Showing papers in "Breast Cancer Research in 2016"

Tumor-associated stromal cells as key contributors to the tumor microenvironment

Abstract: The tumor microenvironment is a heterogeneous population of cells consisting of the tumor bulk plus supporting cells. It is becoming increasingly evident that these supporting cells are recruited by cancer cells from nearby endogenous host stroma and promote events such as tumor angiogenesis, proliferation, invasion, and metastasis, as well as mediate mechanisms of therapeutic resistance. In addition, recruited stromal cells range in type and include vascular endothelial cells, pericytes, adipocytes, fibroblasts, and bone-marrow mesenchymal stromal cells. During normal wound healing and inflammatory processes, local stromal cells change their phenotype to become that of reactive stroma. Under certain conditions, however, tumor cells can co-opt these reactive stromal cells and further transition them into tumor-associated stromal cells (TASCs). These TASCs express higher levels of proteins, including alpha-smooth muscle actin, fibroblast activating protein, and matrix metalloproteinases, compared with their normal, non-reactive counterparts. TASCs are also known to secrete many pro-tumorigenic factors, including IL-6, IL-8, stromal-derived factor-1 alpha, vascular endothelial growth factor, tenascin-C, and matrix metalloproteinases, among others, which recruit additional tumor and pro-tumorigenic cells to the developing microenvironment. Here, we review the current literature pertaining to the origins of recruited host stroma, contributions toward tumor progression, tumor-associated stromal cells, and mechanisms of crosstalk between endogenous host stroma and tumor cells.

Plasma exosome microRNAs are indicative of breast cancer

Abstract: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer.

Cancer stem cell metabolism

Abstract: Cancer is now viewed as a stem cell disease. There is still no consensus on the metabolic characteristics of cancer stem cells, with several studies indicating that they are mainly glycolytic and others pointing instead to mitochondrial metabolism as their principal source of energy. Cancer stem cells also seem to adapt their metabolism to microenvironmental changes by conveniently shifting energy production from one pathway to another, or by acquiring intermediate metabolic phenotypes. Determining the role of cancer stem cell metabolism in carcinogenesis has become a major focus in cancer research, and substantial efforts are conducted towards discovering clinical targets.

Targeting the cyclin-dependent kinases (CDK) 4/6 in estrogen receptor-positive breast cancers

Abstract: Despite significant advances in early detection and treatment, breast cancer still remains a major cause of morbidity and mortality for women. Our understanding of the molecular heterogeneity of the disease has significantly expanded over the past decade and the role of cell cycle signaling in both breast cancer oncogenesis and anti-estrogen resistance has gained increasing attention. The mammalian cell cycle is driven by a complex interplay between cyclins and their associated cyclin-dependent kinase (CDK) partners, and dysregulation of this process is one of the hallmarks of cancer. Despite this, initial results with broadly acting CDK inhibitors were largely disappointing. However, recent preclinical and phase I/II clinical studies using a novel, oral, reversible CDK4/6 inhibitor, palbociclib (PD-0332991), have validated the role of CDK4/6 as a potential target in estrogen receptor-positive (ER+) breast cancers. This review highlights our current understanding of CDK signaling in both normal and malignant breast tissues, with special attention placed on recent clinical advances in inhibition of CDK4/6 in ER+ disease.

Breast cancer brain metastases: biology and new clinical perspectives.

Abstract: Because of improvements in the treatment of patients with metastatic breast cancer, the development of brain metastases (BM) has become a major limitation of life expectancy and quality of life for many breast cancer patients. The improvement of management strategies for BM is thus an important clinical challenge, especially among high-risk patients such as human epidermal growth factor receptor 2-positive and triple-negative patients. However, the formation of BM as a multistep process is thus far poorly understood. To grow in the brain, single tumor cells must pass through the tight blood–brain barrier (BBB). The BBB represents an obstacle for circulating tumor cells entering the brain, but it also plays a protective role against immune cell and toxic agents once metastatic cells have colonized the cerebral compartment. Furthermore, animal studies have shown that, after passing the BBB, the tumor cells not only require close contact with endothelial cells but also interact closely with many different brain residential cells. Thus, in addition to a genetic predisposition of the tumor cells, cellular adaptation processes within the new microenvironment may also determine the ability of a tumor cell to metastasize. In this review, we summarize the biology of breast cancer that has spread into the brain and discuss the implications for current and potential future treatment strategies.

Lymphocyte depletion and repopulation after chemotherapy for primary breast cancer

Abstract: Approximately 30 % of breast cancer patients receive chemotherapy, yet little is known about influences of current regimens on circulating lymphocyte levels and phenotypes. Similarly, clinico-pathological factors that modify these influences, and implications for future immune health remain mainly unexplored. We used flow-cytometry to assess circulating lymphocyte levels and phenotypes in 88 primary breast cancer patients before chemotherapy and at time-points from 2 weeks to 9 months after chemotherapy completion. We examined circulating titres of antibodies against pneumococcal and tetanus antigens using ELISAs. Levels of B, T and NK cells were significantly reduced 2 weeks after chemotherapy (p < 0.001). B cells demonstrated particularly dramatic depletion, falling to 5.4 % of pre-chemotherapy levels. Levels of all cells recovered to some extent, although B and CD4+ T cells remained significantly depleted even 9 months post-chemotherapy (p < 0.001). Phenotypes of repopulating B and CD4+ T cells were significantly different from, and showed no sign of returning to pre-chemotherapy profiles. Repopulating B cells were highly depleted in memory cells, with proportions of memory cells falling from 38 % to 10 % (p < 0.001). Conversely, repopulating CD4+ T cells were enriched in memory cells, which increased from 63 % to 75 % (p < 0.001). Differences in chemotherapy regimen and patient smoking were associated with significant differences in depletion extent or repopulation dynamics. Titres of anti-pneumococcal and anti-tetanus antibodies were both significantly reduced post-chemotherapy and did not recover during the study (p < 0.001). Breast cancer chemotherapy is associated with long-term changes in immune parameters that should be considered during clinical management.

