Progression To Invasive Ductal Carcinoma Research Articles

Transcriptome analysis reveals differences in cell cycle, growth and migration related genes that distinguish fibroblasts derived from pre-invasive and invasive breast cancer.

As the most common form of pre-invasive breast cancer, ductal carcinoma in situ (DCIS) affects over 50,000 women in the US annually. Despite standardized treatment involving lumpectomy and radiation therapy, up to 25% of patients with DCIS experience disease recurrence often with invasive ductal carcinoma (IDC), indicating that a subset of patients may be under-treated. As most DCIS cases will not progress to invasion, many patients may experience over-treatment. By understanding the underlying processes associated with DCIS to IDC progression, we can identify new biomarkers to determine which DCIS cases may become invasive and improve treatment for patients. Accumulation of fibroblasts in IDC is associated with disease progression and reduced survival. While fibroblasts have been detected in DCIS, little is understood about their role in DCIS progression. We sought to determine 1) whether DCIS fibroblasts were similar or distinct from normal and IDC fibroblasts at the transcriptome level, and 2) the contributions of DCIS fibroblasts to breast cancer progression. Fibroblasts underwent transcriptome profiling and pathway analysis. Significant DCIS fibroblast-associated genes were further analyzed in existing breast cancer mRNA databases and through tissue array immunostaining. Using the sub-renal capsule graft model, fibroblasts from normal breast, DCIS and IDC tissues were co-transplanted with DCIS.com breast cancer cells. Through transcriptome profiling, we found that DCIS fibroblasts were characterized by unique alterations in cell cycle and motility related genes such as PKMYT1, TGF-α, SFRP1 and SFRP2, which predicted increased cell growth and invasion by Ingenuity Pathway Analysis. Immunostaining analysis revealed corresponding increases in expression of stromal derived PKMYT1, TGF-α and corresponding decreases in expression of SFRP1 and SFRP2 in DCIS and IDC tissues. Grafting studies in mice revealed that DCIS fibroblasts enhanced breast cancer growth and invasion associated with arginase-1+ cell recruitment. DCIS fibroblasts are phenotypically distinct from normal breast and IDC fibroblasts, and play an important role in breast cancer growth, invasion, and recruitment of myeloid cells. These studies provide novel insight into the role of DCIS fibroblasts in breast cancer progression and identify some key biomarkers associated with DCIS progression to IDC, with important clinical implications.

Proteomics-Based Identification of Dysregulated Proteins and Biomarker Discovery in Invasive Ductal Carcinoma, the Most Common Breast Cancer Subtype

Invasive ductal carcinoma (IDC) is the most common histological subtype of malignant breast cancer (BC), and accounts for 70-80% of all invasive BCs. IDC demonstrates great heterogeneity in clinical and histopathological characteristics, prognoses, treatment strategies, gene expressions, and proteomic profiles. Significant proteomic determinants of the progression from intraductal pre-invasive malignant lesions of the breast, which characterize a ductal carcinoma in situ (DCIS), to IDC, are still poorly identified, validated, and clinically applied. In the era of "6P" medicine, it remains a great challenge to determine which patients should be over-treated versus which need to be actively monitored without aggressive treatment. The major difficulties for designating DCIS to IDC progression may be solved by understanding the integrated genomic, transcriptomic, and proteomic bases of invasion. In this review, we showed that multiple proteomics-based techniques, such as LC-MS/MS, MALDI-ToF MS, SELDI-ToF-MS, MALDI-ToF/ToF MS, MALDI-MSI or MasSpec Pen, applied to in-tissue, off-tissue, BC cell lines and liquid biopsies, improve the diagnosis of IDC, as well as its prognosis and treatment monitoring. Classic proteomics strategies that allow the identification of dysregulated protein expressions, biological processes, and interrelated pathway analyses based on aberrant protein-protein interaction (PPI) networks have been improved to perform non-invasive/minimally invasive biomarker detection of early-stage IDC. Thus, in modern surgical oncology, highly sensitive, rapid, and accurate MS-based detection has been coupled with "proteome point sampling" methods that allow for proteomic profiling by in vivo "proteome point characterization", or by minimal tissue removal, for ex vivo accurate differentiation and delimitation of IDC. For the detection of low-molecular-weight proteins and protein fragments in bodily fluids, LC-MS/MS and MALDI-MS techniques may be coupled to enrich and capture methods which allow for the identification of early-stage IDC protein biomarkers that were previously invisible for MS-based techniques. Moreover, the detection and characterization of protein isoforms, including posttranslational modifications of proteins (PTMs), is also essential to emphasize specific molecular mechanisms, and to assure the early-stage detection of IDC of the breast.

