Ductal Carcinoma In Situ Samples Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Chromosomal copy number alterations for associations of ductal carcinoma in situ with invasive breast cancer.

IntroductionScreening mammography has contributed to a significant increase in the diagnosis of ductal carcinoma in situ (DCIS), raising concerns about overdiagnosis and overtreatment. Building on prior observations from lineage evolution analysis, we examined whether measuring genomic features of DCIS would predict association with invasive breast carcinoma (IBC). The long-term goal is to enhance standard clinicopathologic measures of low- versus high-risk DCIS and to enable risk-appropriate treatment.MethodsWe studied three common chromosomal copy number alterations (CNA) in IBC and designed fluorescence in situ hybridization-based assay to measure copy number at these loci in DCIS samples. Clinicopathologic data were extracted from the electronic medical records of Stanford Cancer Institute and linked to demographic data from the population-based California Cancer Registry; results were integrated with data from tissue microarrays of specimens containing DCIS that did not develop IBC versus DCIS with concurrent IBC. Multivariable logistic regression analysis was performed to describe associations of CNAs with these two groups of DCIS.ResultsWe examined 271 patients with DCIS (120 that did not develop IBC and 151 with concurrent IBC) for the presence of 1q, 8q24 and 11q13 copy number gains. Compared to DCIS-only patients, patients with concurrent IBC had higher frequencies of CNAs in their DCIS samples. On multivariable analysis with conventional clinicopathologic features, the copy number gains were significantly associated with concurrent IBC. The state of two of the three copy number gains in DCIS was associated with a risk of IBC that was 9.07 times that of no copy number gains, and the presence of gains at all three genomic loci in DCIS was associated with a more than 17-fold risk (P = 0.0013).ConclusionsCNAs have the potential to improve the identification of high-risk DCIS, defined by presence of concurrent IBC. Expanding and validating this approach in both additional cross-sectional and longitudinal cohorts may enable improved risk stratification and risk-appropriate treatment in DCIS.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0623-y) contains supplementary material, which is available to authorized users.

Abstract 4747: Genomic alterations in ductal carcinoma in situ compared with Invasive breast cancer: a quantitative real-time PCR study

Abstract Introduction: Several molecular markers have been investigated as possible prognostic factors for ductal carcinoma in situ (DCIS) progression to invasive ductal cancer (IDC). DCIS is regarded as a non-obligate precursor of IDC as it is frequently found adjacent to or surrounded by invasive breast cancer. Previously, we carried out genomic analyses that demonstrated that DCIS has specific genetic alterations that were conserved in invasive disease. Nevertheless, it is still unknown whether DCIS is merely a marker of increased risk, or actually a driver of the progression to IDC.Thus, it is crucial to better understand the genetic aberrations that underlie DCIS progression to IDC. Methodology: We conducted array chromosomal genomic hybridization on pure DCIS, IDC and DCIS paired with IDC, with 30 cases each (all were microdissected). Three chromosomal regions were selected based on the rate of their detection, which was significantly different (p<0.05) between tumors of different receptor status, whether in combination, or single parameter; ER, PgR, HER2. In total, 9 genes were chosen for further validation, spanning Chromosome 4p15.33, Chromosome 5q11.2 and Chromosome 5q31.3. Quantitative real-time polymerase chain reaction (Q-RT-PCR) was used to study the copy number state of the 9 genes in different pure cases, including 22 DCIS, 29 IDC and 10 DCIS paired with IDC (all were microdissected). All areas contained more than 90% carcinoma or control cells. By normalizing values to the housekeeping gene, human albumin, and normal control DNA samples, we obtained ΔΔCt values for each sample. Results: 61 samples were analyzed by comparing DCIS and IDC copy numbers of genes within the selected chromosomal regions. The median relative gene quantities were 1.66(4p15.33), 1.34(5q11.2) and 1.25(5q31.3). In all regions, IDC showed higher levels of amplification than DCIS with varying degrees of significance. Not all differences between DCIS and IDC in individual genes were significant. This observation may be attributed to focal variations in gene copy numbers and noise from Q-RT-PCR methods. 4p15.33 and 5q31.3 regions demonstrated significant gain in IDC compared to DCIS (p = 0.021). Genes within these regions include miRNA 572 (MIR572) and integrin alpha 2 (ITGA2). MIR572 is known to be involved in post-transcriptional regulation of gene expression, whereas ITGA2 encodes the alpha subunit of a transmembrane receptor for collagens. In the Chromosome 5q31.3 region, paired samples of DCIS and IDC also showed a trend in gene copy numbers, with IDC having a higher value than DCIS. Conclusions: Our findings demonstrate that there are specific genetic alterations associated with DCIS progression to IDC. In particular, MIR572 within Chromosome 4p15.33 and ITGA2 within Chromosome 5q31.3 may serve as candidate regions to predict the progression of DCIS to IDC. Note: This abstract was not presented at the meeting. Citation Format: Trillium E. Chang, Keisha Warren, Ranju Nair, Tian Y. Lu, Adewunmi Adeoye, Vladimir Iakovlev, Susan J. Done. Genomic alterations in ductal carcinoma in situ compared with Invasive breast cancer: a quantitative real-time PCR study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4747. doi:10.1158/1538-7445.AM2015-4747

Epiregulin contributes to breast tumorigenesis through regulating matrix metalloproteinase 1 and promoting cell survival.

