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Purpose: Most breast biopsies are diagnosed as benign breast disease (BBD), with 1.5- to fourfold increased breast cancer (BC) risk. Apart from pathologic diagnoses of atypical hyperplasia, few factors aid in BC risk assessment of these patients. We assessed whether a 313-SNP polygenic risk score (PRS) stratifies risk of BBD patients., Patients and Methods: We pooled data from five Breast Cancer Association Consortium case-control studies (mean age = 59.9 years), including 6,706 cases and 8,488 controls. Using logistic regression, we estimated BC risk associations by self-reported BBD history and strata of PRS, with median PRS category among women without BBD as the referent. We assessed interactions and mediation of BBD and PRS with BC risk., Results: BBD history was associated with increased BC risk (OR = 1.48, 95% CI: 1.37-1.60; p < .001). PRS increased BC risk, irrespective of BBD history (p-interaction = 0.48), with minimal evidence of mediation of either factor by the other. Women with BBD and PRS in the highest tertile had over 2-fold increased odds of BC (OR = 2.73, 95% CI: 2.41-3.09) and those with BBD and PRS in the lowest tertile experienced reduced BC risk (OR = 0.79, 95% CI: 0.70-0.91), compared to the reference group. Women with BBD and PRS in the highest decile had a 3.7- fold increase (95% CI: 3.00-4.61) compared to those with median PRS without BBD., Conclusion: BC risks are elevated among women with BBD and increase progressively with PRS, suggesting that optimal combinations of these factors may improve risk stratification., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: To characterize associations of microcalcifications (calcs) with benign breast disease lesion subtypes and assess whether tissue calcs affect risks of ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC)., Methods: We analyzed detailed histopathologic data for 4,819 BBD biopsies from a single institution cohort (2002-2013) followed for DCIS or IBC for a median of 7.4 years for cases (N = 338) and 11.2 years for controls. Natural language processing was used to identify biopsies containing calcs based on pathology reports. Univariable and multivariable regression models were applied to assess associations with BBD lesion type and age-adjusted Cox proportional hazard regressions were performed to model risk of IBC or DCIS stratified by the presence or absence of calcs., Results: Calcs were identified in 2063 (42.8%) biopsies. Calcs were associated with older age at BBD diagnosis (56.2 versus 49.0 years; P < 0.001). Overall, the risk of developing IBC or DCIS did not differ significantly between patients with calcs (HR 1.13, 95% CI 0.90, 1.41) as compared to patients without calcs. Stratification by BBD severity or subtype, age at BBD biopsy, outcomes of IBC versus DCIS, and mammography technique (screen-film versus full-field digital mammography) did not significantly alter association between calcs and risk., Conclusion: Our analysis of calcs in BBD biopsies did not find a significant association between calcs and risk of breast cancer., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The fibrotic tumor microenvironment is a pivotal therapeutic target. Nintedanib, a clinically approved multikinase antifibrotic inhibitor, is effective against lung adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). Previous studies have implicated the secretome of tumor-associated fibroblasts (TAFs) in the selective effects of nintedanib in ADC, but the driving factor(s) remained unidentified. Here we examined the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a tumor-promoting cytokine overproduced in ADC-TAFs. To this aim, we combined genetic approaches with in vitro and in vivo preclinical models based on patient-derived TAFs. Nintedanib reduced TIMP-1 production more efficiently in ADC-TAFs than SCC-TAFs through a SMAD3-dependent mechanism. Cell culture experiments indicated that silencing TIMP1 in ADC-TAFs abolished the therapeutic effects of nintedanib on cancer cell growth and invasion, which were otherwise enhanced by the TAF secretome. Consistently, co-injecting ADC cells with TIMP1-knockdown ADC-TAFs into immunocompromised mice elicited a less effective reduction of tumor growth and invasion under nintedanib treatment compared to tumors bearing unmodified fibroblasts. Our results unveil a key mechanism underlying the selective mode of action of nintedanib in ADC based on the excessive production of TIMP-1 in ADC-TAFs. We further pinpoint reduced SMAD3 expression and consequent limited TIMP-1 production in SCC-TAFs as key for the resistance of SCC to nintedanib. These observations strongly support the emerging role of TIMP-1 as a critical regulator of therapy response in solid tumors., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

While RNA sequencing and multi-omic approaches have significantly advanced cancer diagnosis and treatment, their limitation in preserving critical spatial information has been a notable drawback. This spatial context is essential for understanding cellular interactions and tissue dynamics. Multiplex digital spatial profiling (MDSP) technologies overcome this limitation by enabling the simultaneous analysis of transcriptome and proteome data within the intact spatial architecture of tissues. In breast cancer research, MDSP has emerged as a promising tool, revealing complex biological questions related to disease evolution, identifying biomarkers, and discovering drug targets. This review highlights the potential of MDSP to revolutionize clinical applications, ranging from risk assessment and diagnostics to prognostics, patient monitoring, and the customization of treatment strategies, including clinical trial guidance. We discuss the major MDSP techniques, their applications in breast cancer research, and their integration in clinical practice, addressing both their potential and current limitations. Emphasizing the strategic use of MDSP in risk stratification for women with benign breast disease, we also highlight its transformative potential in reshaping the landscape of breast cancer research and treatment.

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

Breast cancer is a significant cause of morbidity and mortality in women, with over two million new cases reported worldwide each year, the majority of which occur in post-menopausal women. Despite advances in early detection and treatment, approximately one-third of patients diagnosed with breast cancer will develop metastatic disease. The pathogenesis and progression of breast cancer are influenced by a variety of biological and social risk factors, including age, ethnicity, pregnancy status, diet, and genomic alterations. Recent advancements in breast cancer research have focused on harnessing the power of the patient's adaptive and innate immune systems for diagnostic and therapeutic purposes. The breast immune microenvironment plays a critical role in regulating tissue homeostasis and resistance to tumorigenesis. In this review, we explore the dynamic changes in the breast immune microenvironment that occur with age, how these changes impact breast cancer development and progression, and how targeted therapeutic interventions that leverage the immune system can be used to improve patient outcomes. Our review emphasizes the importance of understanding the complex interplay between aging, the immune system, and breast cancer, and highlights the potential of immune-based therapies in the fight against this devastating disease.