Comprehensive transcriptome analysis identifies novel molecular subtypes and subtype-specific RNAs of triple-negative breast cancer

Abstract: Triple-negative breast cancer (TNBC) is a highly heterogeneous group of cancers, and molecular subtyping is necessary to better identify molecular-based therapies. While some classifiers have been established, no one has integrated the expression profiles of long noncoding RNAs (lncRNAs) into such subtyping criterions. Considering the emerging important role of lncRNAs in cellular processes, a novel classification integrating transcriptome profiles of both messenger RNA (mRNA) and lncRNA would help us better understand the heterogeneity of TNBC. Using human transcriptome microarrays, we analyzed the transcriptome profiles of 165 TNBC samples. We used k-means clustering and empirical cumulative distribution function to determine optimal number of TNBC subtypes. Gene Ontology (GO) and pathway analyses were applied to determine the main function of the subtype-specific genes and pathways. We conducted co-expression network analyses to identify interactions between mRNAs and lncRNAs. All of the 165 TNBC tumors were classified into four distinct clusters, including an immunomodulatory subtype (IM), a luminal androgen receptor subtype (LAR), a mesenchymal-like subtype (MES) and a basal-like and immune suppressed (BLIS) subtype. The IM subtype had high expressions of immune cell signaling and cytokine signaling genes. The LAR subtype was characterized by androgen receptor signaling. The MES subtype was enriched with growth factor signaling pathways. The BLIS subtype was characterized by down-regulation of immune response genes, activation of cell cycle, and DNA repair. Patients in this subtype experienced worse recurrence-free survival than others (log rank test, P = 0.045). Subtype-specific lncRNAs were identified, and their possible biological functions were predicted using co-expression network analyses. We developed a novel TNBC classification system integrating the expression profiles of both mRNAs and lncRNAs and determined subtype-specific lncRNAs that are potential biomarkers and targets. If further validated in a larger population, our novel classification system could facilitate patient counseling and individualize treatment of TNBC.

Efficacy and safety of palbociclib in combination with letrozole as first-line treatment of ER-positive, HER2-negative, advanced breast cancer: expanded analyses of subgroups from the randomized pivotal trial PALOMA-1/TRIO-18.

Abstract: Palbociclib is an oral small-molecule inhibitor of cyclin-dependent kinases 4 and 6. In the randomized, open-label, phase II PALOMA-1/TRIO-18 trial, palbociclib in combination with letrozole improved progression-free survival (PFS) compared with letrozole alone as first-line treatment of estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative, advanced breast cancer (20.2 months versus 10.2 months; hazard ratio (HR) = 0.488, 95 % confidence interval (CI) 0.319–0.748; one-sided p = 0.0004). Grade 3–4 neutropenia was the most common adverse event (AE) in the palbociclib + letrozole arm. We now present efficacy and safety analyses based on several specific patient and tumor characteristics, and present in detail the clinical patterns of neutropenia observed in the palbociclib + letrozole arm of the overall safety population. Postmenopausal women (n = 165) with ER+, HER2-negative, advanced breast cancer who had not received any systemic treatment for their advanced disease were randomized 1:1 to receive either palbociclib in combination with letrozole or letrozole alone. Treatment continued until disease progression, unacceptable toxicity, consent withdrawal, or death. The primary endpoint was PFS. We now analyze the difference in PFS for the treatment populations by subgroups, including age, histological type, history of prior neoadjuvant/adjuvant systemic treatment, and sites of distant metastasis, using the Kaplan-Meier method. HR and 95 % CI are derived from a Cox proportional hazards regression model. A clinically meaningful improvement in median PFS and clinical benefit response (CBR) rate was seen with palbociclib + letrozole in every subgroup evaluated. Grade 3–4 neutropenia was the most common AE with palbociclib + letrozole in all subgroups. Analysis of the frequency of neutropenia by grade during the first six cycles of treatment showed that there was a downward trend in Grade 3–4 neutropenia over time. Among those who experienced Grade 3–4 neutropenia, 71.7 % had no overlapping infections of any grade and none had overlapping Grade 3–4 infections. The magnitude of clinical benefit seen with the addition of palbociclib to letrozole in improving both median PFS and CBR rate is consistent in nearly all subgroups analyzed, and consistent with that seen in the overall study population. The safety profile of the combination treatment in all subgroups was also comparable to that in the overall safety population of the study.

In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis

Abstract: Background Although recent models suggest that the detection of Circulating Tumor Cells (CTC) in epithelial-to-mesenchymal transition (EM CTC) might be related to disease progression in metastatic breast cancer (MBC) patients, current detection methods are not efficient in identifying this subpopulation of cells. Furthermore, the possible association of EM CTC with both clinicopathological features and prognosis of MBC patients has still to be demonstrated. Aims of this study were: first, to optimize a DEPArray-based protocol meant to identify, quantify and sort single, viable EM CTC and, subsequently, to test the association of EM CTC frequency with clinical data.

Beyond breast density: a review on the advancing role of parenchymal texture analysis in breast cancer risk assessment

Abstract: The assessment of a woman’s risk for developing breast cancer has become increasingly important for establishing personalized screening recommendations and forming preventive strategies. Studies have consistently shown a strong relationship between breast cancer risk and mammographic parenchymal patterns, typically assessed by percent mammographic density. This paper will review the advancing role of mammographic texture analysis as a potential novel approach to characterize the breast parenchymal tissue to augment conventional density assessment in breast cancer risk estimation. The analysis of mammographic texture provides refined, localized descriptors of parenchymal tissue complexity. Currently, there is growing evidence in support of textural features having the potential to augment the typically dichotomized descriptors (dense or not dense) of area or volumetric measures of breast density in breast cancer risk assessment. Therefore, a substantial research effort has been devoted to automate mammographic texture analysis, with the aim of ultimately incorporating such quantitative measures into breast cancer risk assessment models. In this paper, we review current and emerging approaches in this field, summarizing key methodological details and related studies using novel computerized approaches. We also discuss research challenges for advancing the role of parenchymal texture analysis in breast cancer risk stratification and accelerating its clinical translation. The objective is to provide a comprehensive reference for researchers in the field of parenchymal pattern analysis in breast cancer risk assessment, while indicating key directions for future research.