BackgroundThe epidermal growth factor (EGF) family of ligands has been implicated in promoting breast cancer initiation, growth and progression. The contributions of EGF family ligands and their receptors to breast cancer are complex, and the specific mechanisms through which different ligands regulate breast tumor initiation and growth are not well-defined. These studies focus on the EGF family member epiregulin (EREG) as a mediator of early stage breast tumorigenesis.MethodsEREG expression levels were assessed in both cell lines and human samples of ductal carcinoma in situ (DCIS) using quantitative RT-PCR, ELISA and immunohistochemistry. Gene knock-down approaches using shRNA-based strategies were used to determine the requirement of EREG for growth of MCF10DCIS cells in vivo, and for identifying mechanisms through which EREG promotes tumor cell survival. Experiments were performed using a combination of two-dimensional culture, three-dimensional culture and tumor growth in vivo.ResultsIn comparison with other EGF family members, EREG was induced in MCF10DCIS cells compared with MCF10A and MCF10AT cells and its expression was partially regulated by fibroblast growth factor receptor (FGFR) activity. Reduced EREG expression in MCF10DCIS cells led to decreased tumor growth in vivo, which was associated with reduced cell survival. Furthermore, treatment of MCF10A cells with exogenous EREG enhanced cell survival both in three-dimensional culture and in response to chemotherapeutic agents. Examination of EREG-induced signaling pathways demonstrated that EREG promoted survival of MCF10A cells through regulating expression of matrix metalloproteinase-1 (MMP-1). To determine the relevance of these findings in human tumors, samples of DCIS were analyzed for EREG and MMP-1 expression. EREG was induced in DCIS lesions compared to normal breast epithelium, and EREG and MMP-1 were correlated in a subset of DCIS samples.ConclusionsTogether, these studies lead to identification of a novel pathway involving EREG and MMP-1 that contributes to the formation of early stage breast cancer. Understanding these complex pathways could ultimately lead to the development of novel biomarkers of neoplastic progression and/or new therapeutic strategies for patients with early stage cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0408-z) contains supplementary material, which is available to authorized users.

Abstract P4-06-01: Epiregulin-induced matrix metalloproteinase-1 contributes to early stages of breast tumorigenesis

Abstract The earliest stages of malignant transformation require the acquisition of multiple phenotypes, such as proliferation, survival and migration, which contribute to tumor growth and progression. Identifying the key cellular and molecular factors that drive the formation of early breast cancer lesions will ultimately result in the development of preventive approaches for patients that are at high risk for developing invasive breast cancer. The epidermal growth factor (EGF) family of ligands has been implicated in promoting breast cancer growth and progression. The contributions of EGF family ligands and their receptors to breast cancer are complex, and the specific mechanisms through which different ligands regulate different stages of breast tumor initiation and growth are not well-defined. These studies focus on the EGF family member epiregulin (EREG) as a mediator of early stages of breast tumorigenesis. In comparison with other EGF family members, EREG was found to be highly expressed in MCF10DCIS.com cells compared with the non-transformed MCF10A cells. Increased EREG expression was also found in significantly more patient samples of human ductal carcinoma in situ (DCIS) than in samples of normal epithelium (64.5% vs. 29.4%, p<0.05). Therefore, further studies were performed to identify mechanisms through which EREG promotes the formation of DCIS lesions. Experimental studies demonstrated that treatment of MCF10A cells with exogenous EREG enhanced multiple tumorigenic phenotypes, including proliferation, migration and survival. Examination of EREG-induced signaling pathways demonstrated that EREG promotes survival of MCF10A cells primarily through activation of the signal transducer and activator of transcription 5 (STAT5) pathway. Analysis of potential STAT5 target genes revealed that the EREG-STAT5 pathway induces expression of matrix metalloproteinase-1 (MMP-1), a novel STAT5 target gene that was subsequently found to promote survival in response to EREG treatment. To assess the consequences of loss of EREG expression on tumor formation, EREG was knocked down in MCF10DCIS.com cells using shRNA strategies. Loss of EREG led to decreased tumor growth in vivo, which corresponded with decreased cell survival and decreased MMP-1 expression. To determine the relevance of these findings in human tumors, samples of DCIS were analyzed for both EREG and MMP-1 expression. MMP-1 was significantly induced in DCIS lesions in comparison with normal breast epithelium, and EREG and MMP-1 were significantly correlated in a subset of DCIS samples (p=0.011). Together, these studies suggest that EREG contributes to early stages of breast tumorigenesis through regulation of MMP-1. Based on these studies, we propose that EREG contributes to the formation of preneoplastic lesions in a subset of breast cancers and further studies are focused on characterizing the mechanisms through which the EREG-MMP-1 pathway contributes to tumor initiation and growth. Understanding these mechanisms will ultimately lead to the identification of biomarkers of aggressive disease and novel targets for therapeutic strategies for breast cancer patients. Citation Format: Kathryn L Schwertfeger, Mariya Farooqui, Laura R Bohrer, Andrew C Nelson. Epiregulin-induced matrix metalloproteinase-1 contributes to early stages of breast tumorigenesis [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 P4-06-01.