Abstract: Nonsteroidal anti-inflammatory agents (NSAID) are associated with modest inconsistent reductions in breast cancer risk in population-based cohorts, whereas two focused studies of patients with benign breast disease (BBD) have found lower risk with NSAID use. Given that BBD includes fibroinflammatory lesions linked to elevated breast cancer risk, we assessed whether NSAID use was associated with lower breast cancer risk among patients with BBD.Participants were postmenopausal women in the Cancer Prevention Study-II (CPS-II), a prospective study of cancer incidence and mortality, who completed follow-up surveys in 1997 with follow-up through June 30, 2015. History of BBD, NSAID use, and covariate data were updated biennially. This analysis included 23,615 patients with BBD and 36,751 patients with non-BBD, including 3,896 incident breast cancers over an average of 12.72 years of follow-up among participants. NSAID use, overall and by formulation, recency, duration, and pills per month was analyzed versus breast cancer risk overall and by BBD status using multivariable-adjusted Cox models; BBD status and NSAID use were modeled as time-dependent exposures.Patients with BBD who reported using NSAIDs experienced lower breast cancer risk (HR, 0.87; 95% CI, 0.78-0.97), with similar effects for estrogen receptor (ER)-positive breast cancers [HR, 0.85; 95% confidence interval (CI), 0.74-0.97] and ER-negative breast cancers (HR, 0.87; 95% CI, 0.59-1.29); among women without BBD, NSAID use was unrelated to risk (HR, 1.02; 95% CI, 0.92-1.13; Pinteraction = 0.04). Associations stratified by age, obesity, menopausal hormone use, and cardiovascular disease were similar.Among patients with BBD, NSAID use appears linked to lower breast cancer risk. Further studies to assess the value of NSAID use among patients with BBD are warranted., Prevention Relevance: We examined whether NSAID use, a modifiable exposure, is associated with breast cancer risk in postmenopausal women from the Cancer Prevention Study-II with self-reported benign breast disease, an often inflammatory condition associated with higher rates of breast cancer., (©2023 American Association for Cancer Research.)

Purpose: Breast cancer risk is elevated in pathogenic germline BRCA 1/2 mutation carriers due to compromised DNA quality control. We hypothesized that if immunosurveillance promotes tumor suppression, then normal/benign breast lobules from BRCA carriers may demonstrate higher immune cell densities., Methods: We assessed immune cell composition in normal/benign breast lobules from age-matched women with progressively increased breast cancer risk, including (1) low risk: 19 women who donated normal breast tissue to the Komen Tissue Bank (KTB) at Indiana University Simon Cancer Center, (2) intermediate risk: 15 women with biopsy-identified benign breast disease (BBD), and (3) high risk: 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 mm 2 were compared between groups using Wilcoxon rank-sum tests., Results: Normal/benign breast lobules from BRCA carriers had significantly higher densities of immune cells/mm 2 compared to KTB normal donors (all p < 0.001): CD8 + 354.4 vs 150.9; CD4 + 116.3 vs 17.7; CD68 + 237.5 vs 57.8; and CD11c + (3.5% vs 0.4% pixels positive). BBD tissues differed from BRCA carriers only in CD8 + cells but had higher densities of CD4 + , CD11c + , and CD68 + immune cells compared to KTB donors., Conclusions: These preliminary analyses show that normal/benign breast lobules of BRCA mutation carriers contain increased immune cells compared with normal donor breast tissues, and BBD tissues appear overall more similar to BRCA carriers., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Obesity is associated with increased breast cancer incidence, recurrence, and mortality. Adipocytes and adipose-derived stem cells (ASCs), two resident cell types in adipose tissue, accelerate the early stages of breast cancer progression. It remains unclear whether obesity plays a role in the subsequent escape of malignant breast cancer cells into the local circulation., Methods: We engineered models of human breast tumors with adipose stroma that exhibited different obesity-specific alterations. We used these models to assess the invasion and escape of breast cancer cells into an empty, blind-ended cavity (as a mimic of a lymphatic vessel) for up to sixteen days., Results: Lean and obese donor-derived adipose stroma hastened escape to similar extents. Moreover, a hypertrophic adipose stroma did not affect the rate of adipose-induced escape. When admixed directly into the model tumors, lean and obese donor-derived ASCs hastened escape similarly., Conclusions: This study demonstrates that the presence of adipose cells, independently of the obesity status of the adipose tissue donor, hastens the escape of human breast cancer cells in multiple models of obesity-associated breast cancer., Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00750-y., Competing Interests: Conflict of interestYoseph W. Dance, Mackenzie C. Obenreder, Alex J. Seibel, Tova Meshulam, Joshua W. Ogony, Nikhil Lahiri, Laura Pacheco-Spann, Derek C. Radisky, Matthew D. Layne, Stephen R. Farmer, Celeste M. Nelson, and Joe Tien declare that they have no conflict of interest., (© The Author(s) under exclusive licence to Biomedical Engineering Society 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Background: Few studies have examined detailed features of pregnancy and the postpartum period as potential risk factors for early onset breast cancer (BC) by molecular subtype. These data may have value for improving risk assessment and prevention., Methods: We surveyed parous enrollees in the prospective Mayo Clinic Breast Disease Registry (MCBDR) who had been diagnosed with BC at age <55 years between 2015 and 2020. Summary statistics were used to describe survey responses and reproductive risk factors by BC subtype (defined by estrogen/progesterone receptors and human epidermal growth factor receptor expression, nurse-abstracted from the medical record). Associations were assessed with Kruskal-Wallis and Chi-Square tests, followed by age-adjusted linear and logistic regression models. We compared results from this parous cohort to those from a separate cohort of nulliparous MCBDR participants with BC diagnosed at age <55 years., Results: In 436 parous respondents with subtype data abstracted, we identified a higher frequency of BRCA1 mutation, earlier age at diagnosis, and lower BI in patients with triple negative BC. Comparing parous to nulliparous young women with breast cancer, the proportion with TNBC was larger in the latter (12.2% vs. 15.1%, p = 0.03)., Conclusions: Early age at diagnosis and deleterious BRCA1 mutation were more frequent among TNBC patients. In addition, parous young women with TNBC had a lower BI than those with other BC subtypes, a hypothesis-generating finding that supports the need for additional research on the cycle of pregnancy-lactation-postpartum involution and BC etiology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Background: Formalin-fixed, paraffin-embedded (FFPE) tissues have many advantages for identification of risk biomarkers, including wide availability and potential for extended follow-up endpoints. However, RNA derived from archival FFPE samples has limited quality. Here we identified parameters that determine which FFPE samples have the potential for successful RNA extraction, library preparation, and generation of usable RNAseq data., Methods: We optimized library preparation protocols designed for use with FFPE samples using seven FFPE and Fresh Frozen replicate pairs, and tested optimized protocols using a study set of 130 FFPE biopsies from women with benign breast disease. Metrics from RNA extraction and preparation procedures were collected and compared with bioinformatics sequencing summary statistics. Finally, a decision tree model was built to learn the relationship between pre-sequencing lab metrics and qc pass/fail status as determined by bioinformatics metrics., Results: Samples that failed bioinformatics qc tended to have low median sample-wise correlation within the cohort (Spearman correlation < 0.75), low number of reads mapped to gene regions (< 25 million), or low number of detectable genes (11,400 # of detected genes with TPM > 4). The median RNA concentration and pre-capture library Qubit values for qc failed samples were 18.9 ng/ul and 2.08 ng/ul respectively, which were significantly lower than those of qc pass samples (40.8 ng/ul and 5.82 ng/ul). We built a decision tree model based on input RNA concentration, input library qubit values, and achieved an F score of 0.848 in predicting QC status (pass/fail) of FFPE samples., Conclusions: We provide a bioinformatics quality control recommendation for FFPE samples from breast tissue by evaluating bioinformatic and sample metrics. Our results suggest a minimum concentration of 25 ng/ul FFPE-extracted RNA for library preparation and 1.7 ng/ul pre-capture library output to achieve adequate RNA-seq data for downstream bioinformatics analysis., (© 2022. The Author(s).)

