Immune cells are increased in normal breast tissues of BRCA1/2 mutation carriers.

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 mm2 were compared between groups using Wilcoxon rank-sum tests. RESULTS: Normal/benign breast lobules from BRCA carriers had significantly higher densities of immune cells/mm2 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.

Quality control recommendations for RNASeq using FFPE samples based on pre-sequencing lab metrics and post-sequencing bioinformatics metrics.

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.

Towards defining morphologic parameters of normal parous and nulliparous breast tissues by artificial intelligence.

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.

Serum hormone levels and normal breast histology among premenopausal women.

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.

Automated quantification of levels of breast terminal duct lobular (TDLU) involution using deep learning.

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.

Somatic mutations in benign breast disease tissues and association with breast cancer risk.

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.

Cytotoxic T cell depletion with increasing epithelial abnormality in women with benign breast disease.

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/mm2 (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/mm2 (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.

Bioinformatics and DNA-extraction strategies to reliably detect genetic variants from FFPE breast tissue samples.

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.

A Soft Microenvironment Protects from Failure of Midbody Abscission and Multinucleation Downstream of the EMT-Promoting Transcription Factor Snail.

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 TGFß, 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.

Macrophagic "Crown-like Structures" Are Associated with an Increased Risk of Breast Cancer in Benign Breast Disease.

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 (cm2), 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.

CD56+ immune cell infiltration and MICA are decreased in breast lobules with fibrocystic changes.

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.

NanoString-based breast cancer risk prediction for women with sclerosing adenosis.

PURPOSE: Sclerosing adenosis (SA), found in » 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.

Postlactational involution biomarkers plasminogen and phospho-STAT3 are linked with active age-related lobular involution.

PURPOSE: Breast terminal duct lobular units undergo two distinctive physiological processes of involution: age-related lobular involution (LI), which is gradual and associated with decreased breast cancer risk, and postlactational involution, which is relatively precipitous, occurs with weaning, and has been associated with potentiation of tumor aggressiveness in animal models. Here we assessed whether markers of postlactational involution are associated with ongoing LI in a retrospective tissue cohort. METHODS: We selected 57 women from the Mayo Clinic Benign Breast Disease Cohort who underwent multiple biopsies and who were average age 48 at initial biopsy. Women were classified as having progressive or non-progressive LI between initial and subsequent biopsy. Serial tissue sections were immunostained for plasminogen, matrix metalloproteinase 9 (MMP-9), phospho-STAT3 (pSTAT3), tenascin C, Ki67, CD44, cytokeratin 14 (CK14), cytokeratin 19 (CK19), and c-myc. All but Ki67 were digitally quantified. Associations between maximal marker expression per sample and progressive versus non-progressive LI were assessed using logistic regression and adjusted for potential confounders. RESULTS: While no biomarker showed statistically significant association with LI progression when evaluated individually, lower expression of pSTAT3 (OR 0.35, 95% CI 0.13-0.82, p = 0.01) and higher expression of plasminogen (OR 2.89, 95% CI 1.14-8.81, p = 0.02) were associated with progressive LI in models simultaneously adjusted for all biomarkers. Sensitivity analyses indicated that the strengthening in association for pSTAT3 and plasminogen with progressive LI was due to collinearity between these two markers. CONCLUSIONS: This is the first study to identify biomarkers of active LI. Our findings that plasminogen and pSTAT3 are significantly associated with LI suggest that they may represent signaling nodes or biomarkers of pathways common to the processes of postlactational involution and LI.

Alterations in the Immune Cell Composition in Premalignant Breast Tissue that Precede Breast Cancer Development.

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.

Tissue Stiffness and Hypoxia Modulate the Integrin-Linked Kinase ILK to Control Breast Cancer Stem-like Cells.