Abstract P2-04-06: Demonstration of immune cell and pathway heterogeneity in Singapore DCIS samples using novel hyperplexing method (MultiOmyx®)

Abstract Breast cancer is the most common malignancy in Singapore women with rising incidence across all ethnic groups (Chinese, Malays and Indians). Ductal carcinoma in situ (DCIS) is the earliest non-invasive stage of disease and has been shown to account for approximately 26% of diagnoses in all women participating in the Breast-Screen Singapore program. Despite availability of breast screening, there are still Singapore women presenting with locally advanced breast cancer. The goal of the current study was to investigate pathway and immune cell heterogeneity in low, intermediate and high nuclear grade DCIS using a newly developed method for in situ hyperplexed analysis of multiple proteins in a single FFPE tissue (MultiOmyx). FFPE samples from patients (n= 15) diagnosed with DCIS were provided by Singapore General Hospital. Patients were of Chinese origin, ranged from 50-59 years, were all post-menopausal, and included low (n=5), intermediate (n=5) and high grade (n=5) samples. All histological diagnoses were reviewed by a single pathologist. Following a single antigen retrieval step, DAPI and cytokeratin staining was conducted and imaged at 10X. Based on DAPI, cytokeratin and autofluorescence, an H&E-like image was generated for each sample. Using this image, approximately 30 regions of interest (ROI) were selected per slide/patient, including normal and DCIS regions with and without immune cells. In total, 15 biomarkers were imaged following an iterative process of IF staining and dye inactivation. These included CD4 (T-cells), CD8 (T-cells), CD20 (B-cells), CD68 (macrophages), CD10 (myoepithelial cells), CD44v6 (cancer stem cell), Her2, Her4, EGFR (ErbB family proteins), pmTOR (cell growth), SLC7A5 (Mammostrat) and epithelium and cell segmentation markers (pan-cadherin, pan-cytokeratin, S6 and Na+K+ATPase). Images were evaluated for quality and processed to generate single cell quantification data for each marker in the epithelium and stroma. Single cell data was compared with clinical and histological features (grade, DCIS/ low immune cells, DCIS/ high immune cells). Cell-based k-means clustering was then performed for biomarkers in both the epithelial cells (Her4, Her2, panCK, SLC7A5, EGFR, NaKATPase, pmTOR, and CD10) and stromal cells (CD4, CD8, CD20, CD68). Using consensus clustering, the optimum number of clusters was found to be six in both the epithelium and stroma. No distinct clusters were associated with histological grade. Normal ROIs tended to be associated with higher CD10 and EGFR, moderate CD20 and low or negative for other markers. DCIS with low immune cells tended to have higher Her4, pmTOR and moderate CD68 while DCIS with high immune cells tended to have high Her2 and moderate to high CD4, CD20 and CD68. A larger study is needed to associate the findings with outcome. This technology provides a way to elucidate mechanisms of early disease and illustrates the complex inter-play between grade, pathway activation and immune response. Citation Format: Nicole E LaPlante, Yunxia Sui, Michael J Gerdes, Sean Dinn, Rong Zhang, Sireesha Kaanumalle, Elizabeth McDonough, Christina Lowes, Craig Allred, Fiona Ginty, Thomas Foo, Puay-Hoon Tan. Demonstration of immune cell and pathway heterogeneity in Singapore DCIS samples using novel hyperplexing method (MultiOmyx®) [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 P2-04-06.

Methylation profiling of ductal carcinoma in situ and its relationship to histopathological features.

