over a median follow-up of 9.9 years. Regular non-aspirin NSAID use was associated with lower breast cancer risk [HR = 0.63; 95% confidence interval (CI) = 0.46–0.85; P = 0.002] with trends of lower risk (highest tertiles of use vs. nonuse) for greater number of years used [HR = 0.55; 95% CI = 0.31–0.97; Ptrend = 0.003), days used per month (HR = 0.51; 95% CI = 0.33–0.80; Ptrend = 0.001) and lifetime number of doses taken (HR = 0.53; 95% CI = 0.31–0.89; Ptrend = 0.003). We conclude that nonaspirin NSAID use is associated with statistically significant lower breast cancer risk after a BBD biopsy, including a dose–response effect, suggesting a potential role for NSAIDs in breast cancer prevention among patients with BBD.

PDF file - 28K, Supplemental Table 1: The association between pathologic characteristics of benign breast biopsies and subsequent breast cancer risk in African American women in Detroit, MI by age at biopsy

S1. The top pathways regulated by the 26 common microRNAs and miR-4454 expression in a panel of cells. S2. Validation of miR-4454 overexpression and inhibition in OC cells. S3. Functional effects of stable overexpression of miR-4454 in OVCAR8. S4. Genes and pathways regulated by miR-4454. S5. miR-4454 target validation. Supplementary Table 1 : Clinical characteristics of the 42 epithelial ovarian cancer patients. Supplementary Table 2: List of microRNAs downregulated in both OC patient primary tumor vs. matched metastasis and in Kuramochi/OVCAR4/OVCAR8 cells on 3D omental culture vs. control. Supplementary Table 3: Subset of miR-4454 targets with cancer relevant functions based on a literature search.

Supplementary Tables and Figures

Supplementary Table 4 - Associations between hormone levels and median category of acini counts per TDLU (premenopausal women) and median acini counts per TDLU (postmenopausal women) among women in the KTB.

Background: Mammographic density (MD) is a strong breast cancer risk factor. We previously reported associations of percent mammographic density (PMD) with larger and node-positive tumors across all ages, and estrogen receptor (ER)–negative status among women ages Methods: Data were pooled from six studies including 4,095 breast cancers and 8,558 controls. DA and NDA were assessed from digitized film-screen mammograms and standardized across studies. Breast cancer odds by density phenotypes and age according to histopathologic characteristics and receptor status were calculated using polytomous logistic regression.Results: DA was associated with increased breast cancer risk [OR for quartiles: 0.65, 1.00 (Ref), 1.22, 1.55; Ptrend Ptrend Ptrend < 0.001) but no differences by nodal status. Among women + versus ER− tumors (Phet = 0.02), while NDA was more strongly associated with decreased risk of ER− versus ER+ tumors (Phet = 0.03).Conclusions: DA and NDA have differential associations with ER+ versus ER− tumors that vary by age.Impact: DA and NDA are important to consider when developing age- and subtype-specific risk models. Cancer Epidemiol Biomarkers Prev; 24(5); 798–809. ©2015 AACR.

Supplementary Table 3 - Associations between hormone levels and median TDLU span among women in the KTB.

Distribution of dense area (DA) and non dense area (NDA) phenotypes prior to standardization. The age effect on DA and NDA as well as the differences in the distribution across the studies observed for breast cancer cases.

Supplementary Table 1 - Spearman's rank correlation coefficients (p-values) between TDLU measures from normal breast tissue in the KTB.

Standardization of dense (DA) and non dense area (NDA) phenotypes. The age and study effects are removed in standardized measures.

Background: Terminal duct lobular units (TDLU) are the predominant source of breast cancers. Lesser degrees of age-related TDLU involution have been associated with increased breast cancer risk, but factors that influence involution are largely unknown. We assessed whether circulating hormones, implicated in breast cancer risk, are associated with levels of TDLU involution using data from the Susan G. Komen Tissue Bank (KTB) at the Indiana University Simon Cancer Center (2009–2011).Methods: We evaluated three highly reproducible measures of TDLU involution, using normal breast tissue samples from the KTB (n = 390): TDLU counts, median TDLU span, and median acini counts per TDLU. RRs (for continuous measures), ORs (for categorical measures), 95% confidence intervals (95% CI), and Ptrends were calculated to assess the association between tertiles of estradiol, testosterone, sex hormone–binding globulin (SHBG), progesterone, and prolactin with TDLU measures. All models were stratified by menopausal status and adjusted for confounders.Results: Among premenopausal women, higher prolactin levels were associated with higher TDLU counts (RRT3vsT1:1.18; 95% CI: 1.07–1.31; Ptrend = 0.0005), but higher progesterone was associated with lower TDLU counts (RRT3vsT1: 0.80; 95% CI: 0.72–0.89; Ptrend < 0.0001). Among postmenopausal women, higher levels of estradiol (RRT3vsT1:1.61; 95% CI: 1.32–1.97; Ptrend < 0.0001) and testosterone (RRT3vsT1: 1.32; 95% CI: 1.09–1.59; Ptrend = 0.0043) were associated with higher TDLU counts.Conclusions: These data suggest that select hormones may influence breast cancer risk potentially through delaying TDLU involution.Impact: Increased understanding of the relationship between circulating markers and TDLU involution may offer new insights into breast carcinogenesis. Cancer Epidemiol Biomarkers Prev; 23(12); 2765–73. ©2014 AACR.

PDF - 8738K, Figure S1 H&E staining from three high-grade serous (A, B, C) and three endometrioid carcinomas (D, E, F)

Characteristics of the study populations.

PDF - 59K, Legend for Supplementary Figure 1.

Purpose: Little is known about the role of the immune system in the earliest stages of breast carcinogenesis. We studied quantitative differences in immune cell types between breast tissues from normal donors and those from women with benign breast disease (BBD).Experimental Design: A breast tissue matched case–control study was created from donors to the Susan G. Komen for the Cure Tissue Bank (KTB) and from women diagnosed with BBD at Mayo Clinic (Rochester, MN) who either subsequently developed cancer (BBD cases) or remained cancer-free (BBD controls). Serial tissue sections underwent immunostaining and digital quantification of cell number per mm2 for CD4+ T cells, CD8+ T cells, CD20+ B cells, and CD68+ macrophages and quantification of positive pixel measure for CD11c (dendritic cells).Results: In 94 age-matched triplets, BBD lobules showed greater densities of CD8+ T cells, CD11c+ dendritic cells, CD20+ B cells, and CD68+ macrophages compared with KTB normals. Relative to BBD controls, BBD cases had lower CD20+ cell density (P = 0.04). Nearly 42% of BBD cases had no CD20+ B cells in evaluated lobules compared with 28% of BBD controls (P = 0.02). The absence of CD20+ cells versus the presence in all lobules showed an adjusted OR of 5.7 (95% confidence interval, 1.4–23.1) for subsequent breast cancer risk.Conclusions: Elevated infiltration of both innate and adaptive immune effectors in BBD tissues suggests an immunogenic microenvironment. The reduced B-cell infiltration in women with later breast cancer suggests a role for B cells in preventing disease progression and as a possible biomarker for breast cancer risk. Clin Cancer Res; 23(14); 3945–52. ©2017 AACR.