Breast tumors are stiffer and more hypoxic than nonmalignant breast tissue. Here we report that stiff and hypoxic microenvironments promote the development of breast cancer stem-like cells (CSC) through modulation of the integrin-linked kinase ILK. Depleting ILK blocked stiffness and hypoxia-dependent acquisition of CSC marker expression and behavior, whereas ectopic expression of ILK stimulated CSC development under softer or normoxic conditions. Stiff microenvironments also promoted tumor formation and metastasis in ovo, where depleting ILK significantly abrogated the tumorigenic and metastatic potential of invasive breast cancer cells. We further found that the ILK-mediated phenotypes induced by stiff and hypoxic microenvironments are regulated by PI3K/Akt. Analysis of human breast cancer specimens revealed an association between substratum stiffness, ILK, and CSC markers, insofar as ILK and CD44 were expressed in cancer cells located in tumor regions predicted to be stiff. Our results define ILK as a key mechanotransducer in modulating breast CSC development in response to tissue mechanics and oxygen tension. Cancer Res; 76(18); 5277-87. ©2016 AACR.

Standardized measures of lobular involution and subsequent breast cancer risk among women with benign breast disease: a nested case-control study.

Lesser degrees of terminal duct-lobular unit (TDLU) involution predict higher breast cancer risk; however, standardized measures to quantitate levels of TDLU involution have only recently been developed. We assessed whether three standardized measures of TDLU involution, with high intra/inter pathologist reproducibility in normal breast tissue, predict subsequent breast cancer risk among women in the Mayo benign breast disease (BBD) cohort. We performed a masked evaluation of biopsies from 99 women with BBD who subsequently developed breast cancer (cases) after a median of 16.9 years and 145 age-matched controls. We assessed three metrics inversely related to TDLU involution: TDLU count/mm(2), median TDLU span (microns, which approximates acini content), and median category of acini counts/TDLU (0-10; 11-20; 21-30; 31-50; >50). Associations with subsequent breast cancer risk for quartiles (or categories of acini counts) of each of these measures were assessed with multivariable conditional logistic regression to estimate odds ratios (ORs) and 95 % confidence intervals (CI). In multivariable models, women in the highest quartile compared to the lowest quartiles of TDLU counts and TDLU span measures were significantly associated with subsequent breast cancer diagnoses; TDLU counts quartile4 versus quartile1, OR = 2.44, 95 %CI 0.96-6.19, p-trend = 0.02; and TDLU spans, quartile4 versus quartile1, OR = 2.83, 95 %CI = 1.13-7.06, p-trend = 0.03. Significant associations with categorical measures of acini counts/TDLU were also observed: compared to women with median category of <10 acini/TDLU, women with >25 acini counts/TDLU were at significantly higher risk, OR = 3.40, 95 %CI 1.03-11.17, p-trend = 0.032. Women with TDLU spans and TDLU count measures above the median were at further increased risk, OR = 3.75 (95 %CI 1.40-10.00, p-trend = 0.008), compared with women below the median for both of these metrics. Similar results were observed for combinatorial metrics of TDLU acini counts/TDLU, and TDLU count. Standardized quantitative measures of TDLU counts and acini counts approximated by TDLU span measures or visually assessed in categories are independently associated with breast cancer risk. Visual assessment of TDLU numbers and acini content, which are highly reproducible between pathologists, could help identify women at high risk for subsequent breast cancer among the million women diagnosed annually with BBD in the US.

Natural history of age-related lobular involution and impact on breast cancer risk.

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.

Gene signature model for breast cancer risk prediction for women with sclerosing adenosis.