Background: In the United States, cancer disproportionately impacts Black and African American individuals. Identifying genetic factors underlying cancer disparities has been an important research focus and requires data that are equitable in both quantity and quality across racial groups. It is widely recognized that DNA databases quantitatively underrepresent minorities. However, the differences in data quality between racial groups have not been well studied., Methods: We compared the qualities of germline and tumor exomes between ancestrally African and European patients in The Cancer Genome Atlas of 7 cancers with at least 50 self-reported Black patients in the context of sequencing depth, tumor purity, and qualities of germline variants and somatic mutations., Results: Germline and tumor exomes from ancestrally African patients were sequenced at statistically significantly lower depth in 6 out of the 7 cancers. For 3 cancers, most ancestrally European exomes were sequenced in early sample batches at higher depth, whereas ancestrally African exomes were concentrated in later batches and sequenced at much lower depth. For the other 3 cancers, the reasons of lower sequencing coverage of ancestrally African exomes remain unknown. Furthermore, even when the sequencing depths were comparable, African exomes had disproportionally higher percentages of positions with insufficient coverage, likely because of the known European bias in the human reference genome that impacted exome capture kit design., Conclusions: Overall and positional lower sequencing depths of ancestrally African exomes in The Cancer Genome Atlas led to underdetection and lower quality of variants, highlighting the need to consider epidemiological factors for future genomics studies., (© The Author(s) 2022. Published by Oxford University Press.)

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an important regulator of extracellular matrix turnover that has been traditionally regarded as a potential tumor suppressor owing to its inhibitory effects of matrix metalloproteinases. Intriguingly, this interpretation has been challenged by the consistent observation that increased expression of TIMP-1 is associated with poor prognosis in virtually all cancer types including lung cancer, supporting a tumor-promoting function. However, how TIMP-1 is dysregulated within the tumor microenvironment and how it drives tumor progression in lung cancer is poorly understood. We analyzed the expression of TIMP-1 and its cell surface receptor CD63 in two major lung cancer subtypes: lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), and defined the tumor-promoting effects of their interaction. We found that TIMP-1 is aberrantly overexpressed in tumor-associated fibroblasts (TAFs) in ADC compared to SCC. Mechanistically, TIMP-1 overexpression was mediated by the selective hyperactivity of the pro-fibrotic TGF-β1/SMAD3 pathway in ADC-TAFs. Likewise, CD63 was upregulated in ADC compared to SCC cells. Genetic analyses revealed that TIMP-1 secreted by TGF-β1-activated ADC-TAFs is both necessary and sufficient to enhance growth and invasion of ADC cancer cells in culture, and that tumor cell expression of CD63 was required for these effects. Consistently, in vivo analyses revealed that ADC cells co-injected with fibroblasts with reduced SMAD3 or TIMP-1 expression into immunocompromised mice attenuated tumor aggressiveness compared to tumors bearing parental fibroblasts. We also found that high TIMP1 and CD63 mRNA levels combined define a stronger prognostic biomarker than TIMP1 alone. Our results identify an excessive stromal TIMP-1 within the tumor microenvironment selectively in lung ADC, and implicate it in a novel tumor-promoting TAF-carcinoma crosstalk, thereby pointing to TIMP-1/CD63 interaction as a novel therapeutic target in lung cancer., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among potential targets are secreted proteases, enzymes which in many cancers serve as key drivers of malignant progression. Here, we found that inhibitors of trypsin-like serine proteases suppressed malignant phenotypes of OCCC cell lines. To identify the proteases responsible for malignancy in OCCC, we employed activity-based protein profiling to directly analyze enzyme activity. We developed an activity-based probe featuring an arginine diphenylphosphonate warhead to detect active serine proteases of trypsin-like specificity and a biotin handle to facilitate affinity purification of labeled proteases. Using this probe, we identified active trypsin-like serine proteases within the complex proteomes secreted by OCCC cell lines, including two proteases in common, tissue plasminogen activator and urokinase-type plasminogen activator. Further interrogation of these proteases showed that both were involved in cancer cell invasion and proliferation of OCCC cells and were also detected in in vivo models of OCCC. We conclude the detection of tissue plasminogen activator and urokinase-type plasminogen activator as catalytically active proteases and significant drivers of the malignant phenotype may point to these enzymes as targets for new therapeutic strategies in OCCC. Our activity-based probe and profiling methodology will also serve as a valuable tool for detection of active trypsin-like serine proteases in models of other cancers and other diseases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