Abstract IA035: DCIS to IDC progression - a key step of immune escape

Abstract The cellular and molecular mechanisms underlying ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) transition are poorly understood. We have analyzed genetic, gene expression, epigenetic profiles of cells composing the tumor and identified cell type-specific changes during tumor progression. Importantly, we detected significant changes in the immune cell composition during tumor progression implying immune escape in invasive tumors and active adaptive immune response in DCIS. We demonstrate that these progression stage-specific alterations are also present in a rat model of breast cancer and that combined targeting of multiple components of the tumor ecosystem increases the efficacy of anti-cancer therapies. Our data highlight the role of microenvironmental, especially immune-related changes in driving breast tumor progression and emphasize the importance of studying pre-invasive tumors. Citation Format: Kornelia Polyak. DCIS to IDC progression - a key step of immune escape [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 IA035.

Abstract P5-12-09: Cis-acting super-enhancer lncRNAs as diagnostic markers to early-stage breast cancer

Abstract Background: Increased breast cancer screening over the past four decades has led to a substantial rise in the diagnosis of ductal carcinoma in situ (DCIS).Although DCIS lesions precede invasive ductal carcinoma (IDC),they do not always transform into cancer. The current standard-of-care for DCIS is an aggressive course of therapy to prevent invasive and metastatic disease resulting in over-diagnosis and over-treatment. Thus, there is a critical need to identify functional determinants of progression of DCIS to IDC to allow discrimination between indolent and aggressive disease. Super-enhancers are regulatory regions of DNA that play critical roles in driving expression of genes that define cell fate decisions and importantly, their normal function can become co-opted during tumorigenesis. Recent studies have found that super-enhancers, in addition to promoting other gene transcription, are themselves transcribed producing super-enhancer associated long noncoding RNAs (SE-lncRNAs).These SE-lncRNAs can interact with their associated enhancer regions in cis and influence activities and expression of neighboring genes. Furthermore, they represent a novel, untapped group of therapeutic targets. Methods: The MCF10Abreast cancer progression series is composed of four cell lines that mimic the progression of normal cells (MCF10A)to atypia(AT1),to DCIS (DCIS),and finally, to IDC (CA1).With an integrative analysis of enhancer loci with global expression of SE-lncRNAs in the progression series, we have identified differentially expressed SE-lncRNAs which can identify mechanisms for DCIS to IDC progression. Furthermore, cross-referencing these SE-lncRNAs with patient samples in the TCGA database, we have unveiled 31clinicallyrelevant SE-lncRNAs that potentially interact with their enhancer to regulate nearby gene expression. To complement SE-lncRNA expression studies, we conducted an unbiased global analysis of super-enhancers that are acquired or lost in progression. Results: Here we designate SE-lncRNAs RP11-379F4.4and RP11-465B22.8as potential markers of progression of DCIS to IDC through regulation of the expression of their neighboring genes (RARRES1and miR200brespectively).Moreover, we classified 403super-enhancer regions in MCF10Anormal cells, 627in AT1,1053in DCIS, and 320in CA1cells. Comparison analysis of acquired/lost super-enhancer regions with super-enhancer regions classified in 47ER positive patients, 10TripleNegative Breast Cancer (TNBC)patients, and 11TNBC cell lines reveal critically acquired pathways including STAT signaling and NF-kB signaling. In contrast, protein folding and local estrogen production are identified as major pathways lost in progression. Conclusion: Collectively, these analyses identify differentially expressed SE-lncRNAs and acquired/lost super-enhancers in progression of breast cancer important for promoting DCIS lesions to IDC. Citation Format: Ali S Ropri, Rebecca Devaux, Jonah Eng, Sridar Chittur, Jason Herschkowitz. Cis-acting super-enhancer lncRNAs as diagnostic markers to early-stage breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-09.