Selective elimination of breast cancer surgery in exceptional responders: historical perspective and current trials

Abstract: With improvements in chemotherapy regimens, targeted therapies, and our fundamental understanding of the relationship of tumor subtype and pathologic complete response (pCR), there has been dramatic improvement in pCR rates in the past decade, especially among triple-negative and human epidermal growth factor receptor 2-positive breast cancers. Rates of pCR in these groups of patients can be in the 60 % range and thus question the paradigm for the necessity of breast and nodal surgery in all cases, particularly when the patient will be receiving adjuvant local therapy with radiotherapy. Current practice for patients who respond well to neoadjuvant chemotherapy (NCT) is often to proceed with the same breast and axillary procedures as would have been offered women who had not received NCT, regardless of the apparent clinical response. Given these high response rates in defined subgroups among exceptional responders it is appropriate to question whether surgery is now a redundant procedure in their overall management. Further, definitive radiation without surgical resection with or without systemic therapy has been proven effective for several other malignant disease sites including some stages of esophageal, anal, laryngeal, prostate, cervical, and lung carcinoma. The main impediments for potential elimination of surgery have been the fact that prior and current standard and functional breast imaging methods are incapable of accurate prediction of residual disease and that integrating percutaneous biopsy of the breast primary and nodes following NCT may circumvent this issue. This article highlights historical attempts at omission of surgery following NCT in an earlier era, the current status of breast and nodal imaging to predict residual carcinoma, and ongoing and planned trials designed to identify appropriate patients who might be selected for clinical trials designed to test the safety of selected elimination of breast cancer surgery in percutaneous image-guided biopsy-proven exceptional responders to NCT.

Cholesterol biosynthesis pathway as a novel mechanism of resistance to estrogen deprivation in estrogen receptor-positive breast cancer.

Abstract: Therapies targeting estrogenic stimulation in estrogen receptor-positive (ER+) breast cancer (BC) reduce mortality, but resistance remains a major clinical problem. Molecular studies have shown few high-frequency mutations to be associated with endocrine resistance. In contrast, expression profiling of primary ER+ BC samples has identified several promising signatures/networks for targeting. To identify common adaptive mechanisms associated with resistance to aromatase inhibitors (AIs), we assessed changes in global gene expression during adaptation to long-term estrogen deprivation (LTED) in a panel of ER+ BC cell lines cultured in 2D on plastic (MCF7, T47D, HCC1428, SUM44 and ZR75.1) or in 3D on collagen (MCF7) to model the stromal compartment. Furthermore, dimethyl labelling followed by LC-MS/MS was used to assess global changes in protein abundance. The role of target genes/proteins on proliferation, ER-mediated transcription and recruitment of ER to target gene promoters was analysed. The cholesterol biosynthesis pathway was the common upregulated pathway in the ER+ LTED but not the ER– LTED cell lines, suggesting a potential mechanism dependent on continued ER expression. Targeting the individual genes of the cholesterol biosynthesis pathway with siRNAs caused a 30–50 % drop in proliferation. Further analysis showed increased expression of 25-hydroxycholesterol (HC) in the MCF7 LTED cells. Exogenous 25-HC or 27-HC increased ER-mediated transcription and expression of the endogenous estrogen-regulated gene TFF1 in ER+ LTED cells but not in the ER– LTED cells. Additionally, recruitment of the ER and CREB-binding protein (CBP) to the TFF1 and GREB1 promoters was increased upon treatment with 25-HC and 27-HC. In-silico analysis of two independent studies of primary ER+ BC patients treated with neoadjuvant AIs showed that increased expression of MSMO1, EBP, LBR and SQLE enzymes, required for cholesterol synthesis and increased in our in-vitro models, was significantly associated with poor response to endocrine therapy. Taken together, these data provide support for the role of cholesterol biosynthesis enzymes and the cholesterol metabolites, 25-HC and 27-HC, in a novel mechanism of resistance to endocrine therapy in ER+ BC that has potential as a therapeutic target.

Increased peri-ductal collagen micro-organization may contribute to raised mammographic density.

Abstract: Background High mammographic density is a therapeutically modifiable risk factor for breast cancer. Although mammographic density is correlated with the relative abundance of collagen-rich fibroglandular tissue, the causative mechanisms, associated structural remodelling and mechanical consequences remain poorly defined. In this study we have developed a new collaborative bedside-to-bench workflow to determine the relationship between mammographic density, collagen abundance and alignment, tissue stiffness and the expression of extracellular matrix organising proteins.

Circadian clocks and breast cancer.

Abstract: Circadian clocks respond to environmental time cues to coordinate 24-hour oscillations in almost every tissue of the body. In the breast, circadian clocks regulate the rhythmic expression of numerous genes. Disrupted expression of circadian genes can alter breast biology and may promote cancer. Here we overview circadian mechanisms, and the connection between the molecular clock and breast biology. We describe how disruption of circadian genes contributes to cancer via multiple mechanisms, and link this to increased tumour risk in women who work irregular shift patterns. Understanding the influence of circadian rhythms on breast cancer could lead to more efficacious therapies, reformed public health policy and improved patient outcome.