Supplementary Figure Legends from Dense and Nondense Mammographic Area and Risk of Breast Cancer by Age and Tumor Characteristics

PDF - 100K, Table S1 Demographics from the OTTA cohorts representing the test set and excluded cases Table S2 Antibodies, details of immunohistochemical protocols and scoring cut-off Table S3 Training set revision of the COSP model - Areas under the curve (AUC) by histology and model Table S4 Test for heterogeneity for marker expression between training and testing set Table S5 Pairwise agreement of histological types in the testing set by classification method for A_COSP Table S6 Univariate Cox model in the testing set comparing A_COPS and TB_COSPv2. Table S7 Five-year survival estimates for four different type assignments as defined in the text within histological types Table S8 Demographics of endometrioid carcinomas in the test set classified by different methods Table S9 Prediction of type in test set among cases with unclear original diagnosis (N=71).

Distribution of immune cell densities per sample presented as a five-number summary including the minimum (Min), 25th percentile (Q25), median (Q50), 75th percentile (Q75), and maximum (Max).

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 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.

Background: Women with benign breast disease (BBD) are at increased risk of breast cancer (BC). Classic studies based on film-based mammographic screening and pathology diagnosis of surgical biopsies conducted in the 1980s established a hierarchy of increasing BC risk: non-proliferative (NP) BBD, proliferative BBD without atypia (PDWA) and atypical hyperplasia (AH). Given changes in epidemiological BC risk factors and introduction of percutaneous core needle biopsy (CNB) in mid-1990s, and later, digital mammography, we hypothesized that the patient characteristics and relative frequency of BBD diagnoses have changed over time. Accordingly, we performed a longitudinal analysis of the frequency of patient characteristics and BBD diagnoses in the Mayo BBD cohort. Methods: Utilizing the Mayo Clinic Surgical and Pathology Indices, women ages 18 to 85 who had a BBD biopsy between 1/1/67 and 12/31/13 were identified. Breast pathologists reviewed biopsies masked to diagnoses of incident BC diagnosed in follow-up. Demographic characteristics and BC events were obtained by query of institutional data sources and participant surveys. Trends were evaluated for the following eras: 1: pre-mammogram (1967-1981), 2: pre-CNB (1982-1992), 3: CNB Transition (1993-2001), and 4: CNB (2002-2013). Demographics were formally compared across eras using chi-square tests for categorical variables and analyses of variance (ANOVAs) for continuous variables. Results: From 1967-2013, the cohort includes 19,582 unique women with BBD. The frequency of CNB increased from eras 1-4: 0.04%, 0.6%, 51.3 %, and 88.9%, respectively. Mean age at BBD diagnosis was younger in era 1 (48.0 years) vs eras 2-4 (53.2, 52.0, and 51.8, respectively, p Conclusions: Analysis of this large, single institution BBD cohort for the 46 year period 1967-2013 demonstrates that BC risk factors among BBD patients has changed over time, with subjects demonstrating increasing age, BMI, and family history, and that the percentages of BBD classified as PDWA and AH have increased. Impact on BC risk will be further investigated. Citation Format: Amy Degnim, Karthik Ghosh, Jodi M Carter, Robert A Vierkant, Matthew R Jensen, Stacey J Winham, Tanya L Hoskin, Marlene Frost, Teresa M Allers, Denise L Gehling, Mindy J Kern, Laura M Pacheco-Spann, Celine M Vachon, Derek C Radisky, Daniel W Visscher, Mark E Sherman. Benign breast disease: Temporal trends from 1967 to 2013 [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-11.

We evaluated the association of percent mammographic density (PMD), absolute dense area (DA), and non-dense area (NDA) with risk of “intrinsic” molecular breast cancer (BC) subtypes. We pooled 3492 invasive BC and 10,148 controls across six studies with density measures from prediagnostic, digitized film-screen mammograms. We classified BC tumors into subtypes [63% Luminal A, 21% Luminal B, 5% HER2 expressing, and 11% as triple negative (TN)] using information on estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and tumor grade. We used polytomous logistic regression to calculate odds ratio (OR) and 95% confidence intervals (CI) for density measures (per SD) across the subtypes compared to controls, adjusting for age, body mass index and study, and examined differences by age group. All density measures were similarly associated with BC risk across subtypes. Significant interaction of PMD by age (P = 0.001) was observed for Luminal A tumors, with stronger effect sizes seen for younger women

Salivary duct carcinoma (SDC) commonly expresses androgen receptor (AR) and HER2, giving rise to treatment implications. SDC may also express programmed-death-ligand-1 (PD-L1), a predictive marker of response to checkpoint inhibitors. PD-L1 can be associated with genomic instability and high density of tumor infiltrating lymphocytes (TILs). Evaluation of HER2 immunohistochemistry (IHC) in SDC is not standardized, and relationships between ERBB2 copy numbers, PD-L1 expression and TILs in SDC are unknown. We evaluated 32 SDCs for HER2, AR and PD-L1 expression (IHC), ERBB2 status (FISH) and TILs (slide review). HER2 was scored with three different systems (breast, gastric, proposed salivary gland). PD-L1 was evaluated with the combined positive score. Most patients were older men, presenting at advanced clinical stage with nodal or distant metastases. During follow-up (mean 5 years, range 6 months to 21 years), 25 of the 32 patients (78%) died of SDC. We propose a HER2 IHC scoring system which accurately predicts underlying ERBB2 amplification or increased copy numbers in SDC. Most tumors had increased ERBB2 copy numbers (19/32 amplification, 6/32 aneusomy), a finding associated with higher TIL densities (p = 0.045) and PD-L1 expression (p = 0.025). Patients with TILs ≥ 40% had better prognoses (Log-Rank p = 0.013), with TILs being favorable prognosticators in univariate analysis (Hazard ratio: 0.18, p = 0.024). A subset of SDCs with increased ERBB2 copy numbers have higher TILs and PD-L1 expression. TILs ≥ 40% are associated with better prognosis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12105-020-01163-x) contains supplementary material, which is available to authorized users.