Purpose: Breast terminal duct lobular units (TDLUs) are the main source of breast cancer (BC) precursors. Higher serum concentrations of hormones and growth factors have been linked to increased TDLU numbers and to elevated BC risk, with variable effects by menopausal status. We assessed associations of circulating factors with breast histology among premenopausal women using artificial intelligence (AI) and preliminarily tested whether parity modifies associations., Methods: Pathology AI analysis was performed on 316 digital images of H&E-stained sections of normal breast tissues from Komen Tissue Bank donors ages ≤ 45 years to assess 11 quantitative metrics. Associations of circulating factors with AI metrics were assessed using regression analyses, with inclusion of interaction terms to assess effect modification., Results: Higher prolactin levels were related to larger TDLU area (p < 0.001) and increased presence of adipose tissue proximate to TDLUs (p < 0.001), with less significant positive associations for acini counts (p = 0.012), dilated acini (p = 0.043), capillary area (p = 0.014), epithelial area (p = 0.007), and mononuclear cell counts (p = 0.017). Testosterone levels were associated with increased TDLU counts (p < 0.001), irrespective of parity, but associations differed by adipose tissue content. AI data for TDLU counts generally agreed with prior visual assessments., Conclusion: Among premenopausal women, serum hormone levels linked to BC risk were also associated with quantitative features of normal breast tissue. These relationships were suggestively modified by parity status and tissue composition. We conclude that the microanatomic features of normal breast tissue may represent a marker of BC risk., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development. Here, we used a counter-selective screening strategy for directed evolution of yeast-displayed human TIMP-1 to obtain TIMP-1 variants highly selective for the inhibition of MMP-3 in preference over MMP-10. As MMP-3 and MMP-10 are the most similar MMPs in sequence, structure, and function, our results thus clearly demonstrate the capability for engineering full-length TIMP proteins to be highly selective MMP inhibitors. We show using protein crystal structures and models of MMP-3-selective TIMP-1 variants bound to MMP-3 and counter-target MMP-10 how structural alterations within the N-terminal and C-terminal TIMP-1 domains create new favorable and selective interactions with MMP-3 and disrupt unique interactions with MMP-10. While our MMP-3-selective inhibitors may be of interest for future investigation in diseases where this enzyme drives pathology, our platform and screening strategy can be employed for developing selective inhibitors of additional MMPs implicated as therapeutic targets in disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Convolutional neural networks (CNNs) offer the potential to generate comprehensive quantitative analysis of histologic features. Diagnostic reporting of benign breast disease (BBD) biopsies is usually limited to subjective assessment of the most severe lesion in a sample, while ignoring the vast majority of tissue features, including involution of background terminal duct lobular units (TDLUs), the structures from which breast cancers arise. Studies indicate that increased levels of age-related TDLU involution in BBD biopsies predict lower breast cancer risk, and therefore its assessment may have potential value in risk assessment and management. However, assessment of TDLU involution is time-consuming and difficult to standardize and quantitate. Accordingly, we developed a CNN to enable automated quantitative measurement of TDLU involution and tested its performance in 174 specimens selected from the pathology archives at Mayo Clinic, Rochester, MN. The CNN was trained and tested on a subset of 33 biopsies, delineating important tissue types. Nine quantitative features were extracted from delineated TDLU regions. Our CNN reached an overall dice-score of 0.871 (±0.049) for tissue classes versus reference standard annotation. Consensus of four reviewers scoring 705 images for TDLU involution demonstrated substantial agreement with the CNN method (unweighted κappa = 0.747 ± 0.01). Quantitative involution measures showed anticipated associations with BBD histology, breast cancer risk, breast density, menopausal status, and breast cancer risk prediction scores (p < 0.05). Our work demonstrates the potential to improve risk prediction for women with BBD biopsies by applying CNN approaches to generate automated quantitative evaluation of TDLU involution., (© 2022. The Author(s).)

Background: Benign breast disease (BBD) is a risk factor for breast cancer (BC); however, little is known about the genetic alterations present at the time of BBD diagnosis and how these relate to risk of incident BC., Methods: A subset of a long-term BBD cohort was selected to examine DNA variation across three BBD groups (42 future estrogen receptor-positive (ER+) BC, 36 future estrogen receptor-negative (ER-) BC, and 42 controls cancer-free for at least 16 years post-BBD). DNA extracted from archival formalin fixed, paraffin-embedded (FFPE) tissue blocks was analyzed for presence of DNA alterations using a targeted panel of 93 BC-associated genes. To address artifacts frequently observed in FFPE tissues (e.g., C>T changes), we applied three filtering strategies based on alternative allele frequencies and nucleotide substitution context. Gene-level associations were performed using two types of burden tests and adjusted for clinical and technical covariates., Results: After filtering, the variant frequency of SNPs in our sample was highly consistent with population allele frequencies reported in 1 KG/ExAC (0.986, p < 1e-16). The top ten genes found to be nominally associated with later cancer status by four of 12 association methods(p < 0.05) were MED12, MSH2, BRIP1, PMS1, GATA3, MUC16, FAM175A, EXT2, MLH1 and TGFB1, although these were not statistically significant in permutation testing. However, all 10 gene-level associations had OR < 1 with lower mutation burden in controls compared to cases, which was marginally statistically significant in permutation testing (p = 0.04). Comparing between the three case groups, BBD ER+ cases were closer to controls in mutation profile, while BBD ER- cases were distinct. Notably, the variant burden was significantly higher in controls than in either ER+ or ER- cases. CD45 expression was associated with mutational burden (p < 0.001)., Conclusions: Somatic mutations were more frequent in benign breast tissue from women who did not develop cancer, opening questions of clonal diversity or immune-mediated restraint on future cancer development. CD45 expression was positively associated with mutational burden, most strongly in controls. Further studies in both normal and premalignant tissues are needed to better understand the role of somatic gene mutations and their contribution to future cancer development., (© 2021. The Author(s).)

