DCIS Tissues Research Articles

Abstract 5483: Macrospatial ecotype heterogeneity in early-stage breast cancer identified by spatial nucleus barcoding

Abstract Ductal carcinoma in situ (DCIS), the predominant form of early-stage breast cancer and a precursor to invasive breast cancer, remains inadequately understood in terms of spatial microenvironment reprogramming. In this study, we developed a Spatial Nucleus Barcoding (SNuBar) approach for single-nucleus RNA sequencing (SNuBar-RNA) that utilizes a single oligonucleotide adaptor to preserve spatial information. The accuracy and scalability of SNuBar was validated using cell line mixture experiments. We then applied SNuBar-RNA to investigate the macrospatial ecotype heterogeneity in early-stage breast cancer. By analyzing single cells from different macrodissected spatial regions in normal breast tissues, matched normal and DCIS tissues, we identified 13 major cell types and 43 distinct cell states, co-localizing in diverse combinations across four primary topographic areas (adipose-enriched, normal epithelial, activated stroma and tumor). Spatial analysis revealed the reprogramming of vascular and myoepithelial cells in localized zones near premalignant cells, while fibroblasts, T-cells, and myeloid cells exhibited reprogramming across broader fields. Validation using the MERSCOPE assay confirmed stromal heterogeneity between tumor and normal areas. Collectively, our data shows that many diverse cell types are reprogrammed in spatially-defined areas in the early-stage breast cancer microenvironment, relative to the normal breast tissue regions. Citation Format: Kaile Wang, Zhenna Xiao, Yiyun Lin, Runmin Wei, Shanshan Bai, Jie Ye, Rui Ye, Min Hu, Aatish Thennavan, Emi Sei, Alastair Thompson, Savitri Krishnamurthy, Nicholas Navin. Macrospatial ecotype heterogeneity in early-stage breast cancer identified by spatial nucleus barcoding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5483.

Abstract 76: Reprogramming of the preinvasive breast microenvironment identified by spatial nucleus barcoding

Abstract Ductal-carcinoma-in-situ (DCIS) is the most common early-stage breast cancer and a non-obligate precursor to invasive breast cancer. However our understanding of the spatial microenvironment and the reprogramming of cell types in DCIS remains limited. To address this question, we developed a Spatial Nucleus Barcoding (SNuBar) method that utilizes a single oligonucleotide adaptor to preserve spatial information followed by performing single nucleus RNA sequencing. We validated the accuracy and scalability of SNuBar in cell lines and applied this method to investigate the spatial microenvironment of 4 normal breast tissues, 7 normal adjacent DCIS tissues, and 8 DCIS cancers. The analysis of single cells isolated from 596 macrodissected spatial regions identified 13 major cell types and 43 cell states, that co-localized in different combinations across four main topographic areas. Spatial analysis revealed that vascular and myoepithelial cells were reprogrammed in localized zones near the premalignant cells, while fibroblasts, T-cells and myeloid cells were reprogrammed across broader fields. Our data shows that many diverse cell types are reprogrammed in spatially-defined areas in the DCIS microenvironment, relative to the normal breast tissue regions. Citation Format: Kaile Wang, Zhenna Xiao, Yiyun Lin, Runmin Wei, Jie Ye, Rui Ye, Min Hu, Shanshan Bai, Emi Sei, Aatish Thennavan, Bora Lim, Andrew Futreal, Alastair Thompson, Savitri Krishnamurthy, Nicholas Navin. Reprogramming of the preinvasive breast microenvironment identified by spatial nucleus barcoding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 76.

Abstract F1-2: Clonal evolution of DCIS to invasion

Abstract F1-2: Clonal evolution of DCIS to invasion

Abstract PR010: Characterizing N-glycan profiles of DCIS progression using tissue imaging MALDI mass spectrometry

Abstract Identifying distinct biomarkers of DCIS and determining the invasive potential of DCIS are of great clinical importance. N-linked glycans present on the cell surface of DCIS lesions and surrounding stroma are potential biomarkers of disease and tissue, but remain largely unexplored. An N-glycan targeted imaging mass spectrometry (IMS) approach was initiated to identify N-glycans associated with DCIS and progression in clinical FFPE tissues. A cohort of pathologist annotated DCIS and IDC tissues with a range of disease severity as well as the RAHBT TMA DCIS cohort were evaluated. The RAHBT cohort contains primary DCIS lesions with known long-term outcomes and serves as a good sample set to identify early markers indicating progressive potential, while the DCIS and IDC large tissue biopsies provide information on distinct glycan profiles when DCIS has progressed to IDC. Initially, tissue samples containing DCIS alone or both DCIS and IDC were processed for N-glycan MALDI-IMS analysis, the goal being to establish a consensus panel of each analyte and determine distinct profiles of DCIS when alone vs. when IDC was present. For N-glycans, a peak list of 54 N-glycans was selected for evaluation of each DCIS only tissue, as well as the mixed DCIS/IDC tissues. By highest intensity, the major DCIS-associated glycans were in the high-mannose and paucimannose structure categories. Additionally, a series of tri- and tetra-antennary multi-fucosylated glycans were also detected specifically in the DCIS lesions. In tissues containing both IDC and DCIS lesions, the high mannose glycans were also detected in the IDC regions, and GlcNAc-bisected glycans were seen elevated in these as compared to DCIS-only tissues. For samples in the RAHBT TMA cohort (progressors N=43; non-progressors N=78), the bisected N-glycans seen elevated with IDC were also seen to be significantly increased in the RAHBT samples that would eventually progress to IDC. Additional analyses to evaluate N-glycan isomer distributions for fucosylated and sialylated glycan species are also ongoing. The cumulative N-glycan data will be assessed with the extensive spatial genomic, transcriptomic and immunohistological data already generated for the same samples in the RAHBT cohort. This novel combination of multi-enzymatic digests with histopathology annotations represents an extensive multi-dimensional profile of DCIS and a novel tissue biomarker discovery approach. Citation Format: Elizabeth N. Wallace, Grace Grimsley, Siri H. Strand, Robert Michael Angelo, Graham Colditz, E. Shelley Hwang, Robert West, Jeffrey R. Marks, Peggi M. Angel, Richard R. Drake. Characterizing N-glycan profiles of DCIS progression using tissue imaging MALDI mass spectrometry [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr PR010.