Cis-acting super-enhancer lncRNAs as biomarkers to early-stage breast cancer

BackgroundIncreased breast cancer screening over the past four decades has led to a substantial rise in the diagnosis of ductal carcinoma in situ (DCIS). Although DCIS lesions precede invasive ductal carcinoma (IDC), they do not always transform into cancer. The current standard-of-care for DCIS is an aggressive course of therapy to prevent invasive and metastatic disease resulting in over-diagnosis and over-treatment. Thus, there is a critical need to identify functional determinants of progression of DCIS to IDC to allow discrimination between indolent and aggressive disease. Recent studies show that super-enhancers, in addition to promoting other gene transcription, are themselves transcribed producing super-enhancer associated long noncoding RNAs (SE-lncRNAs). These SE-lncRNAs can interact with their associated enhancer regions in cis and influence activities and expression of neighboring genes. Furthermore, they represent a novel, untapped group of therapeutic targets.MethodsWith an integrative analysis of enhancer loci with global expression of SE-lncRNAs in the MCF10A progression series, we have identified differentially expressed SE-lncRNAs which can identify mechanisms for DCIS to IDC progression. Furthermore, cross-referencing these SE-lncRNAs with patient samples in the The Cancer Genome Atlas (TCGA) database, we have unveiled 27 clinically relevant SE-lncRNAs that potentially interact with their enhancer to regulate nearby gene expression. To complement SE-lncRNA expression studies, we conducted an unbiased global analysis of super-enhancers that are acquired or lost in progression.ResultsHere we designate SE-lncRNAs RP11-379F4.4 and RP11-465B22.8 as potential markers of progression of DCIS to IDC through regulation of the expression of their neighboring genes (RARRES1 and miR-200b, respectively). Moreover, we classified 403 super-enhancer regions in MCF10A normal cells, 627 in AT1, 1053 in DCIS, and 320 in CA1 cells. Comparison analysis of acquired/lost super-enhancer regions with super-enhancer regions classified in 47 ER positive patients, 10 triple negative breast cancer (TNBC) patients, and 11 TNBC cell lines reveal critically acquired pathways including STAT signaling and NF-kB signaling. In contrast, protein folding, and local estrogen production are identified as major pathways lost in progression.ConclusionCollectively, these analyses identify differentially expressed SE-lncRNAs and acquired/lost super-enhancers in progression of breast cancer important for promoting DCIS lesions to IDC.

MiR-646/TET1 mediated demethylation of IRX1 promoter upregulates HIST2H2BE and promotes the progression of invasive ductal carcinoma

BackgroundThis study aimed to explore role of miR-646 in breast IDC. MethodsmiR-646, TET1, IRX1, and HIST2H2BE expression was detected by RT-qPCR and/or Western blot analysis. The methylation status of IRX1 promoter region was evaluated by methylation specific PCR. ChIP assay was used to determine the enrichment of TET1 at IRX1 promoter region. Loss- and gain-of functions were performed to determine the roles of miR-646, TET1, IRX1, and HIST2H2BE in cell proliferation, migration, invasion, and apoptosis. The tumor growth, volume, weight, and apoptosis status were measured. ResultsmiR-646 was upregulated while TET1 was downregulated in IDC tissues. miR-646 targeted TET1. Downregulated TET1 impairs demethylation of IRX1 promoter region resulting in reduced expression of IRX1, which subsequently leads to upregulation of HIST2H2BE in IDC. Consequently, elevated HIST2H2BE promotes progression of IDC. ConclusionOur study has demonstrated that miR-646 facilitates the tumorigenesis of IDC via regulating TET1/IRX1/HIST2H2BE axis.

MiRNA‑490‑3p promotes the metastatic progression of invasive ductal carcinoma.