Background: TNBC is the most aggressive sub-type of breast cancer and has limited targeted therapies. Atezolizumab was recently FDA-approved for patients with advanced PD-L1+ TNBC. In a well-characterized cohort of patients with early-stage TNBC, we used the FDA-approved PD-L1 SP142 companion assay for atezolizumab to score whole tumor sections for PD-L1 expression. We determined the frequency and scoring distribution of PD-L1 in these tumors and evaluated PD-L1 status for associations with clinicopathologic variables and outcomes. Methods: Whole tumor sections from 498 women with surgically-treated, non-(distant) metastatic TNBC (ER/PR 15% (80%). 228/498 (46%) tumors were PD-L1+, distributed as IC score 1-5%: 21%, 6-10%: 13%, 11-20%: 5% and >20%: 7%. 270/498 (54%) were PD-L1-. 43 (8.6%) of tumors had PD-L1 staining in TC (all but 1 were also IC+). PD-L1 IC expression was associated with larger tumor size (p = 0.005), higher nodal stage (≥pN1: 43% vs. 31%, p < 0.001), higher grade (grade 3: 95% vs. 86%, p< 0.001), higher Ki-67 PI (>15%: 88% vs. 69%, p Citation Format: Jodi M Carter, Mei-Yin C Polley, Jason P Sinnwell, Roberto A Leon-Ferre, Fergus J Couch, Krishna R Kalari, Judy C Boughey, Minetta C Liu, Daniel W Visscher, Matthew P Goetz. Frequency, characteristics and prognostic factors of PD-L1+ triple negative breast cancer using the PD-L1 SP142 companion assay [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD1-08.

OBJECTIVE. This study is designed to identify genes and pathways that could promote metastasis to the bowel in high-grade serous ovarian cancer (OC) and evaluate their associations with clinical outcomes. METHODS. We performed RNA sequencing of OC primary tumors (PTs) and their corresponding bowel metastases (n = 21 discovery set; n = 18 replication set). Differentially expressed genes (DEGs) were those expressed at least 2-fold higher in bowel metastases (BMets) than PTs in at least 30% of patients (P < .05) with no increased expression in paired benign bowel tissue and were validated with quantitative reverse transcription PCR. Using an independent OC cohort (n = 333), associations between DEGs in PTs and surgical and clinical outcomes were performed. Immunohistochemistry and mouse xenograft studies were performed to confirm the role of LRRC15 in promoting metastasis. RESULTS. Among 27 DEGs in the discovery set, 21 were confirmed in the replication set: SFRP2, Col11A1, LRRC15, ADAM12, ADAMTS12, MFAP5, LUM, PLPP4, FAP, POSTN, GRP, MMP11, MMP13, C1QTNF3, EPYC, DIO2, KCNA1, NETO1, NTM, MYH13, and PVALB. Higher expression of more than half of the genes in the PT was associated with an increased requirement for bowel resection at primary surgery and an inability to achieve complete cytoreduction. Increased expression of LRRC15 in BMets was confirmed by immunohistochemistry and knockdown of LRRC15 significantly inhibited tumor progression in mice. CONCLUSIONS. We identified 21 genes that are overexpressed in bowel metastases among patients with OC. Our findings will help select potential molecular targets for the prevention and treatment of malignant bowel obstruction in OC.

We describe the histology and the frequency of a histologic entity that we term "hyaline fibrous involution", which is characterized by symmetric and regular deposition of basal lamina-like periacinar hyaline material in association with atrophic epithelium, in breast samples from patients with either benign breast disease or germline BRCA mutation. Women with germline BRCA mutation (n = 93) who underwent prophylactic mastectomy (BRCA group) were compared to an age-matched sample of women who underwent biopsy for benign breast disease (n = 93). Median age was 45 years (range, 25-72 years). A single HE section of each subject's benign breast tissue was reviewed. The total number of terminal duct lobular units and the number of terminal duct lobular units with hyaline fibrous involution were recorded for each case. The presence of any hyaline fibrous involution lobules and the within-sample proportion of hyaline fibrous involution lobules relative to total lobules were compared between groups. Presence of any hyaline fibrous involution was significantly more frequent in the BRCA group compared to the benign breast disease group, 47% vs. 15% (p 0.0001, adjusted for total lobules). In women with any hyaline fibrous involution lobules, these unusual lobules were similarly rare in both groups, with median proportion of hyaline fibrous involution-positive lobules relative to all lobules of 0.03 in BRCA specimens (n = 44) and 0.03 in the benign breast disease group (n = 14). Within the BRCA group, frequency of any hyaline fibrous involution present was significantly higher in the perimenopausal age group (45-55 years: 63%) compared to other age groups (45 years, 44%;55 years, 15%; p = 0.05 and p = 0.02, respectively). Increased presence of hyaline fibrous involution in the setting of BRCA mutation suggests that it may represent a pathologic entity, possibly reflecting abnormal involution or an abnormal response to DNA damage.

Patient-derived xenografts (PDX) are generated in immune deficient mice and demonstrate histologic and molecular features similar to their corresponding human tumors. However, murine tumors (non-human) spontaneously occur in these models. 120 consecutive patients with high-risk primary breast cancer enrolled in the prospective neoadjuvant BEAUTY study had tumor tissue obtained at the time of diagnosis. These tumor cells, including initial tissue and subsequent generations, were injected into either NSG (n = 365) or NOD-SCID (n = 396) female mice. Mice with initial tumor growth sufficient for transfer to the 2nd generation underwent histologic review by pathologists, including Ki67 staining. After passaging the tumors for up to 4 generations, at least one primary mouse tumor was detected from 24 of the 54 PDX-lines, for a frequency of 3.2% (24 mice out of 761 mice), including murine lymphomas (n = 13), mammary tumors (n = 7), osteosarcomas (n = 2), and hemangiosarcomas (n = 2). While true PDX showed scattered strong staining with Ki67, murine tumors were Ki67 negative. No significant differences (p = 0.062) were observed comparing development of murine tumors in NOD-SCID (n = 8) vs NSG mice (n = 16). While PDX are a useful tool in cancer research, there is a potential for spontaneous murine tumors to arise, which could alter results of studies utilizing PDX. Morphologic review by a pathologist, potentially along with Ki67 staining, is necessary to ensure that tumor growth represents the desired PDX prior to use in downstream studies. This study is the first prospective study evaluating the frequency, type, and time frame for development of non-human tumors.

Introduction: The immune microenvironment of the breast is critical in the development and progression of breast cancer. By analyzing the immune components of breast tissues from patients at varying degrees of breast cancer risk, we may gain enhanced understanding of breast cancer risk and mechanisms of carcinogenesis. Methods: We compared the immune cell composition in breast lobules of age-matched patients with normal and benign breast disease (BBD) at progressively lower breast cancer risk: 1) 18 women with germline mutations in (BRCA1/2); 2) women at intermediate risk (BBD), and 3) women at normal risk (Komen Tissue Bank- (KTB)). We performed immunohistochemical stains for T lymphocytes (CD4, CD8); dendritic cells (CD11c); B lymphocytes (CD20) and macrophages (CD68) and quantitated immune cell densities in up to 10 representative lobules per sample. Results: The median age of all groups was 43.8 years (range: 34-67). BRCA carriers and BBD patients had a higher percentage of fibrocystic lobules (50% fibrocystic) compared to KTB samples (15% fibrocystic), p Conclusions: Preliminarily, these data showed that benign prophylactic breast tissues from BRCA mutation carriers contain increased numbers of several immune cell types versus tissues from patients at lower breast cancer risk, including BBD patients and research tissue donors. Further investigation is needed to confirm and expand these findings in relation to breast cancer risk. Table: Pairwise Wilcoxon rank-sum p-values comparing immune cell densities in breast lobules from women with BRCA germline mutations, vs BBD and KTB groups. All Lobules BRCA BBD KTB BRCA vs BBD BRCA vs KTB Median Median Median p-value p-value (IQR) (IQR) (IQR) CD4+, cells/mm2 116.3 63.8 17.7 0.08 Citation Format: Joshua W. Ogony, Tanya L. Hoskin, Melody L. Stallings Mann, Stacey J. Winham, Rushin Brahmbhatt, Muhammad Asad Arshad, Alvaro Pena Jimenez, Teresa M. Allers, Mark E. Sherrman, Daniel W. Visscher, Derek C. Radisky, Amy C. Degnim. Immune biomarkers in breast tissues of patients with BRCA1/2 mutations, benign breast disease, and normal tissue donors [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 5947.