Abstract IA034: Compromised myoepithelial cell differentiation correlates with DCIS to IDC transition

Abstract DCIS, breast cancer confined to mammary ducts, shares genomic and transcriptomic signatures with IDC. However for DCIS patients, standard of care for localized breast cancer remains controversial, as treatment reduces local recurrence but does not reduce risk of distant recurrence or mortality. Further, as the risk of dying from DCIS is ~5-20 fold less than from early stage breast cancer, questions remain concerning treatment harms. The fact that treatment does not reduce DCIS death rates provides indirect evidence that DCIS-stage tumor cells can disseminate and serve as reservoirs for metastatic disease. Since presence of myoepithelium is the clinical determinate of non-invasive disease, death from DCIS also implies that myoepithelial cell loss is not required for tumor cell dissemination. Here, we investigate whether changes in myoepithelial cell differentiation, rather than overt cell loss, associate with DCIS tumor cell dissemination. Using murine DCIS models and human DCIS tissue, our group reported sequential loss of myoepithelial cell differentiation markers p63, calponin, and SMA prior to progression to IDC. In human DCIS lesions delineated into low and high risk (pure DCIS vs mixed DCIS with IDC), loss of myoepithelial p63, calponin and SMA occurred more frequently in high risk DCIS lesions. Mice transgenic for calponin knockdown in the myoepithelium were at increased risk for mammary cancer. Combined, these data implicate loss in myoepithelial cell differentiation as a mediator of DCIS progression and dissemination. In human DCIS, compromised myoepithelium correlates with microinvasion and an immune infiltrate enriched in PD1+CD8 T cells. To address the role of immune cells in myoepithelial cell barrier function, we investigated 6 different immune competent mouse models of intraductal cancer (DCIS). While loss of myoepithelial differentiation markers were evident, IDC lesions developed in the absence of DCIS. Similarly, in immune compromised hosts, only IDC lesions were detected after intraductal injection of murine tumor cells, whereas human tumor cells formed stable DCIS lesions. In vitro, murine mammary tumor cells were more motile than human lines, and when tumor cells were co-cultured with myoepithelial cells, fewer myoepithelial to myoepithelial cell contacts were observed in the presence of the mouse tumor cells. Finally, in women, we find the differentiation state of mammary myoepithelium to be modulated by reproductive state, with myoepithelium during weaning–induced gland involution (an established risk window for breast cancer progression) sharing similarities with myoepithelium of high-risk DCIS lesions. In rodents, recently weaned hosts support increased DCIS to IDC transition. In sum, these studies implicate intrinsic and extrinsic regulators of myoepithelial cells, highlight the need for immune competent mouse models of DCIS, and raise the question of whether species mismatch in xenograft models impacts cellular crosstalk between myoepithelial and tumor cells critical to our understanding of the DCIS to IDC transition. Citation Format: Sarah M. Bernhardt, Elizabeth Mitchell, Tanya Russell, Sonali Jindal, Jayasri Narasimhan, Reuben J. Hoffmann, AeSoon Benson, Pepper Schedin. Compromised myoepithelial cell differentiation correlates with DCIS to IDC transition [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr IA034.

Multi-modal imaging of high-risk ductal carcinoma in situ of the breast using C2Am: a targeted cell death imaging agent

BackgroundDuctal carcinoma in situ (DCIS) is a non-invasive form of early breast cancer, with a poorly understood natural history of invasive transformation. Necrosis is a well-recognized adverse prognostic feature of DCIS, and non-invasive detection of its presence and spatial extent could provide information not obtainable by biopsy. We describe here imaging of the distribution and extent of comedo-type necrosis in a model of human DCIS using C2Am, an imaging agent that binds to the phosphatidylserine exposed by necrotic cells.MethodsWe used an established xenograft model of human DCIS that mimics the histopathological features of the disease. Planar near-infrared and optoacoustic imaging, using fluorescently labeled C2Am, were used to image non-invasively the presence and extent of lesion necrosis.ResultsC2Am showed specific and sensitive binding to necrotic areas in DCIS tissue, detectable both in vivo and ex vivo. The imaging signal generated in vivo using near-infrared (NIR) fluorescence imaging was up to 6-fold higher in DCIS lesions than in surrounding fat pad or skin tissue. There was a correlation between the C2Am NIR fluorescence (Pearson R = 0.783, P = 0.0125) and optoacoustic signals (R > 0.875, P < 0.022) in the DCIS lesions in vivo and the corresponding levels of cell death detected histologically.ConclusionsC2Am is a targeted multi-modal imaging agent that could complement current anatomical imaging methods for detecting DCIS. Imaging the presence and spatial extent of necrosis may give better prognostic information than that obtained by biopsy alone.

Squalene epoxidase expression is associated with breast tumor progression and with a poor prognosis in breast cancer.