MicroRNA (miRNA/mir)-490-3p has been defined as a tumor suppressor in different types of cancer, including breast cancer. However, miR-490-3p has been shown to function as a tumor suppressor and promoter in a context-dependent manner in hepatocellular and lung cancer. Contrary to previous studies, the present study revealed that miR-490-3p expression was significantly higher in invasive ductal carcinoma (IDC) tissue specimens, the most common form of breast cancer, compared to tumor-adjacent normal tissue specimens (n=20). Its expression was also higher in the more metastatic breast cancer cell line, MDA-MB-231, compared to the non-metastatic breast cancer cell line, MCF7, and the moderately metastatic breast cancer cell line, MDA-MB-468. The expression of miR-490-3p was induced following transforming growth factor (TGF)-β-induced epithelial-to-mesenchymal transition (EMT) in MCF10A cells. Gain-and loss-of-function assays revealed that the expression of miR-490-3p regulated the proliferation, colony formation, EMT, migration and invasion in vitro, but not the apoptosis of MDA-MB-468 and MDA-MB-231 cells. The knockdown of miR-490-3p expression in MDA-MB-231 cells inhibited experimental metastasis in a tumor xenograft assay. As in lung cancer, miR-490-3p was found to target and downregulate the expression of the tumor suppressor RNA binding protein poly r(C) binding protein 1 (PCBP1). PCBP1 protein and miR-490-3p expression inversely correlated in patients with ductal carcinoma in situ (DCIS; n=10; no nodal involvement) and IDC (n=10; different stages of metastatic progression) with a significantly higher miR-490-3p expression in patients with IDC compared to those with DCIS. The expression of miR-490-3p was negatively associated with both overall and disease-free survival in the patients with breast cancer included in the present study. On the whole, the results confirm a pro-metastatic role of miR-490-3p in IDC, establishing it as a biomarker for disease progression in these patients.

Classification models for Invasive Ductal Carcinoma Progression, based on gene expression data-trained supervised machine learning

Early detection of breast cancer and its correct stage determination are important for prognosis and rendering appropriate personalized clinical treatment to breast cancer patients. However, despite considerable efforts and progress, there is a need to identify the specific genomic factors responsible for, or accompanying Invasive Ductal Carcinoma (IDC) progression stages, which can aid the determination of the correct cancer stages. We have developed two-class machine-learning classification models to differentiate the early and late stages of IDC. The prediction models are trained with RNA-seq gene expression profiles representing different IDC stages of 610 patients, obtained from The Cancer Genome Atlas (TCGA). Different supervised learning algorithms were trained and evaluated with an enriched model learning, facilitated by different feature selection methods. We also developed a machine-learning classifier trained on the same datasets with training sets reduced data corresponding to IDC driver genes. Based on these two classifiers, we have developed a web-server Duct-BRCA-CSP to predict early stage from late stages of IDC based on input RNA-seq gene expression profiles. The analysis conducted by us also enables deeper insights into the stage-dependent molecular events accompanying IDC progression. The server is publicly available at http://bioinfo.icgeb.res.in/duct-BRCA-CSP.

Downregulation of klotho β is associated with invasive ductal carcinoma progression.

Klotho β (KLB) is a single-pass transmembrane protein measuring 1,043 amino acids in length that shares 41.2% homology with klotho α (KLA). KLB is a co-receptor and key regulator of the fibroblast growth factor receptor 4 (FGFR4) pathway. KLB interacts with FGFR4 to induce apoptosis and inhibit the proliferation of hepatoma cells, and KLA has been demonstrated to be a tumor suppressor in human breast cancer; however, little is known regarding the role of KLB in breast cancer. In the present study, through an immunohistochemical analysis of invasive ductal carcinoma tissue arrays, low KLB expression was identified in invasive ductal carcinoma samples compared with paired adjacent non-tumorous breast tissues (82 cases). In invasive ductal carcinoma tissues, KLB expression was negatively associated with pathological grade and lymph node metastasis. In 42 cases of paired microdissected breast specimens, the condition of the KLB gene allele was examined to determine the loss of heterozygosity (LOH), and selective LOH was identified at the KLB locus in 57.1% of primary tumors. These data suggest that KLB may be associated with the progression and metastasis of invasive ductal carcinoma, and therefore have clinical and therapeutic importance.

The highly expressed COL4A1 genes contributes to the proliferation and migration of the invasive ductal carcinomas.