An amendment to this paper has been published and can be accessed via the original article.

Triple negative breast cancer (TNBC) comprises 15–20% of all invasive breast cancer and is associated with a poor prognosis. As therapy options are limited for this subtype, there is a significant need to identify new targeted approaches for TNBC patient management. The expression of the folate receptor alpha (FRα) is significantly increased in patients with TNBC and is therefore a potential biomarker and therapeutic target. We optimized and validated a FRα immunohistochemistry method, specific to TNBC, to measure FRα expression in a centrally confirmed cohort of 384 patients with TNBC in order to determine if expression of the protein is associated with invasive disease-free survival (IDFS) and overall survival (OS). The FRα IHC demonstrated exceptional performance characteristics with low intra- and interassay variability as well as minimal lot-to-lot variation. FRα expression, which varied widely from sample to sample, was detected in 274 (71%) of the TNBC lesions. In a multivariable model adjusted for baseline characteristics, FRα expression was associated with improved IDFS (HR = 0.63, p = 0.01) but not with OS. The results demonstrate the potential of targeting the FRα in the majority of TNBC patients and suggest that variable expression may point to a need to stratify on FRα expression in clinical studies.

Treatment of ovarian cancer is limited by extensive metastasis and yet it remains poorly understood. We have studied the critical step of metastatic colonization in the context of the productive interactions with the metastatic microenvironment with a goal of identifying key regulators. By combining miRNA expression analysis using an organotypic 3D culture model of early ovarian cancer metastasis with that of matched primary and metastatic tumors from 42 patients with ovarian cancer, we identified miR-4454 as a key regulator of both early colonization and advanced metastasis in patients with ovarian cancer. miR-4454 was downregulated in the metastasizing ovarian cancer cells through paracrine signals from microenvironmental fibroblasts, which promoted migration, invasion, proliferation, and clonogenic growth in ovarian cancer cells as well as their ability to penetrate through the outer layers of the omentum. Stable overexpression of miR-4454 decreased metastasis in ovarian cancer xenografts. Its mechanism of action was through the upregulation of its targets, secreted protein acidic and cysteine rich (SPARC) and BCL2 associated athanogene 5 (BAG5), which activated focal adhesion kinase (FAK) signaling, promoted mutant p53 gain of function by its stabilization, and inhibited apoptosis. Because microenvironment-induced downregulation of miR-4454 is essential for early and advanced metastasis, targeting it could be a promising therapeutic approach. Implications: This study identifies a miRNA, miR-4454, which is downregulated by signals from the microenvironment and promotes early and advanced ovarian cancer metastasis through its effects on FAK activation, mutant p53 stabilization, and apoptosis inhibition.

Purpose: Folate receptor alpha (FR) is overexpressed in several cancers. Endogenous immunity to the FR has been demonstrated in patients and suggests the feasibility of targeting FR with vaccine or other immune therapies. CD4 helper T cells are central to the development of coordinated immunity, and prior work shows their importance in protecting against relapse. Our previous identification of degenerate HLA-class II epitopes from human FR led to the development of a broad coverage epitope pool potentially useful in augmenting antigen-specific immune responses in most patients. Patients and Methods: We conducted a phase I clinical trial testing safety and immunogenicity of this vaccine, enrolling patients with ovarian cancer or breast cancer who completed conventional treatment and who showed no evidence of disease. Patients were initially treated with low-dose cyclophosphamide and then vaccinated 6 times, monthly. Immunity and safety were examined during the vaccine period and up to 1 year later. Results: Vaccination was well tolerated in all patients. Vaccine elicited or augmented immunity in more than 90% of patients examined. Unlike recall immunity to tetanus toxoid (TT), FR T-cell responses developed slowly over the course of vaccination with a median time to maximal immunity in 5 months. Despite slow development of immunity, responsiveness appeared to persist for at least 12 months. Conclusions: The results demonstrate that it is safe to augment immunity to the FR tumor antigen, and the developed vaccine is testable for therapeutic activity in most patients whose tumors express FR, regardless of HLA genotype. Clin Cancer Res; 24(13); 3014–25. ©2018 AACR.

Background More than 1.5 million women per year have a benign breast biopsy resulting in concern about their future breast cancer (BC) risk. This study examined the performance of 2 BC risk models that integrate clinical and histologic findings in this population. Methods The BC risk at 5 and 10 years was estimated with the Breast Cancer Surveillance Consortium (BCSC) and Benign Breast Disease to Breast Cancer (BBD-BC) models for women diagnosed with benign breast disease (BBD) at the Mayo Clinic from 1997 to 2001. Women with BBD were eligible for the BBD-BC model, but the BCSC model also required a screening mammogram. Calibration and discrimination were assessed. Results Fifty-six cases of BC were diagnosed among the 2142 women with BBD (median age, 50 years) within 5 years (118 were diagnosed within 10 years). The BBD-BC model had slightly better calibration at 5 years (0.89; 95% confidence interval [CI], 0.71-1.21) versus 10 years (0.81; 95% CI, 0.70-1.00) but similar discrimination in the 2 time periods: 0.68 (95% CI, 0.60-0.75) and 0.66 (95% CI, 0.60-0.71), respectively. In contrast, among the 1089 women with screening mammograms (98 cases of BC within 10 years), the BCSC model had better calibration (0.94; 95% CI, 0.85-1.43) and discrimination (0.63; 95% CI, 0.56-0.71) at 10 years versus 5 years (calibration, 1.31; 95% CI, 0.94-2.25; discrimination, 0.59; 95% CI, 0.46-0.71) where discrimination was not different from chance. Conclusions The BCSC and BBD-BC models were validated in the Mayo BBD cohort, although their performance differed by 5-year risk versus 10-year risk. Further enhancement of these models is needed to provide accurate BC risk estimates for women with BBD.