Differentially expressed genes (DEGs) have been previously identified using massive parallel RNA sequencing in matched normal, breast cancer (BC) and nodal metastatic tissues. Squalene epoxidase (SQLE), one of these DEGs, is a key enzyme in cholesterol synthesis. The aim of the present study was to investigate the potential involvement of SQLE in the tumorigenic process of BC and to determine its association with the clinical outcome of BC. SQLE mRNA expression was measured using reverse transcription-quantitative PCR in 10 pairs of ductal carcinoma in situ (DCIS) and BC tissues and their adjacent normal tissues. Immunohistochemical staining of SQLE on tissue microarray was performed in 26 normal breast, 79 DCIS and 198 BC samples. The role of SQLE as a prognostic biomarker in patients with BC has been verified using BreastMark. SQLE mRNA expression was significantly increased in DCIS and BC tissues compared with that in their adjacent normal tissues. High SQLE expression was detected in 0, 48.1 and 40.4% of normal breast, DCIS and BC tissues, respectively. SQLE expression in DCIS and BC tissues was significantly higher than that in normal breast tissues. High SQLE expression was observed in DCIS with higher nuclear grade, comedo-type necrosis and HER2 positivity. High SQLE expression in BC was associated with larger tumor size, nodal metastases, higher stage, HER2 subtype and distant metastatic relapse. High SQLE expression was associated with poor disease-free and overall survival, and independently predicted poor disease-free survival in patients with BC. Following BreastMark analysis, high SQLE mRNA expression in BC was significantly associated with a poor prognosis in the ‘all’, lymph node negative, lymph node positive, luminal A subtype and luminal B subtype groups. Therefore, SQLE expression may be upregulated during the tumorigenic process of BC, and high SQLE expression may be a useful biomarker for predicting a poor prognosis in patients with BC.

SOX10 expression in mammary invasive ductal carcinomas and benign breast tissue.

SOX10 immunohistochemistry is used to identify tumors of neural crest origin, including melanocytic neoplasms. SOX10 expression has also been identified in myoepithelial cells of the breast and in a subset of invasive mammary carcinomas. In order to characterize SOX10 expression in ductal carcinomas of the breast, the aim of this study was to characterize the SOX10 in invasive ductal carcinomas according to molecular subtype, DCIS, and benign breast tissue. Forty cases of invasive ductal carcinoma of the breast were retrieved, with ten cases with immunohistochemical profile compatible with luminal A-like, luminal B-HER2-positive, non-luminal HER2-positive, and triple-negative subtypes. Whole tissue sections from each case were stained with SOX10. Six (60%) of ten triple-negative tumors were SOX10+ compared with 1 (3%) of 30 carcinomas of other molecular subtypes. All but one of the positive tumors showed at least moderate expression in at least 40% of tumor cells. All seven cases SOX10+ carcinomas were grade 3 tumors. Of the 13 cases with DCIS available for assessment, one (8%) showed positive SOX10 expression (a case associated with triple-negative carcinoma). Twenty-two cases contained normal breast tissue that showed SOX10 expression in both myoepithelial and luminal cells, predominantly patchy with variable intensity. SOX10 showed incomplete myoepithelial staining compared to other myoepithelial markers. In conclusion, SOX10 IHC cannot reliably differentiate between high-grade triple-negative carcinomas, melanomas, and myoepithelial tumors in the breast. SOX10 is not as robust a myoepithelial marker compared with other established markers.

Abstract B14: Sprouty4 regulates the transition to invasive breast ductal carcinoma through ERK/MAPK signaling

Abstract Breast cancer is one of the most common oncologic developments in U.S. women, with approximately 25% of these cases being in situ disease (DCIS). For many years DCIS lesions have been treated using surgery and radiotherapy with the goal of decreasing the amount of late-stage disease. Unfortunately, a number of reports have since shown that early intervention is not sufficient and have highlighted the likelihood of overtreatment. This is especially true for triple-negative breast cancers, which are more strongly associated with distant recurrence, metastasis, and death compared to other types of invasive breast ductal carcinoma (IDC). These factors have generated a substantial clinical need for reliable biomarkers or molecular determinants to assess disease progression. Sprouty proteins are recognized as important regulators of ERK/MAPK signaling, and have been studied in various cancers. We hypothesize that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling, and its loss or reduced expression is a mechanism by which DCIS lesions progress toward IDC, including triple-negative disease. Using immunohistochemistry we found that Sprouty4 expression was reduced in IDC patient tissue samples compared to normal or DCIS tissues, and that ERK/MAPK activity had an inverse relationship to Sprouty4 expression. These results correspond with immunoblot data from our 3D culture model of breast cancer progression in which Sprouty4 expression was higher during DCIS than in the IDC stage. Efficient overexpression of Sprouty4 reduced both ERK/MAPK activity as well as the aggressive phenotype of MCF10.CA1d IDC cells. Immunofluorescence experiments revealed data consistent with the relocation of E-cadherin back to the cell surface and the restoration of adherens junctions. To determine whether these effects were due to changes in ERK/MAPK signaling, IDC cells were treated with MEK162, an allosteric MEK inhibitor. Nanomolar concentrations of drug produced the restoration of an epithelial-like phenotype similar to Sprouty4 overexpression. From these data we conclude that Sprouty4 may act to control ERK/MAPK signaling in DCIS, thus limiting the progression of these premalignant breast lesions. Citation Format: Ethan J. Brock, Ryan Jackson, Julie L. Boerner, Quanwen Li, Bonnie F. Sloane, Raymond R. Mattingly. Sprouty4 regulates the transition to invasive breast ductal carcinoma through ERK/MAPK signaling [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B14.