Breast cancer biologic and etiologic heterogeneity by young age and menopausal status in the Carolina Breast Cancer Study: a case-control study

Abstract: Young-onset breast cancer (<40 years) is associated with worse prognosis and higher mortality. Breast cancer risk factors may contribute to distinct tumor biology and distinct age at onset, but understanding of these relationships has been hampered by limited representation of young women in epidemiologic studies and may be confounded by menopausal status. We examined tumor characteristics and epidemiologic risk factors associated with premenopausal women’s and young women’s breast cancer in phases I–III of the Carolina Breast Cancer Study (5309 cases, 2022 control subjects). Unconditional logistic regression was used to assess heterogeneity by age (<40 vs. ≥40 years) and menopausal status. In both premenopausal and postmenopausal strata, younger women had more aggressive disease, including higher stage, hormone receptor-negative, disease as well as increased frequency of basal-like subtypes, lymph node positivity, and larger tumors. Higher waist-to-hip ratio was associated with reduced breast cancer risk among young women but with elevated risk among older women. Parity was associated with increased risk among young women and reduced risk among older women, while breastfeeding was more strongly protective for young women. Longer time since last birth was protective for older women but not for young women. In comparison, when we stratified by age, menopausal status was not associated with distinct risk factor or tumor characteristic profiles, except for progesterone receptor status, which was more commonly positive among premenopausal women. Age is a key predictor of breast cancer biologic and etiologic heterogeneity and may be a stronger determinant of heterogeneity than menopausal status. Young women’s breast cancer appears to be etiologically and biologically distinct from that among older women.

COX-2 modulates mammary tumor progression in response to collagen density

Abstract: High breast density is linked to an increased risk of breast cancer, and correlates with changes in collagen. In a mouse model of mammary carcinoma in the context of increased collagen deposition, the MMTV-PyMT/Col1a1 tm1jae , there is accelerated mammary tumor formation and progression. Previous gene expression analysis suggests that increased collagen density elevates expression of PTGS2 (prostaglandin-endoperoxide synthase 2), the gene for cyclooxygenase-2 (COX-2). To understand the role of COX-2 in tumor progression within a collagen-dense microenvironment, we treated MMTV-PyMT or MMTV-PyMT/Col1a1 tm1jae tumors prior to and after tumor formation. Animals received treatment with celecoxib, a specific COX-2 inhibitor, or placebo. Mammary tumors were examined for COX-2, inflammatory and stromal cell components, and collagen deposition through immunohistochemical analysis, immunofluorescence, multiplex cytokine ELISA and tissue imaging techniques. PyMT/Col1a1 tm1jae tumors were larger, more proliferative, and expressed higher levels of COX-2 and PGE2 than PyMT tumors in wild type (WT) mice. Treatment with celecoxib significantly decreased the induced tumor size and metastasis of the PyMT/Col1a1 tumors, such that their size was not different from the smaller PyMT tumors. Celecoxib had minimal effect on the PyMT tumors. Celecoxib decreased expression levels of COX-2, PGE2, and Ki-67. Several cytokines were over-expressed in PyMT/Col1a1 compared to PyMT, and celecoxib treatment prevented their over-expression. Furthermore, macrophage and neutrophil recruitment were enhanced in PyMT/Col1a1 tumors, and this effect was inhibited by celecoxib. Notably, COX-2 inhibition reduced overall collagen deposition. Finally, when celecoxib was used prior to tumor formation, PyMT/Col1a1 tumors were fewer and smaller than in untreated animals. These findings suggest that COX-2 has a direct role in modulating tumor progression in tumors arising within collagen-dense microenvironments, and suggest that COX-2 may be an effective therapeutic target for women with dense breast tissue and early-stage breast cancer.

Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods.

Abstract: High-resolution 3D imaging of intact tissue facilitates cellular and subcellular analyses of complex structures within their native environment. However, difficulties associated with immunolabelling and imaging fluorescent proteins deep within whole organs have restricted their applications to thin sections or processed tissue preparations, precluding comprehensive and rapid 3D visualisation. Several tissue clearing methods have been established to circumvent issues associated with depth of imaging in opaque specimens. The application of these techniques to study the elaborate architecture of the mouse mammary gland has yet to be investigated. Multiple tissue clearing methods were applied to intact virgin and lactating mammary glands, namely 3D imaging of solvent-cleared organs, see deep brain (seeDB), clear unobstructed brain imaging cocktails (CUBIC) and passive clarity technique. Using confocal, two-photon and light sheet microscopy, their compatibility with whole-mount immunofluorescent labelling and 3D imaging of mammary tissue was examined. In addition, their suitability for the analysis of mouse mammary tumours was also assessed. Varying degrees of optical transparency, tissue preservation and fluorescent signal conservation were observed between the different clearing methods. SeeDB and CUBIC protocols were considered superior for volumetric fluorescence imaging and whole-mount histochemical staining, respectively. Techniques were compatible with 3D imaging on a variety of platforms, enabling visualisation of mammary ductal and lobulo-alveolar structures at vastly improved depths in cleared tissue. The utility of whole-organ tissue clearing protocols was assessed in the mouse mammary gland. Most methods utilised affordable and widely available reagents, and were compatible with standard confocal microscopy. These techniques enable high-resolution, 3D imaging and phenotyping of mammary cells and tumours in situ, and will significantly enhance our understanding of both normal and pathological mammary gland development.

Growth of human breast tissues from patient cells in 3D hydrogel scaffolds.

Abstract: Three-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells. Primary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue growth was monitored over time using bright-field and fluorescence microscopy, and maturation was assessed using morphological metrics and by immunostaining for markers of stem cells and differentiated cell types. The hydrogel tissues were also studied by fabricating physical models from confocal images using a 3D printer. When seeded into these 3D hydrogels, primary human breast epithelial cells rapidly self-organized in the absence of stromal cells and within 2 weeks expanded to form mature mammary tissues. The mature tissues contained luminal, basal, and stem cells in the correct topological orientation and also exhibited the complex ductal and lobular morphologies observed in the human breast. The expanded tissues became hollow when treated with estrogen and progesterone, and with the further addition of prolactin produced lipid droplets, indicating that they were responding to hormones. Ductal branching was initiated by clusters of cells expressing putative mammary stem cell markers, which subsequently localized to the leading edges of the tissue outgrowths. Ductal elongation was preceded by leader cells that protruded from the tips of ducts and engaged with the extracellular matrix. These 3D hydrogel scaffolds support the growth of complex mammary tissues from primary patient-derived cells. We anticipate that this culture system will empower future studies of human mammary gland development and biology.