Triple-negative breast cancer (TNBC) is a heterogeneous disease with poor prognosis that lacks targeted therapies, especially in patients with chemotherapy-resistant disease. Since DNA methylation-induced silencing of tumor suppressors is common in cancer, reversal of promoter DNA hypermethylation by 5-aza-2'-deoxycytidine (decitabine), an FDA-approved DNA methyltransferase (DNMT) inhibitor, has proven effective in treating hematological neoplasms. However, its antitumor effect varies in solid tumors, stressing the importance of identifying biomarkers predictive of therapeutic response. Here, we focused on the identification of biomarkers to select decitabine-sensitive TNBC through increasing our understanding of the mechanism of decitabine action. We showed that protein levels of DNMTs correlated with response to decitabine in patient-derived xenograft (PDX) organoids originating from chemotherapy-sensitive and -resistant TNBCs, suggesting DNMT levels as potential biomarkers of response. Furthermore, all 3 methytransferases, DNMT1, DNMT3A, and DNMT3B, were degraded following low-concentration, long-term decitabine treatment both in vitro and in vivo. The DNMT proteins could be ubiquitinated by the E3 ligase, TNF receptor-associated factor 6 (TRAF6), leading to lysosome-dependent protein degradation. Depletion of TRAF6 blocked decitabine-induced DNMT degradation, conferring resistance to decitabine. Our study suggests a potential mechanism of regulating DNMT protein degradation and DNMT levels as response biomarkers for DNMT inhibitors in TNBCs.

Purpose While the role of natural killer (NK) cells in breast cancer therapy has been investigated, little information is known about NK cell function and presence in nonmalignant and premalignant breast tissue. Here, we investigate and quantify NK cell marker CD56 and activating ligand MICA in breast tissue with benign breast disease. Methods Serial tissue sections from 88 subjects, 44 with benign breast disease (BBD) who remained cancer-free, and 44 with BBD who later developed cancer, were stained with H&E, anti-MICA, and anti-CD56. Up to ten representative lobules were identified on each section. Using digital image analysis, MICA and CD56 densities were determined for each lobule, reported as percent of pixels in the lobule that registered as stained by each antibody. Analyses were performed on a per-subject and per-lobule basis. Results Per-subject multivariate analyses showed associations of CD56 and MICA with age: CD56 was increased in older subjects (p = 0.03), while MICA was increased in younger subjects (p = 0.005). Per-lobule analyses showed that CD56 and MICA levels were both decreased in lobules with fibrocystic change, with median levels of CD56 and MICA staining, respectively, at 0.31 and 7.0% in fibrocystic lobules compared to 0.76 and 12.2% in lobules without fibrocystic change (p

Purpose Sclerosing adenosis (SA), found in ¼ of benign breast disease (BBD) biopsies, is a histological feature characterized by lobulocentric proliferation of acini and stromal fibrosis and confers a two-fold increase in breast cancer risk compared to women in the general population. We evaluated a NanoString-based gene expression assay to model breast cancer risk using RNA derived from formalin-fixed, paraffin-embedded (FFPE) biopsies with SA. Methods The study group consisted of 151 women diagnosed with SA between 1967 and 2001 within the Mayo BBD cohort, of which 37 subsequently developed cancer within 10 years (cases) and 114 did not (controls). RNA was isolated from benign breast biopsies, and NanoString-based methods were used to assess expression levels of 61 genes, including 35 identified by previous array-based profiling experiments and 26 from biological insight. Diagonal linear discriminant analysis of these data was used to predict cancer within 10 years. Predictive performance was assessed with receiver operating characteristic area under the curve (ROC-AUC) values estimated from 5-fold cross-validation. Results Gene expression prediction models achieved cross-validated ROC-AUC estimates ranging from 0.66 to 0.70. Performing univariate associations within each of the five folds consistently identified genes DLK2, EXOC6, KIT, RGS12, and SORBS2 as significant; a model with only these five genes showed cross-validated ROC-AUC of 0.75, which compared favorably to risk prediction using established clinical models (Gail/BCRAT: 0.57; BBD-BC: 0.67). Conclusions Our results demonstrate that biomarkers of breast cancer risk can be detected in benign breast tissue years prior to cancer development in women with SA. These markers can be assessed using assay methods optimized for RNA derived from FFPE biopsy tissues which are commonly available. Electronic supplementary material The online version of this article (doi:10.1007/s10549-017-4441-z) contains supplementary material, which is available to authorized users.

Purpose: Little is known about the role of the immune system in the earliest stages of breast carcinogenesis. We studied quantitative differences in immune cell types between breast tissues from normal donors and those from women with benign breast disease (BBD). Experimental Design: A breast tissue matched case–control study was created from donors to the Susan G. Komen for the Cure Tissue Bank (KTB) and from women diagnosed with BBD at Mayo Clinic (Rochester, MN) who either subsequently developed cancer (BBD cases) or remained cancer-free (BBD controls). Serial tissue sections underwent immunostaining and digital quantification of cell number per mm2 for CD4+ T cells, CD8+ T cells, CD20+ B cells, and CD68+ macrophages and quantification of positive pixel measure for CD11c (dendritic cells). Results: In 94 age-matched triplets, BBD lobules showed greater densities of CD8+ T cells, CD11c+ dendritic cells, CD20+ B cells, and CD68+ macrophages compared with KTB normals. Relative to BBD controls, BBD cases had lower CD20+ cell density (P = 0.04). Nearly 42% of BBD cases had no CD20+ B cells in evaluated lobules compared with 28% of BBD controls (P = 0.02). The absence of CD20+ cells versus the presence in all lobules showed an adjusted OR of 5.7 (95% confidence interval, 1.4–23.1) for subsequent breast cancer risk. Conclusions: Elevated infiltration of both innate and adaptive immune effectors in BBD tissues suggests an immunogenic microenvironment. The reduced B-cell infiltration in women with later breast cancer suggests a role for B cells in preventing disease progression and as a possible biomarker for breast cancer risk. Clin Cancer Res; 23(14); 3945–52. ©2017 AACR.

OBJECTIVE: Metaplastic breast cancer (MetaBC) is a rare breast cancer subtype poorly responsive to systemic therapy in the metastatic setting with high recurrence rates in the adjuvant setting. However, limited data exist regarding response to neoadjuvant chemotherapy (NAC). We performed a single institutional study to assess the clinical and pathological complete response rates (pCR) of MetaBC to NAC. METHODS: Mayo Clinic Rochester patients with MetaBC treated with NAC were identified using the institutional medical index. Patient demographics, tumor characteristics, chemotherapy treatment, clinical and pathological response, and long-term outcomes were reviewed. Pathologic response was assessed by direct pathology review (n = 14) or review of outside surgical and pathology reports (n = 4). RESULTS: Women with MetaBC (n = 18) received NAC from January 1991 to June 2014. The mean age was 50 years (range 33–79) with a mean tumor size of 5.1 cm (range 2.3–11 cm) and 6/18 had pathologically confirmed lymph nodes prior to surgery. The majority (13/18; 72%) were estrogen receptor (ER), progesterone receptor (PR) and HER-2 negative (TNBC), and 1/18 (5.5%) was HER-2 positive. Five had BRCA testing and 2/5 were BRCA-2 positive. The chemotherapy regimens included anthracycline/cyclophosphamide (AC) (n = 1), AC/taxane (n = 3), AC/taxane/platinum (n = 8), taxane/platinum-based regimens (n = 4), taxane/cyclophosphamide (n = 1) and taxane/trastuzumab (n = 1). Five of 18 (28%) progressed on initial treatment including two who developed metastatic disease during NAC. The overall pCR rate was 2/18 (11%). CONCLUSION: MetaBC is poorly responsive to NAC, with a pCR rate (11%), that is lower than expected in a predominantly TNBC cohort. MetaBC patients should be considered for clinical trials testing new NAC regimens and in the absence of clinical trial enrollment, MetaBC patients with resectable disease should proceed directly to definitive operative management.