BackgroundInvasive ductal carcinoma is a kind of very typical breast cancer. The goal of our research was to figure out the molecular mechanism of Invasive ductal carcinoma and to find out its potential therapy targets.ResultsThe total amount of 478 differentially expressed genes in Invasive ductal carcinoma which compared with normal breast epithelial cells were recognized. Functional enrichment analysis proved the most part of differentially expressed genes had connection with ECM-receptor interaction. The two genes lists were contrasted in PPI network, and miRNA regulation networks, The most two crucial genes were identified in our study, which may be helpful to improve Invasive ductal carcinoma treatment. Additionally, experimental results shows that the COL4A1 gene, one of identified genes, played important roles in both of proliferation and colony formation in Invasive ductal carcinoma.ConclusionsInvasive ductal carcinoma could have connection with ECM-receptor mutations. These 9 vital genes could be an important part in the progression of Invasive ductal carcinoma and be offered as therapy targets and prognosis indicator. and the experimental results showed that one of the most crucial genes, COL4A1, was the key gene that influence the proliferation and colony formation of the Invasive ductal carcinoma cell.

Hornerin Is Involved in Breast Cancer Progression.

PurposeThe S100 gene family, which comprises over 20 members, including S100A1, S100A2, S100A8, S100A9, profilaggrin, and hornerin encodes low molecular weight calcium-binding proteins with physiological and pathological roles in keratinization. Recent studies have suggested a link between S100 proteins and human cancer progression. The purpose of the present study was to determine the expression levels of hornerin, S100A8, and S100A9 and evaluate their roles in the progression of invasive ductal carcinoma (IDC).MethodsSeventy cases of ductal carcinoma in situ (DCIS), IDC, and metastatic carcinoma in lymph nodes (MCN) were included. Tissue microarrays were constructed from lesions of DCIS, IDC, and MCN from the same patients. Expression of hornerin, S100A8, and S100A9 was analyzed using immunohistochemistry.ResultsThe expression of hornerin was associated with the estrogen receptor-negative (p=0.003) and the human epidermal growth factor receptor 2-positive (p=0.002) groups. The expression of S100A8 was associated with a higher pT stage (p=0.017). A significant (p<0.001) correlation between the expression of S100A9 and S100A8 was also found. The mean percentages of hornerin-positive tumor cells in DCIS, IDC, and MCN were 1.0%±3.3% (mean±standard deviation), 12.0%±24.0%, and 75.3%± 27.6%, respectively. The expression of hornerin significantly (p<0.001) increased with the progression of carcinoma. The mean levels of S100A8 and S100A9 in DCIS, IDC, and MCN were not significantly (p>0.050) different. The expression of hornerin increased in a stepwise manner (DCIS<IDC<MCN).ConclusionOur data suggest that hornerin is involved in breast cancer progression and malignant transformation from preinvasive lesions.

Monitoring morphological alterations during invasive ductal breast carcinoma progression using multiphoton microscopy.

Morphological alteration of cells and matrices is critical for tumor initiation and progression. Monitoring these alterations during tumor progression is vitally important for making real-time histological diagnoses of tumor staging. In this study, 20 pairs of normal and cancerous human breast tissues were imaged by multiphoton microscopy (MPM), and nuclear area and collagen density were quantified by LSM 5 software (version 3.2). Comparison of MPM images from normal breast tissue with low- and high-grade invasive ductal carcinoma (IDC) lesions clearly showed changes in both cellular features and extracellular matrix architecture during IDC development. Moreover, analysis of nuclear area and collagen density established a quantitative link between these two morphological features and progression of IDC. Present results demonstrated that MPM can provide both qualitative and quantitative evaluations of tumor progression. With additional development, this technique has the potential to make real-time histological diagnoses of tumor staging and guide development of efficacious clinical therapies.

Podoplanin expression in cancer-associated fibroblasts enhances tumor progression of invasive ductal carcinoma of the pancreas