An immune stratification reveals a subset of PD-1/LAG-3 double-positive triple-negative breast cancers.

Abstract: Stromal tumor-infiltrating lymphocytes (TILs) are a robust prognostic factor in triple-negative breast cancer (TNBC). However, the clinical significance of TILs may be influenced by the complex landscape of the tumor immune microenvironment. In this study, we aimed to evaluate the composition and the functionality of lymphocytic infiltration and checkpoint receptors in TNBC. Formalin-fixed, paraffin-embedded tissues were retrospectively collected from a cohort of patients with early-stage TNBC treated with adjuvant anthracycline-based chemotherapy (n = 259). Results were validated in an independent cohort of patients with TNBC (n = 104). Stromal TILs were evaluated on hematoxylin-and-eosin-stained sections. The density of CD4+, CD8+, and FOXP3+ lymphocytes, and the expression of the immune checkpoints PD-1 and LAG-3, were assessed by immunohistochemical analysis. The presence of elevated TILs positively correlated with the density of all T cell subtypes, especially cytotoxic CD8+ lymphocytes. We showed that increasing stromal TILs assessed as a continuous variable is an independent prognostic marker of prolonged relapse-free survival and overall survival in TNBC. Among immune subpopulations, CD8+ lymphocytes are the main effectors of anti-tumor immune responses. In two independent cohorts, we found that PD-1 and LAG-3 were concurrently expressed in approximately 15% of patients with TNBC. The expression of both checkpoint receptors positively correlated with the presence of TILs, but was not significantly associated with patient outcome. Overall, our data indicate that the evaluation of stromal TILs remains the most reliable immune prognostic marker in TNBC, and support the clinical evaluation of anti-PD-1/PD-L1 and anti-LAG-3 in a subset of patients with TNBC who have concurrent expression of both checkpoint receptors.

Immune response in breast cancer brain metastases and their microenvironment: the role of the PD-1/PD-L axis

Abstract: A better understanding of immune response in breast cancer brain metastases (BCBM) may prompt new preventive and therapeutic strategies. Immunohistochemical expression of stromal tumor-infiltrating lymphocytes (TILs: CD4, CD8, CTLA4), macrophage/microglial cells (CD68), programmed cell death protein 1 receptor (PD-1), programmed cell death protein 1 receptor ligand (PD-L)1, PD-L2 and glial fibrillary acid protein was assessed in 84 BCBM and their microenvironment. Median survival after BCBM excision was 18.3 months (range 0–99). Median number of CD4+, CD8+ TILs and CD68+ was 49, 69 and 76 per 1 mm2, respectively. PD-L1 and PD-L2 expression in BCBM was present in 53 % and 36 % of cases, and was not related to BCBM phenotype. PD-1 expression on TILs correlated positively with CD4+ and CD8+ TILs (r = 0.26 and 0.33), and so did CD68+ (r = 0.23 and 0.27, respectively). In the multivariate analysis, survival after BCBM excision positively correlated with PD-1 expression on TILs (hazard ratio (HR) = 0.3, P = 0.003), CD68+ infiltration (HR = 0.2, P < 0.001), brain radiotherapy (HR = 0.1, P < 0.001), endocrine therapy (HR = 0.1, P < 0.001), and negatively with hormone-receptor-negative/human epidermal growth factor receptor 2 (HER2)-positive phenotype of primary tumor (HR = 2.6, P = 0.01), HER2 expression in BCBM (HR = 4.9, P = 0.01). PD-L1 and PD-L2 expression is a common occurrence in BCBM, irrespective of primary tumor and BCBM phenotype. Favorable prognostic impact of PD-1 expression on TILs suggests a beneficial effect of preexisting immunity and implies a potential therapeutic role of immune checkpoint inhibitors in BCBM.

Male breast cancer in BRCA1 and BRCA2 mutation carriers: Pathology data from the Consortium of Investigators of Modifiers of BRCA1/2

Abstract: BRCA1 and, more commonly, BRCA2 mutations are associated with increased risk of male breast cancer (MBC). However, only a paucity of data exists on the pathology of breast cancers (BCs) in men with BRCA1/2 mutations. Using the largest available dataset, we determined whether MBCs arising in BRCA1/2 mutation carriers display specific pathologic features and whether these features differ from those of BRCA1/2 female BCs (FBCs). We characterised the pathologic features of 419 BRCA1/2 MBCs and, using logistic regression analysis, contrasted those with data from 9675 BRCA1/2 FBCs and with population-based data from 6351 MBCs in the Surveillance, Epidemiology, and End Results (SEER) database. Among BRCA2 MBCs, grade significantly decreased with increasing age at diagnosis (P = 0.005). Compared with BRCA2 FBCs, BRCA2 MBCs were of significantly higher stage (P for trend = 2 × 10−5) and higher grade (P for trend = 0.005) and were more likely to be oestrogen receptor–positive [odds ratio (OR) 10.59; 95 % confidence interval (CI) 5.15–21.80] and progesterone receptor–positive (OR 5.04; 95 % CI 3.17–8.04). With the exception of grade, similar patterns of associations emerged when we compared BRCA1 MBCs and FBCs. BRCA2 MBCs also presented with higher grade than MBCs from the SEER database (P for trend = 4 × 10−12). On the basis of the largest series analysed to date, our results show that BRCA1/2 MBCs display distinct pathologic characteristics compared with BRCA1/2 FBCs, and we identified a specific BRCA2-associated MBC phenotype characterised by a variable suggesting greater biological aggressiveness (i.e., high histologic grade). These findings could lead to the development of gender-specific risk prediction models and guide clinical strategies appropriate for MBC management.