Intraoperative Resection Guidance with Photoacoustic and Fluorescence Molecular Imaging Using an Anti-B7-H3 Antibody-Indocyanine Green Dual Contrast Agent.

Purpose: Breast cancer often requires surgical treatment including breast-conserving surgical resection. However, with current postsurgical histologic margin analysis, one quarter of breast cancer patients undergo reexcision to achieve negative margins corresponding to decreased local recurrence and better outcomes. Therefore, a method with high resolution and specificity for intraoperative margin assessment is needed.Experimental Design: First, quantitative immunofluorescence staining of B7-H3 expression was assessed in four pathologic stages of breast cancer progression of the MMTV-PyMT transgenic murine model. Next, an antibody-dye contrast agent, B7-H3-ICG, was injected into mice prior to surgical resection of breast cancer. Anatomic ultrasound, spectroscopic photoacoustic (sPA), and fluorescence imaging were used to guide resection of mammary glands suspected of containing cancer. Resected tissues were processed for H&E staining and pathologic assessment and compared with sPA and fluorescence imaging signals.Results: Tissue containing DCIS (46.0 ± 4.8 a.u.) or invasive carcinoma (91.7 ± 21.4 a.u.) showed significantly higher (P < 0.05) B7-H3 expression than normal and hyperplastic tissues (1.3 ± 0.8 a.u.). During image-guided surgical resection, tissue pieces assessed as normal or hyperplastic (n = 17) showed lower average sPA (3.17 ± 0.48 a.u.) and fluorescence signal [6.83E07 ± 2.00E06 (p/s)/(μW/cm²)] than DCIS and invasive carcinoma tissue (n = 63) with an average sPA signal of 23.98 ± 4.88 a.u. and an average fluorescence signal of 7.56E07 ± 1.44E06 (p/s)/(μW/cm²) with AUCs of 0.93 [95% confidence interval (CI), 0.87-0.99] and 0.71 (95% CI, 0.57-0.85), respectively.Conclusions: It was demonstrated that sPA and fluorescence molecular imaging combined with B7-H3-ICG agent can assess the disease status of tissues with high diagnostic accuracy, intraoperatively, with high resolution, sensitivity, and specificity. Clin Cancer Res; 24(15); 3572-82. ©2018 AACR.

Abstract P6-06-02: Low expression levels of hormone receptors (ER-α and PGR) in human breast cancer samples is significantly associated with high-grade human cytomegalovirus-IEA

Abstract P6-06-02: Low expression levels of hormone receptors (ER-α and PGR) in human breast cancer samples is significantly associated with high-grade human cytomegalovirus-IEA

Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis.

BackgroundMicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR–92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR–92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR–92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells.Methodology/Principal FindingsWe used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR–92 expression by qRT-PCR. Expression of ERβ1, a direct miR–92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR–92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR–92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERβ1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR–92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR–92 levels in NFs but not CAFs enhanced invasion of both MCF–7 and MDA-MB–231 breast cancer epithelial cells.ConclusionsmiR–92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERβ1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR–92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERβ1 may not be the most important miR–92 target in breast cancer.

DNA methylation in ductal carcinoma in situ related with future development of invasive breast cancer.

BackgroundDuctal carcinoma in situ (DCIS) is a heterogeneous, pre-invasive lesion associated with an increased risk for future invasive ductal carcinoma. However, accurate risk stratification for development of invasive disease and appropriate treatment decisions remain clinical challenges. DNA methylation alterations are early events in the progression of cancer and represent emerging molecular markers that may predict invasive recurrence more accurately than traditional measures of DCIS prognosis.ResultsWe measured DNA methylation using the Illumina HumanMethylation450K array of estrogen-receptor positive DCIS (n = 40) and adjacent-normal (n = 15) tissues from subjects in the New Hampshire Mammography Network longitudinal breast imaging registry. We identified locus-specific methylation differences between DCIS and matched adjacent-normal tissue (95,609 CpGs, Q < 0.05). Among 40 DCIS cases, 13 later developed invasive disease and we identified 641 CpG sites that exhibited differential DNA methylation (P < 0.01 and median |∆β| > 0.1) in these cases compared with age-matched subjects without invasive disease. The set of differentially methylated CpG loci associated with disease progression was enriched in homeobox-containing genes (P = 1.3E-09) and genes involved with limb morphogenesis (P = 1.0E-05). In an independent cohort, a subset of genes with progression-related differential methylation between DCIS and invasive breast cancer were confirmed. Further, the functional relevance of these genes’ regulation by methylation was demonstrated in early stage breast cancers from The Cancer Genome Atlas database.ConclusionsThis work contributes to the understanding of epigenetic alterations that occur in DCIS and illustrates the potential of DNA methylation as markers of DCIS progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0094-0) contains supplementary material, which is available to authorized users.