The role of active antitumor immunity in hormone receptor-positive (HR + ) breast cancer has been historically underlooked. The aim of this study was to determine the contribution of the immune system to antiprogestin-induced tumor growth inhibition using a hormone-dependent breast cancer model. BALB/c-GFP + bone marrow (BM) cells were transplanted into immunodeficient NSG mice to generate an immunocompetent NSG/BM-GFP + (NSG-R) mouse model. Treatment with the antiprogestin mifepristone (MFP) inhibited growth of 59-2-HI tumors with similar kinetics in both animal models. Interestingly, MFP treatment reshaped the tumor microenvironment, enhancing the production of proinflammatory cytokines and chemokines. Tumors in MFP-treated immunocompetent mice showed increased infiltration of F4/80 + macrophages, natural killer, and CD8 T cells, displaying a central memory phenotype. Mechanistically, MFP induced immunogenic cell death (ICD) in vivo and in vitro , as depicted by the expression and subcellular localization of the alarmins calreticulin and HMGB-1 and the induction of an ICD gene program. Moreover, MFP-treated tumor cells efficiently activated immature dendritic cells, evidenced by enhanced expression of MHC-II and CD86, and induced a memory T-cell response, attenuating tumor onset and growth after re-challenge. Finally, MFP treatment increased the sensitivity of HR + 59-2-HI tumor to PD-L1 blockade, suggesting that antiprogestins may improve immunotherapy response rates. These results contribute to a better understanding of the mechanisms underlying the antitumor effect of hormonal treatment and the rational design of therapeutic combinations based on endocrine and immunomodulatory agents in HR + breast cancer. SIGNIFICANCE: Antiprogestin therapy induces immunogenic tumor cell death in PRA-overexpressing tumors, eliciting an adaptive immune memory response that protects mice from future tumor recurrence and increases sensitivity to PD-L1 blockade. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/5/1375/F1.large.jpg., (©2020 American Association for Cancer Research.)

Metastasis, the leading cause of mortality in cancer patients, depends upon the ability of cancer cells to invade into the extracellular matrix that surrounds the primary tumor and to escape into the vasculature. To investigate the features of the microenvironment that regulate invasion and escape, we generated solid microtumors of MDA-MB-231 human breast carcinoma cells within gels of type I collagen. The microtumors were formed at defined distances adjacent to an empty cavity, which served as an artificial vessel into which the constituent tumor cells could escape. To define the relative contributions of matrix degradation and cell proliferation on invasion and escape, we used pharmacological approaches to block the activity of matrix metalloproteinases (MMPs) or to arrest the cell cycle. We found that blocking MMP activity prevents both invasion and escape of the breast cancer cells. Surprisingly, blocking proliferation increases the rate of invasion but has no effect on that of escape. We found that arresting the cell cycle increases the expression of MMPs, consistent with the increased rate of invasion. To gain additional insight into the role of cell proliferation in the invasion process, we generated microtumors from cells that express the fluorescent ubiquitination-based cell cycle indicator. We found that the cells that initiate invasions are preferentially quiescent, whereas cell proliferation is associated with the extension of invasions. These data suggest that matrix degradation and cell proliferation are coupled during the invasion and escape of human breast cancer cells and highlight the critical role of matrix proteolysis in governing tumor phenotype., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

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; P trend = 0.003), days used per month (HR = 0.51; 95% CI = 0.33-0.80; P trend = 0.001) and lifetime number of doses taken (HR = 0.53; 95% CI = 0.31-0.89; P trend = 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., (©2020 American Association for Cancer Research.)

The etiology of triple-negative breast cancers (TNBC) is poorly understood. As many TNBCs develop prior to the initiation of breast cancer screening or at younger ages when the sensitivity of mammography is comparatively low, understanding the etiology of TNBCs is critical for discovering novel prevention approaches for these tumors. Furthermore, the higher incidence rate of estrogen receptor-negative breast cancers, and specifically, of TNBCs, among young African American women (AAW) versus white women is a source of racial disparities in breast cancer mortality. Whereas immune responses to TNBCs have received considerable attention in relation to prognosis and treatment, the concept that dysregulated immune responses may predispose to the development of TNBCs has received limited attention. We present evidence that dysregulated immune responses are critical in the pathogenesis of TNBCs, based on the molecular biology of the cancers and the mechanisms proposed to mediate TNBC risk factors. Furthermore, proposed risk factors for TNBC, especially childbearing without breastfeeding, high parity, and obesity, are more prevalent among AAW than white women. Limited data suggest genetic differences in immune responses by race, which favor a stronger Thr type 2 (Th2) immune response among AAW than white women. Th2 responses contribute to wound-healing processes, which are implicated in the pathogenesis of TNBCs. Accordingly, we review data on the link between immune responses and TNBC risk and consider whether the prevalence of risk factors that result in dysregulated immunity is higher among AAW than white women., (©2020 American Association for Cancer Research.)