Benign breast disease (BBD) is diagnosed in 1-2 million women/year in the US, and while these patients are known to be at substantially increased risk for subsequent development of breast cancer, existing models for risk assessment perform poorly at the individual level. Here, we describe a DNA-microarray-based transcriptional model for breast cancer risk prediction for patients with sclerosing adenosis (SA), which represent » of all BBD patients. A training set was developed from 86 patients diagnosed with SA, of which 27 subsequently developed cancer within 10 years (cases) and 59 remained cancer-free at 10 years (controls). An diagonal linear discriminate analysis-prediction model for prediction of cancer within 10 years (SA TTC10) was generated from transcriptional profiles of FFPE biopsy-derived RNA. This model was tested on a separate validation case-control set composed of 65 SA patients. The SA TTC10 gene signature model, composed of 35 gene features, achieved a clear and significant separation between case and control with receiver operating characteristic area under the curve of 0.913 in the training set and 0.836 in the validation set. Our results provide the first demonstration that benign breast tissue contains transcriptional alterations that indicate risk of breast cancer development, demonstrating that essential precursor biomarkers of malignancy are present many years prior to cancer development. Furthermore, the SA TTC10 gene signature model, which can be assessed on FFPE biopsies, constitutes a novel prognostic biomarker for patients with SA.

Activation of PI3K/Akt/mTOR signaling in the tumor stroma drives endocrine therapy-dependent breast tumor regression.

Improved efficacy of neoadjuvant endocrine-targeting therapies in luminal breast carcinomas could be achieved with optimal use of pathway targeting agents. In a mouse model of ductal breast carcinoma we identify a tumor regressive stromal reaction that is induced by neoadjuvant endocrine therapy. This reparative reaction is characterized by tumor neovascularization accompanied by infiltration of immune cells and carcinoma-associated fibroblasts that stain for phosphorylated ribosomal protein S6 (pS6), downstream the PI3K/Akt/mTOR pathway. While tumor variants with higher PI3K/Akt/mTOR activity respond well to a combination of endocrine and PI3K/Akt/mTOR inhibitors, tumor variants with lower PI3K/Akt/mTOR activity respond more poorly to the combination therapy than to the endocrine therapy alone, associated with inhibition of stromal pS6 and the reparative reaction. In human breast cancer xenografts we confirm that such differential sensitivity to therapy is primarily determined by the level of PI3K/Akt/mTOR in tumor cells. We further show that the clinical response of breast cancer patients undergoing neoadjuvant endocrine therapy is associated with the reparative stromal reaction. We conclude that tumor level and localization of pS6 are associated with therapeutic response in breast cancer and represent biomarkers to distinguish which tumors will benefit from the incorporation of PI3K/Akt/mTOR inhibitors with neoadjuvant endocrine therapy.

Ki-67 expression in sclerosing adenosis and adjacent normal breast terminal ductal lobular units: a nested case-control study from the Mayo Benign Breast Disease Cohort.

Sclerosing adenosis (SA) increases risk for invasive breast cancer (BC) 2.1 times relative to that in the general population. Here, we sought to evaluate whether the proliferation marker Ki-67 stratifies risk among women with SA. A nested case-control sample of patients with SA was obtained from the Mayo Clinic Benign Breast Disease Cohort. Ki-67 expression was evaluated in SA lesions and in the adjacent normal terminal duct lobular units (TDLU) in women who did (cases, n = 133) or did not (controls, n = 239) develop BC. Ki-67 was scored by intensity and number of positively stained cells per one high-power field (magnification, ×40) (40× HPF) for both SA and normal TDLU. Associations of Ki-67 expression with case-control status were assessed using conditional logistic regression. Higher Ki-67 expression was significantly associated with case-control status in both SA (P = 0.03) and normal background TDLU (P = 0.006). For the SA lesion, >2 average positively stained cells/40× HPF showed an adjusted odds ratio (OR) of 1.9 (95 % CI, 1.1-3.4) compared to samples with an average of ≤2 positively stained cells. For background TDLU, lobules with >2 but ≤6 average positively stained cells showed an adjusted OR of 1.3-1.5, whereas those with an average of >6 positively stained cells had an OR of 2.4 (95 % CI, 1.1-5.3) compared to those with an average of <2 positively stained cells. Among women with SA, increased Ki-67 expression in either the SA lesion or the normal background TDLU carried an approximately twofold increased odds of subsequent BC as compared to lower Ki-67 expression.