Abstract P4-05-11: CENPU acts as a new proto-oncogene to regulate tumorigenesis and cancer metastasis in breast cancer

Abstract Background: Centromere protein (CENPU) gene locus is at chromosome 4q35.1, encoding a protein with some classic functional domains; recently some research groups proved CENPU protein is a constitutive kinetochore component and regulates cell-cycle status by recruitment of function-specific proteins. Homologous gene in murine hematopoiesis system represents early erythroblasts-specific expression. Our previous research had found that about 25 % of transgenic mice amplified CENPU would suffer breast cancer in body surface which induce us to hypothesize that CENPU gene and its protein played an important role in breast cancer occurrence and development. Methods: CENPU protein expression was examined in normal mammary tissue, DCIS tissue, primary invasive breast cancer and different human breast cancer cell lines. Cell type and epitope-dependent subcellular-specific CENPU staining pattern in normal mammary gland epithelium and cancer biopsies were correlated to molecular and clinical parameters. A CENPU-specific small inhibitory RNA (siRNA) was introduced into MDA-MB-231 human breast cancer cell lines to investigate its effect on cancer cell growth, migration and invasion. Distribution of cell cycles were examined with flow cytometry. Test the tumorigenicities of si-control and si-CENPU cells by soft agar colony formation assay. Migration was observed by wound healing and transwell migration assays. The expression of related pathway proteins were determined by Western blotting analysis. Results: Compared with normal mammary and DCIS tissues, CENPU protein was highest expression in primary invasive breast cancer tissue (P&amp;lt;0.001).Then we compared CENPU expression lever between no metastasis and metastasis patients during three year, and found that CENPU expression in recurrence group is higher than no-metastasis group (P&amp;lt;0.01). CENPU protein expression of different human breast cancer cell lines were detected, which found the CENPU in triple negative breast cancer cell line, MDA-MB-231, was the highest expression. Knockdown of CENPU by specific siRNA in MDA-MB-231 reduced the ability of colony formation and induced the G2/M phase arrest. Moreover, the migration rate of si-CENPU MDA-MB-231 cancer cells was significantly reduced as compared with si-controls (P&amp;lt;0.01).With the knockdown of CENPU, the E-cadherin expression was up-regulated and the N-cadherin, AKT1 and NF-κB were downp-regulated. Conclusion: We propose that CENPU functions as a novel proto-oncogene to regulate oncogenesis and metastasis. In further, in vivo study is needed to evaluate the biologaical importance of CENPU in breast cancer. Citation Format: Jin Zhang, Xiaobei Zhang, Yunhui Hu, Jiao Li, Jingjing Liu, Sheng Zhang, Wei Su. CENPU acts as a new proto-oncogene to regulate tumorigenesis and cancer metastasis in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-11.

Abstract P2-05-15: Exploring type II microcalcifications in benign, premalignant and malignant breast lesions by shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS)

Abstract Background: Type II microcalcifications, most often seen in proliferative lesions, including both benign and malignant pathologies. But whether the emergence of type II microcalcifications associated with cell canceration is still not clear now. Raman spectroscopy is a powerful, non-invasive analytical tool which can provide detailed and meaningful information about biochemical composition of tissues at molecular level. Our aim was to find the differences and relationships of type II microcalcifications between fibroadenoma, ADH tissues, and DCIS, IDC in breast based on their various biochemical characteristics by Raman spectroscopy. Methods: The frozen sections were collected from 15 patients (all female; ages 25-57) who underwent surgical resection or mammotome biopsy at the Department of Breast Surgery, the First Hospital of Jilin University. After operation the samples were immediately frozen at -20∼-25° and two contiguous sections (6 µm thickness) were cut from a sample by freezing microtome. One was stained with haematoxylin and eosin for routine histopathological analysis and found the microcalcification locations by three expert breast pathologists; the same position of the other section was detected by Microscopic confocal Raman spectrometer (HORIBA JY Lab800, 633nm) with its mirror image (the H&amp;E section). After the spectra we needed had been obtained, the Au@SiO2 shell-isolated nanoparticles (SHINs) were added to the surface of frozen sections immediately and then the spectra with SHINs were collected. Results: A total of 122 Raman spectra and 119 SHINERS spectra were obtained from the microcalcifications in 3 fibroadenoma tissues, 3 ADH, and 5 DCIS, 4 IDC breast tissues. The Raman signals were significantly enhanced by SHINs. Except the major peak at 958-960 cm-1 which attributed to the vibrational modes of calcium hydroxyapatite, the results show no calcium oxalate dihydrate peaks but several other chemical species. The peaks of these species appear at 1002, 1072, 1126, 1446, 1556, and 1657 cm-1, these are attributed to amino acid residue (phenylalanine), nucleic acids, lipids, carotenoids, and Amide I, respectively. The fibroadenoma and ADH microcalcifications mean spectral have the same peak at 1072cm-1, which belongs to the O-P-O stretch of nucleic acids, but in DCIS and IDC tissues the peak changes to 1078cm-1. The ADH have more obvious peak at 1657 cm-1 which assigned to different vibrational modes of the backbone and Amide I, but in DCIS and IDC tissues, the Amide I bands were disappeared. In the DCIS microcalcifications mean spectral, the peaks attributed to amino acid residue (phenylalanine) at 1003, 1031 cm-1 and lipids (CH2 and CH3 bending) at 1301, 1441 cm-1 show more stronger. Meanwhile, compare with DCIS, the IDC spectral shows more stronger nucleic acids peaks and weaker lipids peaks. Conclusion: We have demonstrated the potential of SHINERS to differentiate Type II microcalcifications found in fibroadenoma, ADH, and DCIS, IDC breast tissues. The results presented in this paper provide the biochemical characteristics of the Type II microcalcifications among these tissues, and may represent a key factor responsible for mechanisms of carcinogenesis. Citation Format: Haipeng Zhang, Bing Han, Chao Zheng, Ye Du, Lijia Liang, Weiqing Xu, Zhimin Fan. Exploring type II microcalcifications in benign, premalignant and malignant breast lesions by shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-15.