IntroductionDNA methylation is a well-studied biomarker in invasive breast cancer, but its role in ductal carcinoma in situ (DCIS) is less well characterized. The aims of this study are to assess the methylation profile in DCIS for a panel of well-characterized genes that are frequently methylated in breast cancer, to investigate the relationship of methylation with pathological features, and to perform a proof-of-principle study to evaluate the practicality of methylation as a biomarker in diagnostic DCIS material.MethodsPromoter CpG island methylation for a panel of 11 breast cancer-related genes was performed by methylation-sensitive high resolution melting (MS-HRM). Formalin-fixed, paraffin-embedded (FFPE) biopsies from 72 samples of pure DCIS (DCIS occurring in the absence of synchronous invasive carcinoma), 10 samples of mixed DCIS (DCIS adjacent to invasive carcinoma), and 18 samples of normal breast epithelium adjacent to a DCIS lesion were micro-dissected prior to DNA extraction.ResultsMethylation was seen for all the tested genes except BRCA1. RASSF1A was the most frequently methylated gene (90% of DCIS samples) and its methylation was associated with comedo necrosis (p = 0.018). Cluster analysis based on the methylation profile revealed four groups, the highly methylated cluster being significantly associated with high nuclear grade, HER2 amplification, negative estrogen receptor (ER) α status, and negative progesterone receptor (PgR) status, (p = 0.038, p = 0.018, p <0.001, p = 0.001, respectively). Methylation of APC (p = 0.017), CDH13 (p = 0.017), and RARβ (p <0.001) was associated with negative ERα status. Methylation of CDH13 (p <0.001), and RARβ (p = 0.001) was associated with negative PgR status. Methylation of APC (p = 0.013) and CDH13 (p = 0.026) was associated with high nuclear grade. Methylation of CDH13 (p = 0.009), and RARβ (p = 0.042) was associated with HER2-amplification.ConclusionsDNA methylation can be assessed in FFPE-derived samples using suitable methodologies. Methylation of a panel of genes that are known to be methylated in invasive breast cancer was able to classify DCIS into distinct groups and was differentially associated with phenotypic features in DCIS.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0423-9) contains supplementary material, which is available to authorized users.

Abstract LB-153: Influence of macrophages and p38α/β signaling on early metastatic dissemination of premalignant ErbB2+ mammary epithelial cells

Abstract Metastases originate from tumor cells that disseminate from the primary site through a process aided by macrophages. This was proposed to occur only during late invasive stages of cancer. However, the presence of tumor cells in the bone marrow of patients with ductal carcinoma in situ (DCIS), a pre-invasive form of breast cancer, argues that these events can take place earlier. The mechanisms driving this process are poorly understood. Our lab showed that in the MMTV-ErbB2 breast cancer model, inhibition of p38 signaling accelerated disease progression and enhances early dissemination. Using this same model and knowing that macrophages are key for mammary gland development and remodeling, we investigated whether these inflammatory cells might actively participate in early dissemination. We now show that F4/80+ macrophages associate with mammary ducts in the periphery of the myoepithelial layer but are never inside the ducts in wild type or in pre-malignant 18-20 week old MMTV-ErbB2 mice mammary glands. We found that macrophages are recruited into epithelial layers of pre-malignant lesions, when the ErbB2 oncogene is amplified and p38 activity is lost. This event showed a high correlation with enhanced dissemination of ErbB2+CK8/18+ mammary epithelial cells (MECs) detected in circulation or the bone marrow. Sites where macrophages had invaded the ducts showed disruption of the myoepithelium and reduced E-Cadherin expression in MECs. Depletion of macrophages from pre-malignant MMTV-Neu mice abolished p38 driven early dissemination. Importantly, DCIS samples from patients frequently contained intra-epithelial macrophages. These data suggest that macrophages may actively aid early dissemination from pre-malignant tissue, but only in the context of p38 inhibition and ErbB2 activation. Further, in areas of macrophage infiltration, luminal ductal cells may downregulate E-cadherin, which could foster cell migration/invasion. Our data in human DCIS samples further underscores the relevance of our findings and indicates that analysis of macrophages in DCIS lesions might be useful to better identify patients at high risk of carrying disseminated disease. Citation Format: Nina Linde, Arthur Mortha, Soledad Sosa, Miriam Merad, Julio Aguirre-Ghiso. Influence of macrophages and p38α/β signaling on early metastatic dissemination of premalignant ErbB2+ mammary epithelial cells. [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 LB-153. doi:10.1158/1538-7445.AM2014-LB-153

Genome-wide DNA methylation profiles in progression to in situand invasive carcinoma of the breast with impact on gene transcription and prognosis

BackgroundDuctal carcinoma in situ (DCIS) of the breast is a precursor of invasive breast carcinoma. DNA methylation alterations are thought to be an early event in progression of cancer, and may prove valuable as a tool in clinical decision making and for understanding neoplastic development.ResultsWe generate genome-wide DNA methylation profiles of 285 breast tissue samples representing progression of cancer, and validate methylation changes between normal and DCIS in an independent dataset of 15 normal and 40 DCIS samples. We also validate a prognostic signature on 583 breast cancer samples from The Cancer Genome Atlas. Our analysis reveals that DNA methylation profiles of DCIS are radically altered compared to normal breast tissue, involving more than 5,000 genes. Changes between DCIS and invasive breast carcinoma involve around 1,000 genes. In tumors, DNA methylation is associated with gene expression of almost 3,000 genes, including both negative and positive correlations. A prognostic signature based on methylation level of 18 CpGs is associated with survival of breast cancer patients with invasive tumors, as well as with survival of patients with DCIS and mixed lesions of DCIS and invasive breast carcinoma.ConclusionsThis work demonstrates that changes in the epigenome occur early in the neoplastic progression, provides evidence for the possible utilization of DNA methylation-based markers of progression in the clinic, and highlights the importance of epigenetic changes in carcinogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0435-x) contains supplementary material, which is available to authorized users.