A major clinical challenge of ovarian cancer is the development of malignant ascites accompanied by widespread peritoneal metastasis. In ovarian clear cell carcinoma (OCCC), a challenging subtype of ovarian cancer, this problem is compounded by near-universal primary chemoresistance; patients with advanced stage OCCC thus lack effective therapies and face extremely poor survival rates. Here we show that tumor-cell-expressed serine protease inhibitor Kazal type 1 (SPINK1) is a key driver of OCCC progression and metastasis. Using cell culture models of human OCCC, we find that shRNA silencing of SPINK1 sensitizes tumor cells to anoikis and inhibits proliferation. Knockdown of SPINK1 in OCCC cells also profoundly suppresses peritoneal metastasis in mouse implantation models of human OCCC. We next identify a novel autocrine signaling axis in OCCC cells whereby tumor-cell-produced interleukin-6 (IL-6) regulates SPINK1 expression to stimulate a common protumorigenic gene expression pattern leading to anoikis resistance and proliferation of OCCC cells. We further demonstrate that this signaling pathway can be successfully interrupted with the IL-6Rα inhibitor tocilizumab, sensitizing cells to anoikis in vitro and reducing metastasis in vivo. These results suggest that clinical trials of IL-6 pathway inhibitors in OCCC may be warranted, and that SPINK1 might offer a candidate predictive biomarker in this population.

Purpose: We quantified cytotoxic T cells in nonmalignant breast tissues from women with and without subsequent breast cancer to assess evidence of whether immunosurveillance may be suppressed prior to tumor development., Methods: We used an age-matched set of breast tissues from women with benign breast disease (BBD) who subsequently developed breast cancer (BBD with later BC), women with BBD who remained cancer free (BBD cancer-free), and normal Komen Tissue Bank (KTB) tissue donors (KTB controls). We evaluated terminal duct lobular units (lobules) for degree of epithelial abnormality and density of dual-positive CD8/CD103 T cells, as CD103+ cells are thought to be a subset of CD8+ cytotoxic T cells located primarily in the intraepithelial compartment., Results: In 10 sets of age-matched women, 256 breast lobules were studied: 85 in BBD women with later BC, 85 in BBD cancer-free women, and 86 in KTB donors. The majority of all lobules were histologically normal (N = 143, 56%), with 65 (25%) nonproliferative fibrocystic change, and 48 (19%) proliferative epithelial change (with or without atypia). In BBD women with later BC, median CD8+/CD103+ cell density was 39.6, 31.7, and 10.5 cells/mm 2 (p = 0.002) for normal, nonproliferative, and proliferative lobules. In BBD cancer-free women, median CD8+/CD103+ cell density values were 46.7, 14.3, and 0 cells/mm 2 (p = 0.004) respectively. In KTB donors, CD8+/CD103+ cell density was not significantly different across the lobule types (medians 0, 5.8, 10.7, p = 0.43)., Conclusion: In women with BBD, breast lobules with increasing epithelial abnormality show significant decreases in cytotoxic T cells as measured by CD8/CD103 staining, suggesting that impaired immunosurveillance may be a component of the earliest stages of breast cancer development.

During branching morphogenesis, a simple cluster of cells proliferates and branches to generate an arborized network that facilitates fluid flow. The overall architecture of the mouse lung is established by domain branching, wherein new branches form laterally off the side of an existing branch. The airway epithelium develops concomitantly with a layer of smooth muscle that is derived from the embryonic mesenchyme. Here, we examined the role of smooth muscle differentiation in shaping emerging domain branches. We found that the position and morphology of domain branches are highly stereotyped, as is the pattern of smooth muscle that differentiates around the base of each branch. Perturbing the pattern of smooth muscle differentiation genetically or pharmacologically causes abnormal domain branching. Loss of smooth muscle results in ectopic branching and decreases branch stereotypy. Increased smooth muscle suppresses branch initiation and extension. Computational modeling revealed that epithelial proliferation is insufficient to generate domain branches and that smooth muscle wrapping is required to shape the epithelium into a branch. Our work sheds light on the physical mechanisms of branching morphogenesis in the mouse lung., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background: Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples., Results: As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different DNA extraction methods. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (< 5%), and collectively shared a "C > T|G > A" mutational signature known to be representative of FFPE artifacts resulting from cytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates., Conclusions: Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

Background: A better definition of biomarkers and biological processes related to local recurrence and disease progression is highly warranted for ductal breast carcinoma in situ (DCIS). Stromal-epithelial interactions are likely of major importance for the biological, clinical, and pathological distinctions between high- and low-risk DCIS cases., Methods: Stromal platelet derived growth factor receptor (PDGFR) was immunohistochemically assessed in two DCIS patient cohorts (n = 458 and n = 80). Cox proportional hazards models were used to calculate the hazard ratios of recurrence. The molecular mechanisms regulating stromal PDGFR expression were investigated in experimental in vitro co-culture systems of DCIS cells and fibroblasts and analyzed using immunoblot and quantitative real-time PCR. Knock-out of JAG1 in DCIS cells and NOTCH2 in fibroblasts was obtained through CRISPR/Cas9. Experimental data were validated by mammary fat pad injection of DCIS and DCIS-JAG1 knock-out cells (10 mice per group). All statistical tests were two-sided., Results: PDGFRα(low)/PDGFRβ(high) fibroblasts were associated with increased risk for recurrence in DCIS (univariate hazard ratio = 1.59, 95% confidence interval [CI] = 1.02 to 2.46; P = .04 Wald test; multivariable hazard ratio = 1.78, 95% CI = 1.07 to 2.97; P = .03). Tissue culture and mouse model studies indicated that this fibroblast phenotype is induced by DCIS cells in a cell contact-dependent manner. Epithelial Jagged1 and fibroblast Notch2 were identified through loss-of-function studies as key juxtacrine signaling components driving the formation of the poor prognosis-associated fibroblast phenotype., Conclusions: A PDGFRα(low)/PDGFRβ(high) fibroblast subset was identified as a marker for high-risk DCIS. The Jagged-1/Notch2/PDGFR stroma-epithelial pathway was described as a novel signaling mechanism regulating this poor prognosis-associated fibroblast subset. In general terms, the study highlights epithelial-stromal crosstalk in DCIS and contributes to ongoing efforts to define clinically relevant fibroblast subsets and their etiology., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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 H&E 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.