Background Obesity and elevated breast density are common risk factors for breast cancer, and their effects may vary by estrogen receptor (ER) subtype. However, their joint effects on ER subtype-specific risk are unknown. Understanding this relationship could enhance risk stratification for screening and prevention. Thus, we assessed the association between breast density and ER subtype according to body mass index (BMI) and menopausal status. Methods We conducted a case-control study nested within two mammography screening cohorts, the Mayo Mammography Health Study and the San Francisco Bay Area Breast Cancer SPORE/San Francisco Mammography Registry. Our pooled analysis contained 1538 ER-positive and 285 ER-negative invasive breast cancer cases and 4720 controls matched on age, menopausal status at time of mammogram, and year of mammogram. Percent density was measured on digitized film mammograms using computer-assisted techniques. We used polytomous logistic regression to evaluate the association between percent density and ER subtype by BMI subgroup (normal/underweight

Contains fulltext : 202591.pdf (Publisher’s version ) (Closed access) OBJECTIVE: Poly(ADP-ribose) polymerase (PARP) inhibitors have shown substantial activity in homologous recombination- (HR-) deficient ovarian cancer and are undergoing testing in other HR-deficient tumors. For reasons that are incompletely understood, not all patients with HR-deficient cancers respond to these agents. Preclinical studies have demonstrated that changes in alternative DNA repair pathways affect PARP inhibitor (PARPi) sensitivity in ovarian cancer models. This has not previously been assessed in the clinical setting. METHODS: Clonogenic and plasmid-based HR repair assays were performed to compare BRCA1-mutant COV362 ovarian cancer cells with or without 53BP1 gene deletion. Archival biopsies from ovarian cancer patients in the phase I, open-label clinical trial of PARPi ABT-767 were stained for PARP1, RAD51, 53BP1 and multiple components of the nonhomologous end-joining (NHEJ) DNA repair pathway. Modified histochemistry- (H-) scores were determined for each repair protein in each sample. HRD score was determined from tumor DNA. RESULTS: 53BP1 deletion increased HR in BRCA1-mutant COV362 cells and decreased PARPi sensitivity in vitro. In 36 women with relapsed ovarian cancer, responses to the PARPi ABT-767 were observed exclusively in cancers with HR deficiency. In this subset, 7 of 18 patients (39%) had objective responses. The actual HRD score did not further correlate with change from baseline tumor volume (r=0.050; p=0.87). However, in the HR-deficient subset, decreased 53BP1 H-score was associated with decreased antitumor efficacy of ABT-767 (r=-0.69, p=0.004). CONCLUSION: Differences in complementary repair pathways, particularly 53BP1, correlate with PARPi response of HR-deficient ovarian cancers.

548 Background: sTILs are a well-established prognostic and predictive biomarker in patients with operable TNBC receiving pre or postoperative systemic therapy. We1 and others2,3 have also shown that sTILs are prognostic in patients who did not receive adjuvant chemotherapy. Here, we detail the outcomes of systemically untreated patients with N0 TNBC according to sTIL score. We focused on the N0 subset as a group of patients who may be candidates for future prospective therapy de-escalation trials. Methods: From a clinically annotated cohort of 605 patients with centrally confirmed TNBC (ER/PR < 1% and HER2 negative) with long-term outcomes data, we identified 182 patients treated with locoregional therapy only (breast surgery +/- radiation therapy and no chemotherapy). The clinicopathological characteristics of this cohort have previously been published1. In this analysis, we report the 5- and 10-year invasive disease-free survival (iDFS) and overall survival (OS) rates of patients with N0 TNBC according to sTIL levels. IDFS and OS were defined as per the STEEP classification and estimated using the Kaplan–Meier method. Comparisons of the survival distributions between groups were assessed by the log-rank test. sTILs were assessed as a continuous parameter according to the International TIL Working Group guidelines. For comparisons of outcomes between groups, tumors were classified as lymphocyte-predominant TNBC (defined as containing ≥50% sTILs) vs non-lymphocyte-predominant ( < 50% sTILs). Results: Of 182 systemically untreated patients, 149 (82%) were N0 and most (78%) were post-menopausal. T stage distribution was T1: 68%, T2: 28%, T3/4: 4%. Among N0 patients, 31 (21%) had lymphocyte-predominant TNBC, and in this group the 5-year iDFS and OS were 89% (95% CI 76-100) and 96% (95% CI 89-100), while the 10-year iDFS and OS were 89% (95% CI 76-100) and 87% (95% CI 73-100), respectively. In contrast, outcomes for patients with non-lymphocyte predominant TNBC were significantly worse. For this group, 5-year iDFS and OS were 62% (95% CI 53-73) and 78% (95% CI 71-86) while the 10-year iDFS and OS were 45% (95% CI 36-58) and 66% (95% CI 68-76), respectively ( log-rank p = 0.02 for iDFS and log-rank p = 0.03 for OS). Conclusions: sTIL quantification identifies a subset of patients with early-stage N0 TNBC with an exceedingly good prognosis, even in the absence of adjuvant chemotherapy. These data provide support for the evaluation of sTILs as part of prospective investigation of systemic therapy de-escalation strategies in N0 TNBC. References:1Leon-Ferre et al, Breast Cancer Res Treat (2018) 167:89-99 2Park et al, Ann Oncol (2019) 12:1941-1949 3De Jong et al, ESMO 2020

Terminal duct lobular units (TDLUs) are the predominant source of future breast cancers, and lack of TDLU involution (higher TDLU counts, higher acini count per TDLU and the product of the two) is a breast cancer risk factor. Numerous breast cancer susceptibility single nucleotide polymorphisms (SNPs) have been identified, but whether they are associated with TDLU involution is unknown. In a pooled analysis of 872 women from two studies, we investigated 62 established breast cancer SNPs and relationships with TDLU involution. Poisson regression models with robust variance were used to calculate adjusted per-allele relative risks (with the non-breast cancer risk allele as the referent) and 95% confidence intervals between TDLU measures and each SNP. All statistical tests were two-sided; P