The target for statins, HMG-CoA reductase, is expressed in ductal carcinoma-in situ and may predict patient response to radiotherapy.

Patients with ductal carcinoma-in-situ (DCIS) are currently not prescribed adjuvant systemic treatment after surgery and radiotherapy. Prediction of DCIS patients who would benefit from radiotherapy is warranted. Statins have been suggested to exert radio-sensitizing effects. The target for cholesterol-lowering statins is HMG-CoA reductase (HMGCR), the rate-limiting enzyme in the mevalonate pathway. The aim of this study was to examine HMGCR expression in DCIS and study its treatment predictive value. A population-based cohort including 458 women diagnosed with primary DCIS between 1986 and 2004 were followed until November 2011 to study long-term survival. Tumor tissue microarrays were constructed, and immunohistochemical analyses were performed to detect cytoplasmic protein expression of HMGCR. The association between DCIS HMGCR expression and invasive breast cancer recurrence-free survival (RFSinv) and overall survival (OS) was analyzed by Kaplan-Meier curves, log rank test, and Cox proportional hazard analysis. HMGCR was strongly expressed in 24 % of the assessed DCIS samples, moderately expressed in 46 %, and weakly expressed in 23 %; no expression was detected in 7 % of the samples. During the follow-up time (median 13.8 years), 61 patients were diagnosed with an invasive breast cancer recurrence, and 80 patients died. A crude analysis showed no survival benefit from radiotherapy. However, patients with strong HMGCR expression showed an improved RFSinv (log rank, p = 0.03) and OS (log rank, p = 0.04) after radiotherapy. No statistically significant interaction was observed for HMGCR and radiotherapy (RFSinv p = 0.69 and OS p = 0.29). This study demonstrates HMGCR expression in DCIS and suggests HMGCR as a predictive marker of response to postoperative radiotherapy in DCIS, although the test for interaction was nonsignificant. Future DCIS studies addressing the potential of statin treatment targeting HMGCR are warranted.

Emerging Patterns in the Distribution of Trace Elements in Ovarian, Invasive and In-Situ Breast Cancer

Breast cancer is the most common cancer and ovarian cancer is the 8th most common cancer affecting women worldwide. This study highlights the changes of trace element levels accompanied by the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast, using micro probe Synchrotron Radiation X-ray Fluorescence (μSRXRF). The average values for the increase in Ca, Fe and Zn in tumour regions with respect to surrounding regions for the DCIS samples were significantly higher compared to the increase in the IDC samples (P <0.01).This study was also carried out to find a connection between ovarian cancer and breast cancer with respect to the cellular distribution of Ca, Cu, Fe, and Zn. For IDC, DCIS and ovarian cases, the statistical analysis reveals a significant increase in the levels of Ca, Cu and Zn concentrations in cancer tissue when compared to the normal surrounding tissue. For Fe, the differences between tumour regions with respect to surrounding regions were found to be not significant in IDC and ovarian cases. In DCIS cases, the results reveal a significant increase in the levels of Fe in cancer tissue when compared to the surrounding normal breast tissue (P <0.01).

Matricellular protein CCN1 (CYR61) expression is associated with high-grade ductal carcinoma in situ.

Cysteine-rich protein 61, connective tissue growth factor, and nephroblastoma overexpressed gene (CCN) comprise a family of matricellular proteins that have multiple physiologic functions including development, tissue repair, cell adhesion, migration, and proliferation. The expression of CCN1, cyclin D1, β-catenin, and p53 was explored by immunohistochemistry in different grades of ductal carcinoma in situ (DCIS) cases. These cases did not contain any infiltrating carcinoma components. In addition, all cysteine-rich protein 61 gene exons (encoding the CCN1 protein) were sequenced in 30 samples. Allred and H-scores were calculated for expression in both DCIS and the surrounding benign breast tissue. All cases of DCIS showed degrees of cytoplasmic CCN1 staining with median H-scores of 170, 160, and 60 in grades 3, 2, and 1, respectively (P = .043). Twelve of 28 DCIS 3, 1 of 15 DCIS 2, and 0 of 18 DCIS 1 also showed nuclear staining for CCN1. The cytoplasmic staining difference was preserved when the cases were divided into estrogen receptor (ER)+/DCIS grade 1, ER+/DCIS 2 and 3, and ER-/DCIS 2 and 3 by the H-score (P = .037). Cyclin D1 expression was positively correlated with the CCN1 cytoplasmic H-score in all DCIS samples (P = .038). Membranous β-catenin expression correlated with the grade of intraepithelial carcinoma by both H-score (P = .047) and Allred score (P = .026). Our results suggest that CCN1 has a role in the development of intraepithelial carcinoma. CCN1 expression correlates with grade of DCIS independent of ER status. It can induce cell cycle progression through cyclin D1. It is warranted to study high expression of CCN1 in DCIS as an independent risk factor in a larger cohort.