Abstract P6-08-30: The thrombin clotting pathway is upregulated in the stroma of pre-invasive breast cancer and further upregulated in aggressive invasive breast cancer phenotypes

Abstract BACKGROUND Components of the thrombin (extrinsic) clotting pathway are upregulated in cancer. The clotting pathway factors tissue factor (TF) and Thrombin promote tumour progression through protease activated receptors PAR2 and PAR1 respectively. AIMS To determine if tumour expression (epithelial and stromal) of a procoagulant phenotype is associated with aggressive breast cancer phenotypes and reduced survival. METHODS Tumour expression of TF, thrombin, PAR1 and PAR2 was determined by immunohistochemistry in two cohorts. PROSPECTIVE STUDY Early invasive breast cancer (n=199), ductal carcinoma in situ (DCIS, n=42) and normal breast tissue samples (n=121). RETROSPECTIVE STUDY Early invasive breast cancer patients (n=144) with median follow-up of 69 (range 4 to 91) months. Procoagulant phenotype expression was correlated with tumour grade, proliferation (Ki67), ER and HER2 status (both cohorts), survival and recurrence (retrospective cohort). RESULTS PROSPECTIVE STUDY Epithelium Thrombin (p&amp;lt;0.01) but not TF, PAR1 or PAR2 was increased in invasive cancer compared to DCIS and normal breast tissue. Stroma TF, Thrombin, PAR1 and PAR2 were increased in the stroma of DCIS compared to normal breast stroma (p&amp;lt;0.05, all). In invasive breast cancer, TF was increased in invasive cancer compared to DCIS and compared to normal breast tissue (p&amp;lt;0.01, both). Thrombin, PAR1 and PAR2 were increased in invasive cancer compared to normal breast tissue (p&amp;lt;0.01, all). TF, thrombin, PAR1 and PAR2 were increased in high proliferating (p&amp;lt;0.01, all) and high grade cancer (p&amp;lt;0.01, all). TF (p=0.02) and PAR1 (p&amp;lt;0.01) were increased in ER negative cancer. TF, thrombin and PAR2 was increased in HER2 positive cancer (p&amp;lt;0.01, all). RETROSPECTIVE STUDY Stroma As with the prospective study, thrombin and PAR2 expression was increased in high proliferating cancer (p&amp;lt;0.05, both) and thrombin was increased in high grade cancer (p&amp;lt;0.05). PAR1, TF and thrombin expression was increased in ER negative cancer (p&amp;lt;0.05, all) and PAR2 was increased in HER2 positive cancer (p=0.05). Overall (OS) and disease-free survival (DFS) PAR1 stromal expression was an independent predictor of reduced OS (HR 3.3, 95% CI 1.3-8.3, p=0.01) but did not correlate with DFS. There was no association between epithelial PAR1 expression or epithelial or stromal TF, thrombin or PAR2 expression and DFS or OS. CONCLUSION Stromal upregulation of the thrombin pathway occurs in in-situ cancer, implying cancer-stromal communication at the pre-invasive stage. Stromal thrombin pathway components may have a role in the transition of pre-invasive to invasive cancer. Stromal (but not epithelial) thrombin pathway upregulation is associated with aggressive invasive breast cancer phenotypes and reduced survival. The thrombin pathway may provide a novel therapeutic target, particularly in ER negative, HER2 positive breast cancer. Citation Format: Hudhaifah Shaker, Nigel J Bundred, Harith Albadry, Sarah L Nicholson, Susan Pritchard, Karin Jirström, Goran Landberg, Cliona C Kirwan. The thrombin clotting pathway is upregulated in the stroma of pre-invasive breast cancer and further upregulated in aggressive invasive breast cancer phenotypes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-30.

Abstract 412: DNA methylation in ductal carcinoma in situ and its relation with disease progression

Abstract Ductal carcinoma in situ (DCIS) of the breast is a non-invasive lesion associated with a ten-fold increased relative risk for future invasive ductal cancer (IDC). DNA methylation is recognized as an important epigenetic regulator of gene expression and DNA methylation alterations are widespread in breast cancer. In addition, not all patients with DCIS will later present with invasive disease and epigenetic alterations in DCIS associated with disease progression are not clear. To investigate the relation of methylation with progression from DCIS to IDC we first identified patients with DCIS and a subsequent diagnosis of IDC through the New Hampshire Mammography Network (n=13). Next, we identified age-matched subjects with DCIS who did not have a subsequent diagnosis of IDC over a similar follow up period (n=27). Slides from all 40 patients underwent central pathology review by a breast pathologist, and 2 or 3 mm tissue cores were taken from archived FFPE blocks from selected areas of DCIS and adjacent normal tissue for DNA extraction and bisulfite modification. We measured DNA methylation genome-wide using the Illumina HumanMethylation450 BeadChip and processed the data using the ChAMP pipeline for quality control and normalization, after which 380,000 CpG sites remained in the data set. To investigate the relation of methylation with progression from DCIS to IDC, locus-specific differences in DNA methylation of 40 DCIS samples were examined using linear regression models fit to each of 380,000 CpG sites, modeling the logit-transformed methylation beta-values and adjusting for subject age and DCIS grade. Over 1,000 CpG sites exhibited large differences in DNA methylation levels (probes with a median Δβ &amp;gt; 0.2), and had a nominal P-value &amp;lt; 0.05, between patients with and without a subsequent diagnosis of IDC. Among the 1,007 CpG loci with significant and sizable changes in methylation, the majority (87.1%) exhibited increased methylation in women with a subsequent diagnosis of IDC compared to those without. We also sought to determine the relation of DNA methylation with DCIS grade among CpG loci that track to genes previously associated with DCIS grade, modeling the methylation as the dependent variable and DCIS grade (low/intermediate or high) as the independent variable. Comparing DCIS samples of low/intermediate grade with high grade we found that 15 CpG loci located near the transcriptional start site of the APC gene demonstrated significant (P &amp;lt; 0.05) hypermethylation in high grade DCIS compared to low/intermediate grade DCIS. Ongoing analyses are highlighting profound differences between DCIS and available matched adjacent-normal samples (n=15) and examining the enrichment of regulatory features among differentially methylated loci. This work contributes to the understanding of epigenetic alterations that occur in earlier stages of disease and illustrates the potential of DNA methylation as a predictive marker in DCIS. Citation Format: Kevin C. Johnson, Panpan Chen, Devin Koestler, Julia E. Weiss, Erik G. Jenson, Jonathan D. Marotti, Tracy Onega, Brock C. Christensen. DNA methylation in ductal carcinoma in situ and its relation with disease progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 412. doi:10.1158/1538-7445.AM2014-412