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes that contribute to cancer and many inflammatory and degenerative diseases. The TIMP N-terminal domain binds and inhibits an MMP catalytic domain, but the role of the TIMP C-terminal domain in MMP inhibition is poorly understood. Here, we employed yeast surface display for directed evolution of full-length human TIMP-1 to develop MMP-3-targeting ultrabinders. By simultaneously incorporating diversity into both domains, we identified TIMP-1 variants that were up to 10-fold improved in binding MMP-3 compared with WT TIMP-1, with inhibition constants ( K i ) in the low picomolar range. Analysis of individual and paired mutations from the selected TIMP-1 variants revealed cooperative effects between distant residues located on the N- and C-terminal TIMP domains, positioned on opposite sides of the interaction interface with MMP-3. Crystal structures of MMP-3 complexes with TIMP-1 variants revealed conformational changes in TIMP-1 near the cooperative mutation sites. Affinity was strengthened by cinching of a reciprocal "tyrosine clasp" formed between the N-terminal domain of TIMP-1 and proximal MMP-3 interface and by changes in secondary structure within the TIMP-1 C-terminal domain that stabilize interdomain interactions and improve complementarity to MMP-3. Our protein engineering and structural studies provide critical insight into the cooperative function of TIMP domains and the significance of peripheral TIMP epitopes in MMP recognition. Our findings suggest new strategies to engineer TIMP proteins for therapeutic applications, and our directed evolution approach may also enable exploration of functional domain interactions in other protein systems.

Serine proteases have been implicated as key drivers and facilitators of lung cancer malignancy, and while these proteins represent straightforward targets for therapeutic inhibitors, identification of optimal points for intervention has been complicated by the complex networks in which these enzymes function. Here we implicate a signaling pathway consisting of PRSS3/mesotrypsin and kallikrein-related peptidase 5 (KLK5) in lung adenocarcinoma malignancy. We show that elevated PRSS3/mesotrypsin expression is prognostic for poor outcome for patients with lung adenocarcinoma, and that genetic or pharmacologic targeting of PRSS3/mesotrypsin reduces lung adenocarcinoma cell invasiveness and proliferation. We further show that genetic targeting of KLK5, a known target of PRSS3/mesotrypsin, phenocopies the effect of PRSS3/mesotrypsin knockdown, and also that elevated expression of KLK5 is similarly prognostic for outcome in lung adenocarcinoma. Finally, we use transcriptional profiling experiments to show that PRSS3/mesotrypsin and KLK5 control a common malignancy-promoting pathway. These experiments implicate a potential PRSS3/mesotrypsin-KLK5 signaling module in lung adenocarcinoma and reveal the potential therapeutic benefit of selectively targeting these pathways.

Dimethylarginine dimethylaminohydrolases (DDAHs) are known to degrade asymmetric dimethylarginine, an endogenous inhibitor of NOS, and maintain vascular homeostasis; however, the regulatory pathways of DDAHs remain unclear. In this study, we aimed to define the role of transmembrane glycoprotein neuropilin-1 (NRP1) in the expression of DDAHs and investigate the potential roles of NRP1 in regulation of blood pressure. Short hairpin RNA-mediated knockdown of NRP1 reduced the level and mRNA stability of DDAH1 but not DDAH2 in HUVECs, whereas overexpression of NRP1 increased the mRNA stability of DDAH1. Meanwhile, mesenteric arteries and lung vascular endothelial cells of tamoxifen-inducible endothelial cell-specific NRP1 knockout mice exhibited decreased expression of DDAH1 and slightly increased expression of DDAH2. Mechanistically, the regulation of NRP1 on DDAH1 expression is mediated by a posttranscriptional mechanism involving miR-219-5p in HUVECs. Although the endothelial cell-specific NRP1 knockout mice did not exhibit any significant change in blood pressure at the basal level, they were more sensitive to low-dose angiotensin II infusion-induced increases in blood pressure. Our results show that NRP1 is required for full expression of DDAH1 in endothelial cells and that NRP1 contributes to protection from low-dose angiotensin II-induced increases in blood pressure.-Wang, Y., Wang, E., Zhang, Y., Madamsetty, V. S., Ji, B., Radisky, D. C., Grande, J. P., Misra, S., Mukhopadhyay, D. Neuropilin-1 maintains dimethylarginine dimethylaminohydrolase 1 expression in endothelial cells, and contributes to protection from angiotensin II-induced hypertension.

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., (© 2018 American Cancer Society.)

Purpose Women with atypical hyperplasia (AH) on breast biopsy have an aggregate increased risk of breast cancer (BC), but existing risk prediction models do not provide accurate individualized estimates of risk in this subset of high-risk women. Here, we used the Mayo benign breast disease cohort to develop and validate a model of BC risk prediction that is specifically for women with AH, which we have designated as AH-BC. Patients and Methods Retrospective cohorts of women age 18 to 85 years with pathologically confirmed benign AH from Rochester, MN, and Nashville, TN, were used for model development and external validation, respectively. Clinical risk factors and histologic features of the tissue biopsy were selected using L1-penalized Cox proportional hazards regression. Identified features were included in a Fine and Gray regression model to estimate BC risk, with death as a competing risk. Model discrimination and calibration were assessed in the model-building set and an external validation set. Results The model-building set consisted of 699 women with AH, 142 of whom developed BC (median follow-up, 8.1 years), and the external validation set consisted of 461 women with 114 later BC events (median follow-up, 11.4 years). The final AH-BC model included three covariates: age at biopsy, age at biopsy squared, and number of foci of AH. At 10 years, the AH-BC model demonstrated good discrimination (0.63 [95% CI, 0.57 to 0.70]) and calibration (0.87 [95% CI, 0.66 to 1.24]). In the external validation set, the model showed acceptable discrimination (0.59 [95% CI, 0.51 to 0.67]) and calibration (0.91 [95% CI, 0.65 to 1.42]). Conclusion We have created a new model with which to refine BC risk prediction for women with AH. The AH-BC model demonstrates good discrimination and calibration, and it validates in an external data set.