Altered Microenvironment Promotes Progression of Preinvasive Breast Cancer: Myoepithelial Expression of αvβ6 Integrin in DCIS Identifies High-risk Patients and Predicts Recurrence

This study investigated the functional and clinical significance of integrin αvβ6 upregulation in myoepithelial cells of ductal carcinoma in situ (DCIS). Archival samples of DCIS and DCIS with associated invasion (n = 532) were analyzed for expression of αvβ6 by immunohistochemistry and ability to predict recurrence and progression assessed in an independent, unique cohort of DCIS cases with long-term follow-up. Primary myoepithelial cells and myoepithelial cell lines, with and without αvβ6 expression, were used to measure the effect of αvβ6 on growth and invasion of tumor cell lines in vitro and in a xenograft mouse model. Involvement of TGFβ signaling was established using mink lung epithelial cell (MLEC) assay and antibody inhibition, and expression and activation of matrix metalloproteinase (MMP)-9 established by Real Time-PCR and zymography. Expression of αvβ6 is significantly associated with progression to invasive cancer (P < 0.006) and with recurrence over a median follow-up of 114 months in a series of matched DCIS cases treated with local excision. We show that expression of αvβ6 drives myoepithelial cells to promote tumor cell invasion in vitro and enhances mammary tumor growth in vivo. The tumor-promoting effect of αvβ6-positive myoepithelial cells is dependent on TGFβ-driven upregulation of MMP9 and can be abrogated by inhibiting this pathway. These findings indicate that altered myoepithelial cells in DCIS predict disease progression and recurrence and show that upregulation of αvβ6 on myoepithelial cells generates a tumor promoter function through TGFβ upregulation of MMP-9. These data suggest that expression of αvβ6 may be used to stratify patients with DCIS.

Abstract C23: The quest for biomarkers of progressive DCIS using archived tissue

Abstract Background: Presently, an indolent ductal carcinoma in situ (DCIS) cannot be distinguished from a progressive tumor, making the appropriate treatment of DCIS patients a major clinical dilemma. Here, we tested if measuring protein levels in archived tissue could facilitate the development of biomarkers (BMs) accurately discriminating progressive from non-invasive DCIS. We theorized that BMs of progressive lesion are measurable parameters that distinguish pure DCIS from DCIS with concurrent invasive breast cancer (IBC). To test the hypothesis, we studied the expression of the insulin-like growth factor I receptor (IGF-IR), Ras oncogene –like protein 1 (Rap1), and oncoprotein Vav2 implicated in the progression of IBC. Methods: We developed and applied a new quantitative imaging-based uniplex (IBU) method to measure in-tissue protein expression. The quantification was performed on a continuous scale, and multiple repeated measurements of relative (rather than absolute) intensity of immunohistochemical staining took into account fluctuations of background. Protein profiling was completed in 211 breast samples: 42 histologically normal, 71 cancer in situ (with/without associated invasion), and 98 IBC. Groups were compared using one-way analysis of variance (ANOVA) followed by pair-wise t-tests with Tukey's correction for multiple testing. Receiver operating characteristic (ROC) curves were constructed to evaluate how well each BM can predict tumor type. Results: A pair-wise comparison among tissue groups revealed statistically significantly higher levels of IGF-IR and Rap1 in the in situ and IBC groups than in the normal group (P &amp;lt; 0.0001). Strikingly, Vav2 levels in the in situ group were similar to the normal group but remained significantly lower than in the IBC group (P &amp;lt; 0.0001). To account for variations in the invasive potential of DCIS, we further stratified DCIS samples into pure (n=35), with microinvasive carcinoma (DCIS/T1mic, n=11), and with IBC (DCIS/IBC, n=25) subgroups. Compared to normal epithelium, significant increases in IGF-IR (P = 0.0025) and Rap1 (P = 0.0069) were found in pure DCIS but not in DCIS /T1mic and DCIS/IBC. The predictive abilities of IGF-IR and Rap1 measurements for discriminating DCIS subgroups were low. Remarkably, Vav2 levels in the pure DCIS subgroup were similar to the normal group, but were increased in DCIS /T1mic and further significantly increased in DCIS /IBC (P = 0.0347). Moreover, the area under the ROC curve indicated the predictive ability of Vav2 measurements to distinguish progressive from non-invasive DCIS (AUC=0.71; with OR=2.42, 95% CI, 1.26-4.65) Conclusions: The application of our IBU method identified previously undetected changes in IGF-IR, Rap1, and Vav2 that co-evolve with IBC progression. Vav2 may be a valuable BM for the diagnosis of progressive DCIS. Improvement in earlier detection of DCIS with aggressive phenotype would go a long way in advancing efforts towards prevention of IBC. Citation Format: Marina A. Guvakova, YunQing Jiang, Indira Prabakaran, Fei Wan, Nandita Mitra, Douglas L. Fraker, Paul J. Zhang. The quest for biomarkers of progressive DCIS using archived tissue. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C23.

Abstract B40: The quest for biomarkers of progressive DCIS using archived tissue.