BACKGROUND Women with atypical hyperplasia (AH) on breast biopsy have a substantially increased risk of breast cancer (BC). Here the BC risk for the extent and subtype of AH is reported for 2 separate cohorts. METHODS All samples containing AH were included from 2 cohorts of women with benign breast disease (Mayo Clinic and Nashville). Histology review quantified the number of foci of atypical ductal hyperplasia (ADH) and atypical lobular hyperplasia (ALH). The BC risk was stratified for the number of AH foci within AH subtypes. RESULTS The study included 708 Mayo AH subjects and 466 Nashville AH subjects. In the Mayo cohort, an increasing number of foci of AH was associated with a significant increase in the risk of BC both for ADH (relative risks of 2.61, 5.21, and 6.36 for 1, 2, and ≥3 foci, respectively; P for linear trend = .006) and for ALH (relative risks of 2.56, 3.50, and 6.79 for 1, 2, and ≥3 foci, respectively; P for linear trend = .001). In the Nashville cohort, the relative risks of BC for ADH were 2.70, 5.17, and 15.06 for 1, 2, and ≥3 foci, respectively (P for linear trend < .001); for ALH, the relative risks also increased but not significantly (2.61, 3.48, and 4.02, respectively; P = .148). When the Mayo and Nashville samples were combined, the risk increased significantly for 1, 2, and ≥3 foci: the relative risks were 2.65, 5.19, and 8.94, respectively, for ADH (P < .001) and 2.58, 3.49, and 4.97, respectively, for ALH (P = .001). CONCLUSIONS In 2 independent cohort studies of benign breast disease, the extent of atypia stratified the long-term BC risk for ADH and ALH. Cancer 2016. © 2016 American Cancer Society.

Age-related lobular involution (LI) is a physiological process in which the terminal duct lobular units of the breast regress as a woman ages. Analyses of breast biopsies from women with benign breast disease (BBD) have found that extent of LI is negatively associated with subsequent breast cancer development. Here we assess the natural course of LI within individual women, and the impact of progressive LI on breast cancer risk. The Mayo Clinic BBD cohort consists of 13,455 women with BBD from 1967 to 2001. The BBD cohort includes 1115 women who had multiple benign biopsies, 106 of whom had developed breast cancer. Within this multiple biopsy cohort, the progression of the LI process was examined by age at initial biopsy and time between biopsies. The relationship between LI progression and breast cancer risk was assessed using standardized incidence ratios and by Cox proportional hazards analysis. Women who had multiple biopsies were younger age and had a slightly higher family history of breast cancer as compared with the overall BBD cohort. Extent of LI at subsequent biopsy was greater with increasing time between biopsies and for women age 55 + at initial biopsy. Among women with multiple biopsies, there was a significant association of higher breast cancer risk among those with involution stasis (lack of progression, HR 1.63) as compared with those with involution progression, p = 0.036. The multiple biopsy BBD cohort allows for a longitudinal study of the natural progression of LI. The majority of women in the multiple biopsy cohort showed progression of LI status between benign biopsies, and extent of progression was highest for women who were in the perimenopausal age range at initial biopsy. Progression of LI status between initial and subsequent biopsy was associated with decreased breast cancer risk. Electronic supplementary material The online version of this article (doi:10.1007/s10549-016-3691-5) contains supplementary material, which is available to authorized users.

Introduction/Objective Non-mass enhancement (NME) in breast tissue is an area of enhancement on MRI that appears distinct from background breast parenchyma, but lacks definitive features of a mass. NME is characterized by its internal enhancement pattern and distribution, and can represent benign, high risk, or malignant pathology. Given this considerable overlap, a core biopsy is often necessary for diagnosis and management. We aimed to elucidate the most frequent histologic findings found on breast biopsies for MRI NME. Methods Using our institutional database we identified 70 female patients with high risk status for breast cancer with MRI screen detected NME (BIRADS-4-suspicious for malignancy) that underwent subsequent biopsy procedure during the period of 01/2016-12/2017. Primary pathologic diagnoses were subcategorized as follows: malignant, atypical, benign mass-like lesions, fibrocystic changes (proliferative, nonproliferative), or “other” primary diagnoses. Results The median age of patients was 48 years (range: 22-76 years). Of the 70 patients, 66 underwent MRI-guided core biopsy, 3 underwent ultrasound-guided core biopsy and 1 underwent excisional biopsy. The primary diagnosis was analyzed. Of these 70 cases, 8 (11.4%) were malignant (7 with ductal carcinoma in situ and 1 with invasive ductal carcinoma), 1 (1.4%) had atypical lobular hyperplasia, and the remaining 61 (87.1%) showed benign findings (36 with fibrocystic changes (FCC), 22 benign mass-like lesions, 3 with other non-specific findings). The FCC were subcategorized as proliferative (usual ductal hyperplasia, columnar cell change, incidental radial scar, incidental intraductal papilloma, sclerosing adenosis, focal pseudoangiomatous stromal hyperplasia (PASH)) or nonprolifeative (stromal fibrosis, duct ectasia, apocrine metaplasia). Majority (61.1%) of FCC were both proliferative and nonproliferative, 22.2% proliferative only and 16.7% nonproliferative only. Benign mass-like lesions included PASH (45.4%), fibroadenomatoid nodule (22.7%), fat necrosis (18.2 %) and remaining had the diagnosis of clustered apocrine cysts, papillomatosis, and radial sclerosing lesion. Conclusion Less than a third of cases showed malignant findings and more than two-third of cases showed benign findings with a high rate of detection of proliferative lesions and PASH.

Background: Biopsy diagnosis of benign breast disease (BBD) based on the most severe lesion in a sample predicts future breast cancer risk and has implications for screening and management. Lobules are the functional unit of the breast and the structures from which BBD arises. We developed and preliminarily validated an automated computational pathology algorithm to discriminate normal from BBD breast lobules as a step toward automated comprehensive characterization of benign biopsies. Methods: 152 BBD biopsies (27 training, 125 validation) from the Mayo Clinic were examined using scanned digital images of H&E stained sections. For each image, a pathologist annotated up to 10 representative lobules as normal or BBD using standard pathology criteria. A deep learning algorithm to quantify lobular features was developed using 129 lobules from 27 subjects. Nine features were identified that discriminate normal vs BBD lobules, expressing lobule size, acini size and number, acinar lumen size, proportion of lobular stroma, and capillaries. Here, we validate their performance to discriminate normal vs BBD lobules in a set of 1250 lobules from 125 subjects using area under the ROC curve (AUC) analysis. Random forest analysis was used for multivariable modeling; model performance was assessed with a tenfold cross-validation approach. Results: Median subject age was 52 years. Among the 125 validation subjects, BBD findings were nonproliferative in 39%, proliferative in 45%, and atypical hyperplasia in 16%. Sections included 552 (44%) normal lobules and 698 BBD (56%) lobules, with representation of both lobule types on each section. In univariate analyses, four individual features showed good discrimination between normal vs. BBD lobules, yielding the following AUCs: lobule size= 0.74, mean acini size= 0.75, epithelial area= 0.75, and number of acini with large lumens= 0.76. Lobule size and epithelial area were highly correlated (Spearman rank correlation r = 0.95), but both of these features showed lower correlation with mean acini size and number of acini with large lumens (each r < 0.5). With random forest modeling, number of acini with large lumens was the strongest discriminating feature, followed by mean acini size, lobule size, and epithelial area. With ten-fold cross validation of the multivariable random forest model, the overall AUC was 0.82 (95% CI: 0.79-0.85). Conclusion: Our validation showed that automated quantitative computational pathology assessment of breast lobules can discriminate normal versus BBD on a per lobule basis. This finding supports the feasibility of developing automated algorithms to classify every lesion in breast biopsies, expanding beyond visual assessment. Further studies using deep learning may reveal novel pathology features for classifying BBD biopsies with the potential to strengthen estimation of breast cancer risk. Citation Format: Amy C. Degnim, Thomas de Bel, Mark E. Sherman, Derek C. Radisky, Stacey J. Winham, Tanya L. Hoskin, Melody L. Stallings Mann, Marlene Frost, Robert A. Vierkant, Brendan T. Broderick, Ethan P. Heinzen, Rushin Brahmbhatt, Muhammad Arshad, Celine M. Vachon, Jodi M. Carter, Lori A. Denison, Daniel W. Visscher, Jeroen van der Laak. Discrimination of benign breast disease from normal lobules using an automated computational pathology algorithm [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2113.