British Society of Breast Radiology Annual Scientific Meeting 2016 : Manchester, UK. 6-8 November 2016

Abstract: Jennifer Ferguson2, Guy Stevens1, Robert Hills2, Kate Gower Thomas1 1Breast Test Wales, Cardiff, UK; 2Cardiff University School of Medicine, Cardiff, UK Correspondence: Jennifer Ferguson Introduction To determine the rate and classification of interval breast cancer (IC) in the two years preceeding and following the introduction of digital mammography in 2011 in a national screening programme. Methods Two 2-year bands were used to ensure completed data for pre and post digital groups. Retrospective analysis of a prospectively collected database of IC was undertaken, identifying women who had undergone screening in the two years prior to and after the introduction of digital screening mammography (1.1.10 until 31.12.11 and 1.1.12 until 31.12.13). Chi squared analysis was used to compare the rate between the two groups and the rate of false negative (FN) and minimal signs (MS) categories considered together. Results Of 87,868 screened before digital between 1.1.10 and 31.12.11, 103 developed IC (0.12%) and of 100,672 screened with digital between 1.1.12 and 31.12.13, 145 developed IC (0.14%) (p = 0.12). Those women with FN and MS interval cancer numbered 17 (16.5%) predigital and 41 (28.3%) with digital; (p = 0.04). The number of true IC was similar in the pre (49.5%) and post (46.9%) groups. Conclusion The rate if IC is similar to the NHSBSP standard (1.2 per 1000 women screened). Despite earlier groups indicating that digital technology will lessen the number of IC, especially those ‘missed’ (FN and MS groups) which are often asymmetric densities and poorly defined masses, this has not been found in our study. The reasons for this will be discussed and examples will be shown.

Circulating cell-free DNA-based epigenetic assay can detect early breast cancer

Abstract: Circulating cell-free DNA (cfDNA) has recently been recognized as a resource for biomarkers of cancer progression, treatment response, and drug resistance. However, few have demonstrated the usefulness of cfDNA for early detection of cancer. Although aberrant DNA methylation in cfDNA has been reported for more than a decade, its diagnostic accuracy remains unsatisfactory for cancer screening. Thus, the aim of the present study was to develop a highly sensitive cfDNA-based system for detection of primary breast cancer (BC) using epigenetic biomarkers and digital PCR technology. Array-based genome-wide DNA methylation analysis was performed using 56 microdissected breast tissue specimens, 34 cell lines, and 29 blood samples from healthy volunteers (HVs). Epigenetic markers for BC detection were selected, and a droplet digital methylation-specific PCR (ddMSP) panel with the selected markers was established. The detection model was constructed by support vector machine and evaluated using cfDNA samples. The methylation array analysis identified 12 novel epigenetic markers (JAK3, RASGRF1, CPXM1, SHF, DNM3, CAV2, HOXA10, B3GNT5, ST3GAL6, DACH1, P2RX3, and chr8:23572595) for detecting BC. We also selected four internal control markers (CREM, GLYATL3, ELMOD3, and KLF9) that were identified as infrequently altered genes using a public database. A ddMSP panel using these 16 markers was developed and detection models were constructed with a training dataset containing cfDNA samples from 80 HVs and 87 cancer patients. The best detection model adopted four methylation markers (RASGRF1, CPXM1, HOXA10, and DACH1) and two parameters (cfDNA concentration and the mean of 12 methylation markers), and, and was validated in an independent dataset of 53 HVs and 58 BC patients. The area under the receiver operating characteristic curve for cancer-normal discrimination was 0.916 and 0.876 in the training and validation dataset, respectively. The sensitivity and the specificity of the model was 0.862 (stages 0-I 0.846, IIA 0.862, IIB-III 0.818, metastatic BC 0.935) and 0.827, respectively. Our epigenetic-marker-based system distinguished BC patients from HVs with high accuracy. As detection of early BC using this system was comparable with that of mammography screening, this system would be beneficial as an optional method of screening for BC.

Intratumoral heterogeneity as a source of discordance in breast cancer biomarker classification

Abstract: Spatial heterogeneity in biomarker expression may impact breast cancer classification. The aims of this study were to estimate the frequency of spatial heterogeneity in biomarker expression within tumors, to identify technical and biological factors contributing to spatial heterogeneity, and to examine the impact of discordant biomarker status within tumors on clinical record agreement. Tissue microarrays (TMAs) were constructed using two to four cores (1.0 mm) for each of 1085 invasive breast cancers from the Carolina Breast Cancer Study, which is part of the AMBER Consortium. Immunohistochemical staining for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) was quantified using automated digital imaging analysis. The biomarker status for each core and for each case was assigned using clinical thresholds. Cases with core-to-core biomarker discordance were manually reviewed to distinguish intratumoral biomarker heterogeneity from misclassification of biomarker status by the automated algorithm. The impact of core-to-core biomarker discordance on case-level agreement between TMAs and the clinical record was evaluated. On the basis of automated analysis, discordant biomarker status between TMA cores occurred in 9 %, 16 %, and 18 % of cases for ER, PR, and HER2, respectively. Misclassification of benign epithelium and/or ductal carcinoma in situ as invasive carcinoma by the automated algorithm was implicated in discordance among cores. However, manual review of discordant cases confirmed spatial heterogeneity as a source of discordant biomarker status between cores in 2 %, 7 %, and 8 % of cases for ER, PR, and HER2, respectively. Overall, agreement between TMA and clinical record was high for ER (94 %), PR (89 %), and HER2 (88 %), but it was reduced in cases with core-to-core discordance (agreement 70 % for ER, 61 % for PR, and 57 % for HER2). Intratumoral biomarker heterogeneity may impact breast cancer classification accuracy, with implications for clinical management. Both manually confirmed biomarker heterogeneity and misclassification of biomarker status by automated image analysis contribute to discordant biomarker status between TMA cores. Given that manually confirmed heterogeneity is uncommon (<10 % of cases), large studies are needed to study the impact of heterogeneous biomarker expression on breast cancer classification and outcomes.