Abstract Background: Presently, an indolent ductal carcinoma in situ (DCIS) cannot be distinguished from a progressive tumor, making the appropriate treatment of DCIS patients a major clinical dilemma. Here, we tested if measuring protein levels in archived tissue could facilitate the development of biomarkers (BMs) accurately discriminating progressive from non-invasive DCIS. We theorized that BMs of progressive lesion are measurable parameters that distinguish pure DCIS from DCIS with concurrent invasive breast cancer (IBC). To test the hypothesis, we studied the insulin-like growth factor I receptor (IGF-IR), Ras oncogene-like protein 1 (Rap1), and oncoprotein Vav2 implicated in the progression of IBC. Methods: We developed and applied a new quantitative imaging-based uniplex (IBU) method to measure in-tissue protein expression. The quantification was performed on a continuous scale, and multiple repeated measurements of relative (rather than absolute) intensity of immunohistochemical staining took into account fluctuations of background. Protein profiling was completed in 211 breast samples: 42 histologically normal, 71 cancer in situ (with/without associated invasion), and 98 IBC. Groups were compared using one-way analysis of variance (ANOVA) followed by pair-wise t-tests with Tukey's correction for multiple testing. Receiver operating characteristic (ROC) curves were constructed to evaluate how well each BM can predict tumor type. Results: A pair-wise comparison among tissue groups revealed statistically significantly higher levels of IGF-IR and Rap1 in the in situ and IBC groups than in the normal group (P &amp;lt; 0.0001). Strikingly, Vav2 levels in the in situ group were similar to the normal group but remained significantly lower than in the IBC group (P &amp;lt; 0.0001). To account for variations in the invasive potential of DCIS, we further stratified DCIS samples into pure (n=35), with microinvasive carcinoma (DCIS/T1mic, n=11), and with IBC (DCIS/IBC, n=25) subgroups. Compared to normal epithelium, significant increases in IGF-IR (P = 0.0025) and Rap1 (P = 0.0069) were found in pure DCIS without further changes in DCIS /T1mic and DCIS/IBC. The predictive abilities of IGF-IR and Rap1 measurements for discriminating DCIS subgroups were low. Remarkably, Vav2 levels in the pure DCIS subgroup were similar to the normal group, but were increased in DCIS /T1mic and also significantly increased in DCIS /IBC (P = 0.0347). Moreover, the area under the ROC curve indicated the predictive ability of Vav2 measurements to distinguish progressive from non-invasive DCIS (AUC=0.71; 95% CI, 1.26-4.65). Conclusions: The application of our IBU method identified previously undetected changes in IGF-IR, Rap1, and Vav2 that co-evolve with IBC progression. Vav2 may be a valuable BM for the diagnosis of progressive DCIS. Improvement in earlier detection of DCIS with aggressive phenotype would go a long way in advancing efforts towards prevention of IBC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B40. Citation Format: Marina A. Guvakova, YunQing Jiang, Indira Prabakaran, Fei Wan, Nandita Mitra, Douglas L. Fraker, Paul J. Zhang. The quest for biomarkers of progressive DCIS using archived tissue. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B40.

Diagnostic accuracy of fiberoptic ductoscopy plus in vivo iodine staining for intraductal proliferative lesions

Background Iodine staining during endoscopy has been successfully used to detect early carcinomatous and precancerous lesions in the esophagus, cervix, and oral cavity. The objective of this study was to determine the diagnostic accuracy of fiberoptic ductoscopy (FDS) plus in vivo iodine staining for intraductal proliferative lesions of the breast. Methods We performed periodic acid-Schiff (PAS) and in vitro iodine staining on 52 and 64 specimens of benign mammary hyperplasia, respectively, and 57 and 53 specimens of ductal carcinoma in situ (DCIS), respectively. Next, FDS was performed on 177 recurrent nipple discharge patients who were randomly divided into two groups. One group was iodine-staining group in which 92 patients were randomly selected to undergo iodine staining during FDS, and the remaining 85 were assigned to the control group. Biopsy specimens of suspicious lesions were obtained and subjected to histopathological examination. Results Following PAS staining, benign mammary hyperplasia lesions were positively stained, while negligible PAS positivity was observed in the DCIS lesions (P &lt;0.05). Following in vitro iodine staining, benign mammary hyperplasia specimens appeared dark brown, whereas DCIS samples appeared significantly lighter or unstained. Compared with the pathological examination results, FDS with iodine staining showed an agreement rate in the diagnosis of ductal intraepithelial neoplasia (DIN), sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and Youden index of 97.82%, 98.83%, 83.33%, 5.93, 0.014, and 0.8216, respectively; the corresponding values for FDS without iodine staining were 88.24%, 89.16%, 50.00%, 1.78, 0.217, and 0.3916, respectively. Conclusion FDS with iodine staining was superior to conventional FDS for the diagnosis of DIN and is valuable for breast cancer prevention.