Abstract 2499: Application of a multiplexed fluorescence microscopy method (MultiOmyxTM) to dissect proteomic biomarkers of (18)F-fluorodeoxy-glucose ((18)FDG) uptake in breast cancer

Abstract Background: While routinely collected human tumor specimen can now readily be examined for genetic alterations on a genome wide scale, multi-parameter measurements of protein expression remain challenging. We developed a multiplexed fluorescence microscopy method (MultiOmyxTM) for the quantitative characterization of multiple analytes in formalin-fixed paraffin-embedded tissue (PMID23818604). We now applied this platform to human breast cancer samples to determine the expression of 25 proteins at a single-cell level and examine their relationship to tumor uptake of the Positron Emission Tomography (PET) radiotracer (18)F-fluorodeoxy-glucose ((18)FDG). Methodology: We stained a single 5 µm section of breast carcinoma from each of 18 patients with antibodies against members of growth factor signaling pathways (HER2, IGF1R, PTEN, p-EGFR, p-PDK1, p-ERK1/2, p-S6 Ribosomal Protein, p-4EBP1, p-eIF4E), the glycolysis pathway (Glut-1, HK2, LDH-A), hormone receptors (ER, PR, AR), tumor cell proliferation markers (KI-67 and phospho-histone H3), and markers of hypoxia (HIF-1α, CA IX), and angiogenesis (CD31). 28-30 representative fields of view (FOVs) were randomly selected in each tumor, each comprising 300-500 cells. Images were automatically separated into subcellular and histopathological compartments based on the staining of tumor cells with a panel of segmentation markers (NaK-ATPase, pan-Cadherin, pan-cytokeratin, S6, and DAPI). A breast pathologist assessed and annotated the histologic composition of each FOV. Further analysis focused on FOVs (n=390) containing only invasive ductal carcinoma without admixed DCIS or normal breast tissue. Multivariate analysis between marker expression and FDG uptake was performed using logistic regression and Cox proportional hazard models. All patients had (18)-FDG PET within four weeks prior to collection of the tumor specimen. Results: Staining results with our MultiOmyxTM platform correlated closely with the results from CLIA-certified single marker biomarker assays (e.g., ER IHC and HER2 FISH assay) performed independently on the same set of samples (PMID: 21646475). Nuclear ER staining was associated with low FDG uptake (p=0.02). KI-67 was higher in tumors with high FDG uptake (p=0.04). K-median clustering identified molecular breast cancer subtypes. Conclusions: Our study illustrates the feasibility of quantitative proteomic measurements using a new in-situ multiplexed fluorescence microscopy platform on only a single routinely collected FFPE tissue section. Quantitative staining results from many thousand cells per tumor support the previously reported relationship between hormone receptor status, tumor cell proliferation, and FDG-uptake in breast cancer. Multiple protein markers showed a high degree of intra- and inter-tumoral heterogeneity. Citation Format: Anup Sood, Alexandra M. Miller, Fiona Ginty, Elizabeth McDonough, Yunxia Sui, Alexander Bordwell, Qing Li, Sireesha Kaanumalle, Zhengyu Pang, Franklin Torres, Edi Brogi, Steven Larson, Ingo Mellinghoff. Application of a multiplexed fluorescence microscopy method (MultiOmyxTM) to dissect proteomic biomarkers of (18)F-fluorodeoxy-glucose ((18)FDG) uptake in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2499. doi:10.1158/1538-7445.AM2014-2499

Abstract P4-01-20: Explore the differences and relationships between normal, ADH tissues and DCIS, IDC in breast by shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS)