Multinucleation is found in more than one third of tumors and is linked to increased tolerance for mutation, resistance to chemotherapy, and invasive potential. The integrity of the genome depends on proper execution of the cell cycle, which can be altered through mechanotransduction pathways as the tumor microenvironment stiffens during tumorigenesis. Here, we show that signaling downstream of matrix metalloproteinase-3 (MMP3) or TGFβ, known inducers of epithelial-mesenchymal transition (EMT), also promotes multinucleation in stiff microenvironments through Snail-dependent expression of the filament-forming protein septin-6, resulting in midbody persistence, abscission failure, and multinucleation. Consistently, we observed elevated expression of Snail and septin-6 as well as multinucleation in a human patient sample of metaplastic carcinoma of the breast, a rare classification characterized by deposition of collagen fibers and active EMT. In contrast, a soft microenvironment protected mammary epithelial cells from becoming multinucleated by preventing Snail-induced upregulation of septin-6. Our data suggest that tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Significance: These findings reveal tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Cancer Res; 78(9); 2277-89. ©2018 AACR ., (©2018 American Association for Cancer Research.)

In breast adipose tissue, macrophages that encircle damaged adipocytes form "crown-like structures of breast" (CLS-B). Although CLS-B have been associated with breast cancer, their role in benign breast disease (BBD) and early carcinogenesis is not understood. We evaluated breast biopsies from three age-matched groups ( n = 86 each, mean age 55 years), including normal tissue donors of the Susan G. Komen for the Cure Tissue Bank (KTB), and subjects in the Mayo Clinic Benign Breast Disease Cohort who developed cancer (BBD cases) or did not develop cancer (BBD controls, median follow-up 14 years). Biopsies were classified into histologic categories, and CD68-immunostained tissue sections were evaluated for the frequency and density of CLS-B. Our data demonstrate that CLS-B are associated with BBD: CLS-B-positive samples were significantly less frequent among KTB biopsies (3/86, 3.5%) than BBD controls (16/86 = 18.6%, P = 0.01) and BBD cases (21/86 = 24%, P = 0.002). CLS-B were strongly associated with body mass index (BMI); BMI < 25: 7% CLS-B positive, BMI 25-29: 13%, and BMI ≥ 30: 29% ( P = 0.0005). Among BBD biopsies, a high CLS-B count [>5 CLS-B/sample: 10.5% (BBD cases) vs 4.7% (BBD controls), P = 0.007] conferred a breast cancer OR of 6.8 (95% CI, 1.4-32.4), P = 0.02, after adjusting for adipose tissue area (cm 2 ), histologic impression, and BMI. As high CLS-B densities are independently associated with an increased breast cancer risk, they may be a promising histologic marker of breast cancer risk in BBD. Cancer Prev Res; 11(2); 113-9. ©2017 AACR ., (©2017 American Association for Cancer Research.)

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 < 0.001 for each). Among fibrocystic lobules, proliferative/atypical lobules showed significantly lower expression compared to nonproliferative lobules for MICA (p = 0.02) but not for CD56 (p = 0.80)., Conclusion: Levels of CD56+ NK cells and activating ligand MICA were decreased in breast lobules with fibrocystic change, and MICA levels showed a significant stepwise decrease with increasing histopathologic abnormality. MICA levels were also significantly decreased in older subjects, who generally have higher risk of developing cancer. These findings advance a model in which MICA promotes cytotoxic activity in CD56+ NK cells to protect against tumorigenesis in breast lobules, and suggest further research is warranted.

Background: Over 40% of women undergoing breast screening have mammographically dense breasts. Elevated mammographic breast density (MBD) is an established breast cancer risk factor and is known to mask tumors within the dense tissue. However, the association of MBD with high risk benign breast disease (BBD) is unknown., Method: We analyzed data for 3400 women diagnosed with pathologically confirmed BBD in the Mayo Clinic BBD cohort from 1985-2001, with a clinical MBD measure (either parenchymal pattern (PP) or Breast Imaging Reporting and Data Systems (BI-RADS) density) and expert pathology review. Risk factor information was collected from medical records and questionnaires. MBD was dichotomized as dense (PP classification P2 or DY, or BI-RADS classification c or d) or non-dense (PP classification N1 or P1, or BI-RADS classification a or b). Associations of clinical and histologic characteristics with MBD were examined using logistic regression analysis to estimate odds ratios (ORs) with 95% confidence intervals (CIs)., Results: Of 3400 women in the study, 2163 (64%) had dense breasts. Adjusting for age and body mass index (BMI), there were positive associations of dense breasts with use of hormone therapy (HT), lack of lobular involution, presence of atypical lobular hyperplasia (ALH), histologic fibrosis, columnar cell hyperplasia/flat epithelia atypia (CCH/FEA), sclerosing adenosis (SA), cyst, usual ductal hyperplasia, and calcifications. In fully adjusted multivariate models, HT (1.3, 95% CI 1.1-1.5), ALH (1.5, 95% CI 1.0-2.2), lack of lobular involution (OR 1.6, 95% CI 1.2-2.1, compared to complete involution), fibrosis (OR 2.2, 95% CI 1.9-2.6) and CCH/FEA (OR 1.3, 95% CI 1.0-1.6) remained significantly associated with high MBD., Conclusion: Our findings support an association between high risk BBD and high MBD, suggesting that risks associated with the latter may act early in breast carcinogenesis.

Mechanical forces are increasingly recognized to regulate morphogenesis, but how this is accomplished in the context of the multiple tissue types present within a developing organ remains unclear. Here, we use bioengineered 'microfluidic chest cavities' to precisely control the mechanical environment of the fetal lung. We show that transmural pressure controls airway branching morphogenesis, the frequency of airway smooth muscle contraction, and the rate of developmental maturation of the lungs, as assessed by transcriptional analyses. Time-lapse imaging reveals that branching events are synchronized across distant locations within the lung, and are preceded by long-duration waves of airway smooth muscle contraction. Higher transmural pressure decreases the interval between systemic smooth muscle contractions and increases the rate of morphogenesis of the airway epithelium. These data reveal that the mechanical properties of the microenvironment instruct crosstalk between different tissues to control the development of the embryonic lung., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)