Metastasis causes poor prognosis and yet remains the least understood aspect of cancer. To identify key regulators of ovarian cancer metastasis that drive early colonization and are also essential for advanced metastasis, we have applied a novel, comprehensive approach of combining the analysis of an organotypic 3D culture model of early metastasis with ovarian cancer patient tumors. Using matched primary and metastatic tumors from 42 OC patients, we have compared the fully formed metastatic tumors with the primary tumor of the same patient. Since this is an end-point analysis and reveals little about the intermediated regulatory steps, we have also studied the changes occurring during early metastatic colonization as a result of the interactions with the metastatic microenvironment by using an organotypic 3D culture model of omental metastasis. By combining these two approaches, we have identified key master regulators of metastasis that are essential for early metastatic colonization and remain important in the fully formed metastatic tumor. We specifically focused on microRNAs because of their pleotropic effects through translational inhibition of multiple targets, and also our previous studies have demonstrated their important role in metastasis. Using this approach, we identified a novel microRNA miR-4454, downregulated in the metastasizing ovarian cancer cells through their specific paracrine interactions with fibroblasts in the metastatic microenvironment. The downregulation of miR-4454 promoted high-grade serous ovarian cancer cell migration, invasion, proliferation, and clonogenic growth as well as metastasis in xenografts. The targets of miR-4454 were identified by RNA-seq in high-grade serous ovarian cancer cells overexpressing the microRNA. SPARC and BAG5 were found to be the functional effectors of miR-4454. SPARC activated FAK signaling and promoted metastasis, while BAG5 inhibited apoptosis and helped the metastasizing cancer cells tide over adverse conditions in the microenvironment. Since miR-4454 is essential for early metastatic colonization as well as in advanced metastasis, targeting it is a promising approach to treat metastatic ovarian cancer. Citation Format: Subramanyam Dasari, Taruni Pandhiri, Tommaso Grassi, Daniel W. Visscher, Francesco Multinu, Komal Agarwal, Andrea Mariani, Viji Shridhar, Anirban K. Mitra. Paracrine interactions with microenvironmental fibroblasts promote ovarian cancer metastasis through downregulation of miR-4454 [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B66.

Background The Circulating Cell-free Genome Atlas (CCGA; NCT02889978) study is a prospective, multi-center, observational, case-control study with longitudinal follow-up to support the development of a plasma cfDNA-based multi-cancer early detection assay. We previously reported that the fraction of tumor-derived cfDNA fragments in plasma (estimated tumor fraction [TF]) is associated with detection of multiple cancer types using a prototype whole-genome bisulfite sequencing (WGBS) assay in a pre-specified substudy. In addition, detectability of breast cancer varied by hormone-receptor (HR) status: HR negative (HR-) breast cancer had a higher detection rate than the overall breast cancer cohort. Here, we performed a planned in-depth analysis of the breast cancer cohort to understand the main clinical and biological determinants of detectability using our WGBS assay. Methods Blood samples were prospectively collected from participants (pts) with newly-diagnosed, untreated breast cancer. In a pre-specified CCGA substudy, 511 (31.4%) of 1,628 pts had a clinicopathologic diagnosis of breast cancer (any stage, excluding stage 0) and a WGBS result. Pre-specified biological and clinical factors were assessed for correlation with TF: clinical stage, pathologic N stage, lesion size (by imaging), T stage, histologic grade, hormone receptor (HR) status, and proliferative rate (Ki-67 by IHC [MIB-1 clone]) via univariate and multivariate analyses. Ki-67 was obtained from pathology reports provided by enrollment sites and also assessed centrally (Mayo Clinic [Rochester, MN]). Stage-specific differences in detection rates between HR positive (HR+) and HR- breast cancers due to biological differences such as histologic grade and/or proliferative rate was also assessed. Results Of 511 pts, 94 (18.4%; 48.9% HR- and 12.3% in HR+) had WGBS-detected cancer. Higher TF was significantly associated with cancer detection (p Conclusions Using a prototype methylation-based plasma cfDNA cancer detection assay, TF was found to be associated with detection, and with clinical and biological features of breast cancer. Features routinely used to assess clinical aggressiveness (eg, HR- status and histologic grade) also demonstrated stage-specific associations with TF. Given the known relationship between TF and detection, these data suggest that clinical and biological cancer features may also provide insight into the variability of cancer detection using plasma-based cfDNA tests. Citation Format: Minetta C. Liu, Jodi M. Carter, Daniel W. Visscher, Karla Kopp, Rita Shaknovich, Xiaoji Chen, Kathryn N. Kurtzman, Shilpen Patel, Jacqueline D. Brooks, Carlo R. Cosenza, Jafi A. Lipson, Donald A. Richards, Fergus J. Couch, Zhao Dong, Hai Liu, Oliver Venn, Joerg Bredno, Eric T. Fung, Anne-Renee Hartman. Blood-based cancer detection in plasma cell-free DNA (cfDNA): Evaluating clinical and pathologic tumor characteristics in participants with breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-01.

Background Fibroadenomas are common benign breast lesions, and studies of European American women indicate a persistent, increased risk of breast cancer after diagnosing a fibroadenoma on biopsy. This association has not been independently assessed in African American women, despite reports that these women are more likely to present with fibroadenomas. Methods The study cohort included 3853 African American women with a breast biopsy completed between 1997 and 2010 in metropolitan Detroit. Biopsies were microscopically reviewed for benign breast lesions, including fibroadenoma, proliferative disease, and atypia. Risk of breast cancer within the cohort was estimated using relative risk ratios and 95% CIs calculated using multivariable log-binomial regression. Relative risk of breast cancer in this cohort compared with African American women in the broader metropolitan Detroit population was estimated using standardized incidence ratios (SIRs). Results Fibroadenomas occurred more frequently in biopsies of younger women, and other types of benign breast lesions were less likely to occur when a fibroadenoma was present (p = 0.008 for lobular hyperplasia; all other p values