Abstract Background: The mechanism that ADH changes to DCIS and even IDC is still not clear now. Raman spectroscopy is a powerful, non-invasive analytical tool which can provide detailed and meaningful information about biochemical composition of tissues at molecular level. Our aim was to find the differences and relationships between normal, ADH tissues and DCIS, IDC in breast based on their various biochemical characteristics by Raman spectroscopy. Methods: The frozen sections were collected from 39 patients (all female; ages 27-74) who underwent surgical resection or mammotome biopsy at the Department of Breast Surgery, the First Hospital of Jilin University. After operation the samples were immediately frozen at -20∼-25°C and two contiguous sections (6 μm thickness) were cut from a sample by freezing microtome. One was stained with haematoxylin and eosin for routine histopathological analysis; the other was detected by Microscopic confocal Raman spectrometer (HORIBA JY Lab800, 633nm) with its mirror image (the H&amp;E section). After the spectra we needed had been obtained, the Au@SiO2 shell-isolated nanoparticles (SHINs) were added to the surface of frozen sections immediately and then the spectra with SHINs were collected. Results: A total of 475 Raman spectra and 470 SHINERS spectra were obtained from 9 normal breast tissues, 7 ADH, 8 DCIS, and 15 IDC breast tissues. The Raman signals were significantly enhanced by SHINs. The main spectral features of normal tissues at around 1301, 1442, 1654, and 1743 cm−1 are attributed to lipids. Meanwhile, no strong protein peaks emerged. The peaks of ADH, DCIS and IDC tissues located at 1004, 1033, 1610 and 1658 cm−1 from vibrational modes of proteins shows more stronger. 830, 880, 970, 1090 and 1157cm−1, representing DNA feature peaks, hold the same rules. Table 1: Peak assignments of the Raman spectra of Breast tissues (wavenumbers in cm-1)NBADHDCISIDCMajor Assignments 880878880O-P-O stretch, DNA1003100410041003phenylalanine1092109210911090O-P-O stretch, DNA1301,14421302,14421303,14431303,1442CH2 twist and CH2 deformation, lipids 15261527C = C stretch of carotene 165816581658Amide I, α-helix1743174717451747C = O stretch, lipids These show that diseased tissues contained more proteins and nucleic acids. The spectra of DCIS and IDC tissues contained 1527 cm−1, stemming from C = C stretching of carotene. The spectral features between ADH, DCIS and IDC tissues are described as followings: firstly, the concentrations of proteins, nucleic acids and carotene increased gradually but lipids content reduced from ADH to IDC. Secondly, ADH and DCIS tissues have extraordinary strong band of amide I, but in IDC tissues the peak almost cannot observed. Additionally, DCIS tissues have the red shifted and broad peak of CH2, which explains acyl backbone of lipids and proteins has fractured. Conclusion: The changes including content and molecular structure of lipids, carotene, proteins and nucleic acids accompany with deterioration, indicating the Raman spectroscopy can be used to explore the differences and relationships between normal, ADH tissues and DCIS, IDC in breast. Simultaneously, it might be used to discover the mechanism that ADH convert to DCIS. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-01-20.

Abstract B127: Loss of miR-92 expression in breast epithelial cells is associated with cancer progression

Abstract MicroRNAs (miR) are a class of short non-coding RNAs that regulate gene expression and are commonly dysregulated in cancers, including those of the breast. MiR-92 is thought to function as an oncogene, promoting cell proliferation and reducing apoptosis. We have previously shown that expression of ERβ1 is negatively regulated by miR-92 in unselected non-microdissected breast cancers, providing a mechanism for down-regulation of this putative tumour suppressor gene. Here, we aimed to confirm the function of ERβ1 in breast cancer cells and examined expression of miR-92, specifically in the breast epithelium, during breast cancer progression. We also investigated the role of miR-92 in fibroblasts and examined whether differential expression in these cells might modify the behaviour of breast cancer epithelial cells. Stable ERβ1 overexpression was achieved using a MoMLV-based vector (pFBneo) or silenced by siRNA and effects were examined using various functional assays. We used laser microdissection (LMD) to isolate matched normal, co-incident DCIS and invasive tissue from 10 breast cancer patients. RNA was extracted, cDNA synthesised and preamplified using MegaPlex PreAmp Primers. MiR-92 expression was analysed by real-time PCR. Expression of ERβ1 protein was concurrently analysed for each patient by immunohistochemistry. LMD was also used to isolate matched normal and cancer-associated fibroblasts from 21 breast cancer patients and miR-92 expression was examined as above. Loss and gain of miR-92 in fibroblasts was achieved using anti-miR-92 miRNA and miRNASelect pEP-hsa-mir-92a-1 expression vector, respectively. The invasive potential of these fibroblasts was examined using a 2D modified Boyden chamber assay. ERβ1 overexpression decreased growth and induced cell death in T47D and BT-20 breast cancer cells. Surprisingly, we found that whilst absolute miR-92 expression levels varied across patients the expression pattern of miR-92 was consistent in all cases; miR-92 levels decreased during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p&amp;lt;0.01) and lowest in invasive breast tissue (p&amp;lt;0.01). Expression of ERβ1 and miR-92 were not inversely correlated as expected. Further examination of the staining patterns of ERβ1 showed nuclear expression in normal breast epithelium with increased cytoplasmic immunoreactivity during progression to DCIS and invasive breast cancer. ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues, however miR-92 expression levels remained unchanged between matched microdissected adjacent normal and cancer-associated fibroblasts. Despite this, we demonstrated that fibroblasts can contribute to breast tumour progression as manipulation of miR-92 levels influenced the invasive potential of breast cancer epithelial cells. In summary, whilst confirming a potential tumour suppressor role for ERβ1 we have shown that expression is not inversely correlated with its negative regulator miR-92 in breast epithelium, suggesting other methods of regulation. Expression of miR-92 was lost, rather than gained, in breast epithelial cells during breast cancer progression correlating with a shift in ERβ1 staining from nuclei to the cytoplasm. While miR-92 expression levels remained unchanged in stromal fibroblasts, our data support a functional role in fibroblasts and show that differential miR-92 expression in these cells can influence the invasive capacity of breast cancer epithelial cells. Finally, our study highlights the importance of isolating specific cell types prior to expression analyses since changes in the epithelium may be masked by expression from the stroma and vice versa. Citation Format: Laura Smith, Euan Baxter, Andrew Hanby, Thomas Hughes, Rebecca Millican-Slater, Eldo Verghese, Valerie Speirs. Loss of miR-92 expression in breast epithelial cells is associated with cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B127.