Quantitative assessment of the spatial heterogeneity of tumor-infiltrating lymphocytes in breast cancer

Abstract: Tumor-infiltrating lymphocyte (TIL) count in breast cancer carries prognostic information and represents a potential predictive marker for emerging immunotherapies. However, the distribution of the lymphocyte subpopulations is not well defined. The goals of this study were to examine intratumor heterogeneity in TIL subpopulation counts in different fields of view (FOV) within each section, in different sections from the same biopsy, and between biopsies from different regions of the same cancer using quantitative immunofluorescence (QIF). We used multiplexed QIF to quantify cytokeratin-positive epithelial cells, and CD3-positive, CD8-positive and CD20-positive lymphocytes in tissue sections from multiple biopsies obtained from different areas of 31 surgically resected primary breast carcinomas (93 samples total). Log2-transformed QIF scores or concordance and variance component analyses with linear mixed-effects models were used. Cohen’s kappa index [k] of high versus low scores, defined as above and below the median, was used to measure sample similarity between areas. We found a strong positive correlation between CD3 and CD8 levels across all patients (Pearson correlation coefficient [CC] = 0.827). CD3 and CD8 showed a weaker but significant association with CD20 (CC = 0.446 and 0.363, respectively). For each marker, the variation between different FOVs in the same section was higher than the variation between sections or between biopsies of the same cancer. The intraclass correlation coefficients (ICC) were 0.411 for CD3, 0.324 for CD8, and 0.252 for CD20. In component analysis, 66–69 % of the variance was attributable to differences between FOVs in the same section and 30–33 % was due to differences between biopsies from different areas of the same cancer. Section to section differences were negligible. Concordance for low versus high marker status assignment in single biopsies compared to all three biopsies combined yielded k = 0.705 for CD3, k = 0.655 for CD8, and k = 0.603 for CD20. T and B lymphocytes show more heterogeneity across the dimensions of a single section than between different sections or regions of a given breast tumor. This observation suggests that the average lymphocyte score from a single biopsy of a tumor is reasonably representative of the whole cancer.

Naringenin prevents TGF-β1 secretion from breast cancer and suppresses pulmonary metastasis by inhibiting PKC activation

Abstract: Targeting the TGF-β1 pathway for breast cancer metastasis therapy has become an attractive strategy. We have previously demonstrated that naringenin significantly reduced TGF-β1 levels in bleomycin-induced lung fibrosis and effectively prevented pulmonary metastases of tumors. This raised the question of whether naringenin can block TGF-β1 secretion from breast cancer cells and inhibit their pulmonary metastasis. We transduced a lentiviral vector encoding the mouse Tgf-β1 gene into mouse breast carcinoma (4T1-Luc2) cells and inoculated the transformant cells (4T1/TGF-β1) into the fourth primary fat pat of Balb/c mice. Pulmonary metastases derived from the primary tumors were monitored using bioluminescent imaging. Spleens, lungs and serum (n = 18–20 per treatment group) were analyzed for immune cell activity and TGF-β1 level. The mechanism whereby naringenin decreases TGF-β1 secretion from breast cancer cells was investigated at different levels, including Tgf-β1 transcription, mRNA stability, translation, and extracellular release. In contrast to the null-vector control (4T1/RFP) tumors, extensive pulmonary metastases derived from 4T1/TGF-β1 tumors were observed. Administration of the TGF-β1 blocking antibody 1D11 or naringenin showed an inhibition of pulmonary metastasis for both 4T1/TGF-β1 tumors and 4T1/RFP tumors, resulting in increased survival of the mice. Compared with 4T1/RFP bearing mice, systemic immunosuppression in 4T1/TGF-β1 bearing mice was observed, represented by a higher proportion of regulatory T cells and myeloid-derived suppressor cells and a lower proportion of activated T cells and INFγ expression in CD8+ T cells. These metrics were improved by administration of 1D11 or naringenin. However, compared with 1D11, which neutralized secreted TGF-β1 but did not affect intracellular TGF-β1 levels, naringenin reduced the secretion of TGF-β1 from the cells, leading to an accumulation of intracellular TGF-β1. Further experiments revealed that naringenin had no effect on Tgf-β1 transcription, mRNA decay or protein translation, but prevented TGF-β1 transport from the trans-Golgi network by inhibiting PKC activity. Naringenin blocks TGF-β1 trafficking from the trans-Golgi network by suppressing PKC activity, resulting in a reduction of TGF-β1 secretion from breast cancer cells. This finding suggests that naringenin may be an attractive therapeutic candidate for TGF-β1 related diseases.