A Multigene Expression Assay to Predict Local Recurrence Risk for Ductal Carcinoma In Situ

A Multigene Expression Assay to Predict Local Recurrence Risk for Ductal Carcinoma In Situ

Combined Inhibition of ErbB1/2 and Notch Receptors Effectively Targets Breast Ductal Carcinoma In Situ (DCIS) Stem/Progenitor Cell Activity Regardless of ErbB2 Status

Pathways involved in DCIS stem and progenitor signalling are poorly understood yet are critical to understand DCIS biology and to develop new therapies. Notch and ErbB1/2 receptor signalling cross talk has been demonstrated in invasive breast cancer, but their role in DCIS stem and progenitor cells has not been investigated. We have utilised 2 DCIS cell lines, MCF10DCIS.com (ErbB2-normal) and SUM225 (ErbB2-overexpressing) and 7 human primary DCIS samples were cultured in 3D matrigel and as mammospheres in the presence, absence or combination of the Notch inhibitor, DAPT, and ErbB1/2 inhibitors, lapatinib or gefitinib. Western blotting was applied to assess downstream signalling. In this study we demonstrate that DAPT reduced acini size and mammosphere formation in MCF10DCIS.com whereas there was no effect in SUM225. Lapatinb reduced acini size and mammosphere formation in SUM225, whereas mammosphere formation and Notch1 activity were increased in MCF10DCIS.com. Combined DAPT/lapatinib treatment was more effective at reducing acini size in both DCIS cell lines. Mammosphere formation in cell lines and human primary DCIS was reduced further by DAPT/lapatinib or DAPT/gefitinib regardless of ErbB2 receptor status. Our pre-clinical human models of DCIS demonstrate that Notch and ErbB1/2 both play a role in DCIS acini growth and stem cell activity. We report for the first time that cross talk between the two pathways in DCIS occurs regardless of ErbB2 receptor status and inhibition of Notch and ErbB1/2 was more efficacious than either alone. These data provide further understanding of DCIS biology and suggest treatment strategies combining Notch and ErbB1/2 inhibitors should be investigated regardless of ErbB2 receptor status.

Lectin Histochemistry Reveals Changes in Carbohydrate Expression on Morphological Types of Breast Ductal Carcinoma in situ

Breast Ductal Carcinoma in situ (DCIS) show a high degree of architectural heterogeneity and what kind of DCIS lesions will progress to invasive disease remains unknown. Thus, its characterization remains a complex process. In addition, in the same tumor we can find different types of lesions that presented different biological behaviour and morphology. Carbohydrate expression changes play crucial roles in carcinogenesis of the breast, becoming the study these changes a useful tool for an accurate predictor. In this context, this work aimed analyzed the differential expression of specific carbohydrates among the DCIS lesion through lectin histochemistry and its correlation with its behaviors. Glycobiology expression of 218 DCIS samples of human breast cancer was investigated by MAL-II, PNA, WGA, PHA-L and SNA lectins. MAL-II was more expressed (p=0.0191) in cribriform (positive in 100%) than in comedo (positive in 41.2%) and solid lesions (p=0.0440). However, PNA profile showed a higher reactivity in comedo (p=0.0052) and micropapillary (p=0.0010) lesions when compared to solid one. The other lectins showed different expression degrees in all lesions analyzed but none was statistically relevant. Our results revels different carbohydrate patterns concerning different DCIS morphological subtypes indicating that these lesions could act as independent biological entities.

NEW MARKERS FOR BREAST CANCER

Background: The ductal carcinoma in situ (DCIS) of the mammary gland represents an early, pre-invasive stage in the development of invasive breast carcinoma. Since DCIS is a curable disease, it would be highly desirable to identifymolecularmarkers that allowearly detection.Mice transgenic for theWAP-SV40 early genome region were used as a model for DCIS development. Gene expression profiling was carried out on DCIS-bearing mice and control animals. Additionally, a set of human DCIS and invasive mammary tumors were analyzed in a similar fashion. Enhanced expression of these marker genes in human and murine samples was validated by quantitative RT-PCR. Besides, marker gene expression was also validated by immunohistochemistry of human samples. Furthermore in silico analyses using an online microarray database were performed. Results: In DCIS-mice seven genes were identified that were significantly up-regulated in DCIS: DEPDC1, NUSAP1, EXO1, RRM2, FOXM1, MUC1 and SPP1. A similar up-regulation of homologues of the murine genes was observed in human DCIS samples. Enhanced expression of these genes in DCIS and IDC (invasive ductal carcinoma) was validated by quantitative RT-PCRand immunohistochemistry. Conclusions: By comparingmurinemarkers for the ductal carcinoma in situ (DCIS) of the mammary gland with genes up-regulated in human DCIS-sampleswe were able to identify a set of genes whichmight allowearly detection of DCIS and invasive carcinomas in the future. The similarities between gene expression in DCIS andinvasive carcinomas in our data suggest that the early detection and treatment of DCIS is of utmost relevance for the survival of patients who are at high risk of developing breast carcinomas.