Does primary neoadjuvant systemic therapy eradicate minimal residual disease? Analysis of disseminated and circulating tumor cells before and after therapy

Abstract: Patients with breast cancer (BC) undergoing neoadjuvant chemotherapy (NACT) may experience metastatic relapse despite achieving a pathologic complete response. We analyzed patients with BC before and after NACT for disseminated tumor cells (DTCs) in the bone marrow(BM); comprehensively characterized circulating tumor cells (CTCs), including stem cell–like CTCs (slCTCs), in blood to prove the effectiveness of treatment on these cells; and correlated these findings with response to therapy, progression-free survival (PFS), and overall survival (OS). CTCs (n = 135) and slCTCs (n = 91) before and after NACT were analyzed using the AdnaTest BreastCancer, AdnaTest TumorStemCell, and epithelial–mesenchymal transition (QIAGEN Hannover GmbH Germany). The expression of estrogen receptor, progesterone receptor, and the resistance marker excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1), nuclease were studied in separate single-plex reverse transcription polymerase chain reaction experiments. DTCs were evaluated in 142 patients before and 165 patients after NACT using the pan-cytokeratin antibody A45-B/B3 for immunocytochemistry. The positivity rates for DTCs, CTCs, and slCTCs were 27 %, 24 %, and 51 % before and 20 %, 8 %, and 20 % after NACT, respectively. Interestingly, 72 % of CTCs present after therapy were positive for ERCC1, and CTCs before (p = 0.005) and after NACT (p = 0.05) were significantly associated with the presence of slCTCs. Whereas no significant associations with clinical parameters were found for CTCs and slCTCs, DTCs were significantly associated with nodal status (p = 0.03) and histology (0.046) before NACT and with the immunohistochemical subtype (p = 0.02) after NACT. Univariable Cox regression analysis revealed that age (p = 0.0065), tumor size before NACT (p = 0.0473), nodal status after NACT (p = 0.0137), and response to NACT (p = 0.0136) were significantly correlated with PFS, whereas age (p = 0.0162) and nodal status after NACT (p = 0.0243) were significantly associated with OS. No significant correlations were found for DTCs or any CTCs before and after therapy with regard to PFS and OS. Although CTCs were eradicated more effectively than DTCs, CTCs detected after treatment seemed to be associated with tumor cells showing tumor stem cell characteristics as well as with resistant tumor cell populations that might indicate a worse outcome in the future. Thus, these patients might benefit from additional second-line treatment protocols including bisphosphonates for the eradication of DTCs.

Raised mammographic density: Causative mechanisms and biological consequences

Abstract: High mammographic density is the most important risk factor for breast cancer, after ageing. However, the composition, architecture, and mechanical properties of high X-ray density soft tissues, and the causative mechanisms resulting in different mammographic densities, are not well described. Moreover, it is not known how high breast density leads to increased susceptibility for cancer, or the extent to which it causes the genomic changes that characterise the disease. An understanding of these principals may lead to new diagnostic tools and therapeutic interventions.

Mammographic density and breast cancer risk: a mediation analysis

Abstract: High mammographic density (MD) is a strong risk factor for breast cancer. However, it is unclear whether high MD is an intermediate phenotype or whether breast cancer risk factors influence breast cancer risk and MD independently. Our study population included 1290 invasive breast cancer cases and 3422 controls from the Nurses’ Health Studies. We estimated the percent of the total association between the risk factor and breast cancer that was mediated by MD. In both pre- and postmenopausal women, the association between history of biopsy-confirmed benign breast disease and risk was partially mediated by percent MD (percent mediated (PM) = 17 %, p < 0.01 and PM = 33 %, p = 0.04, respectively). In premenopausal women, the associations between early life body size (adolescent somatotype and BMI at age 18) and breast cancer risk were substantially mediated by percent MD (PM = 73 %, p = 0.05 and PM = 82 %, p = 0.04, respectively). In postmenopausal women, the proportion of the associations of childhood somatotype and adolescent somatotype that were mediated by percent MD were lower (PM = 26 %, p = 0.01 for both measures). Hormone therapy use at mammogram was significantly mediated by percent MD in postmenopausal women (PM = 22 %, p < 0.01). Associations with other risk factors, such as age at menarche or family history of breast cancer, were not mediated by percent MD. Percent MD partially mediated some of the associations between risk factors and breast cancer, though the magnitude varied by risk factor and menopausal status. These findings suggest that high MD may be an intermediate in some biological pathways for breast cancer development.

An integrated genomics analysis of epigenetic subtypes in human breast tumors links DNA methylation patterns to chromatin states in normal mammary cells

Abstract: Aberrant DNA methylation is frequently observed in breast cancer. However, the relationship between methylation patterns and the heterogeneity of breast cancer has not been comprehensively characterized. Whole-genome DNA methylation analysis using Illumina Infinium HumanMethylation450 BeadChip arrays was performed on 188 human breast tumors. Unsupervised bootstrap consensus clustering was performed to identify DNA methylation epigenetic subgroups (epitypes). The Cancer Genome Atlas data, including methylation profiles of 669 human breast tumors, was used for validation. The identified epitypes were characterized by integration with publicly available genome-wide data, including gene expression levels, DNA copy numbers, whole-exome sequencing data, and chromatin states. We identified seven breast cancer epitypes. One epitype was distinctly associated with basal-like tumors and with BRCA1 mutations, one epitype contained a subset of ERBB2-amplified tumors characterized by multiple additional amplifications and the most complex genomes, and one epitype displayed a methylation profile similar to normal epithelial cells. Luminal tumors were stratified into the remaining four epitypes, with differences in promoter hypermethylation, global hypomethylation, proliferative rates, and genomic instability. Specific hyper- and hypomethylation across the basal-like epitype was rare. However, we observed that the candidate genomic instability drivers BRCA1 and HORMAD1 displayed aberrant methylation linked to gene expression levels in some basal-like tumors. Hypomethylation in luminal tumors was associated with DNA repeats and subtelomeric regions. We observed two dominant patterns of aberrant methylation in breast cancer. One pattern, constitutively methylated in both basal-like and luminal breast cancer, was linked to genes with promoters in a Polycomb-repressed state in normal epithelial cells and displayed no correlation with gene expression levels. The second pattern correlated with gene expression levels and was associated with methylation in luminal tumors and genes with active promoters in normal epithelial cells. Our results suggest that hypermethylation patterns across basal-like breast cancer may have limited influence on tumor progression and instead reflect the repressed chromatin state of the tissue of origin. On the contrary, hypermethylation patterns specific to luminal breast cancer influence gene expression, may contribute to tumor progression, and may present an actionable epigenetic alteration in a subset of luminal breast cancers.