BackgroundInteractions between cancer cells and surrounding cancer-associated fibroblasts (CAFs) play an important role in cancer progression. Invasive ductal carcinoma (IDC) of the pancreas is characterized by abundant fibrous connective tissue called desmoplasia. Podoplanin (PDPN) is a lymphatic vessel marker (D2-40), and expression of PDPN by stromal CAFs has been reported to be a prognostic indicator in various types of cancer.MethodsExpression of PDPN in pancreatic IDCs was assessed by immunohistochemical examination in 105 patients who underwent pancreatic resection. Primary CAFs were established from pancreatic cancer tissue obtained by surgery. Quantitative reverse transcription-polymerase chain reaction and flow cytometric analysis were performed to investigate PDPN expression in CAFs. We sorted CAFs according to PDPN expression, and analyzed the functional differences between PDPN+ CAFs and PDPN– CAFs using indirect co-culture with pancreatic cancer cell lines. We also investigated the culture conditions to regulate PDPN expression in CAFs.ResultsPDPN expression in stromal fibroblasts was associated with lymphatic vessel invasion (P = 0.0461), vascular invasion (P = 0.0101), tumor size ≥3 cm (P = 0.0038), histological grade (P = 0.0344), Union for International Cancer Control classification T stage (P = 0.029), and shorter survival time (P < 0.0001). Primary CAFs showed heterogeneous PDPN expression in vitro. Moreover, migration and invasion of pancreatic cancer cell lines (PANC-1 and SUIT-2) were associated with PDPN expression in CAFs (P < 0.01) and expression of CD10, matrix metalloproteinase (MMP) 2, and MMP3. In cultured CAFs, PDPN positivity changed over time under several conditions including co-culture with cancer cells, different culture media, and addition of growth factor.ConclusionsPDPN-expressing CAFs enhance the progression of pancreatic IDC, and a high ratio of PDPN-expressing CAFs is an independent predictor of poor outcome. Understanding the regulation of the tumor microenvironment is an important step towards developing new therapeutic strategies.

Tu1760 Podoplanin Expressing Fibroblasts Enhance the Tumor Progression of Invasive Ductal Carcinoma of Pancreas, and Podoplanin Expression Was Affected by Cultured Condition

Tu1760 Podoplanin Expressing Fibroblasts Enhance the Tumor Progression of Invasive Ductal Carcinoma of Pancreas, and Podoplanin Expression Was Affected by Cultured Condition

Fibroblasts expressing podoplanin enhance the tumor progression of invasive ductal carcinoma of pancreas

Fibroblasts expressing podoplanin enhance the tumor progression of invasive ductal carcinoma of pancreas

Modified primary tumour/vessel tumour/nodal tumour classification for patients with invasive ductal carcinoma of the breast

Background:We previously reported that the primary tumour/vessel tumour/nodal tumour (PVN) classification is significantly superior to the UICC pTNM classification and the Nottingham Prognostic Index for accurately predicting the outcome of patients with invasive ductal carcinoma of the breast in a manner that is independent of the nodal status and the hormone receptor status.Methods:The purpose of the present study was to compare the outcome predictive power of a modified PVN classification to that of the newly devised pathological UICC pTNM classification and the reclassified Nottingham Prognostic Index in a different group of patients with invasive ductal carcinoma (n=1042) using multivariate analyses by the Cox proportional hazard regression model.Results:The modified PVN classification clearly exhibited a superior significant power, compared with the other classifications, for the accurate prediction of tumour recurrence and tumour-related death among patients with invasive ductal carcinoma in a manner that was independent of the nodal status, the hormone receptor status, and adjuvant therapy status.Conclusion:The modified PVN classification is a useful classification system for predicting the outcome of invasive ductal carcinoma of the breast.

Abstract P4-06-17: MRE11/RAD50/NBS1 (MRN) Complex Functions in Invasion in Breast Cells and Tumor Progression

Abstract Background: The MRN complex is essential in conserving genetic integrity. It recruits ATM kinase to damage sites, and distinguishes and activates homologous recombinational repair of DNA double-strand breaks. Individuals with mutations in the MRE11 and NBS1 genes experience radiation sensitivity and are associated with a higher cancer rate. Previously, we showed that some genes involved in DNA repair are involved in regulating cell invasiveness. Since the MRN complex is downregulated in invasive breast cancer, we hypothesize that change in MRN complex expression may contribute to the pre-invasive to invasive progression of breast cancer. Materials and Methods: The HMT-3522 series of breast epithelial cell lines containing non-invasive S1, pre-invasive S3-C, and invasive T4-2 counterparts were used in 2D or 3D culture. Western blot and immunohistochemistry (IHC) were used to determine MRE11, RAD50, and NBS1 expression levels. Breast tumor tissues each containing benign, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) from 48 patients were used after IRB approval. Two pathologists and Ariol, an automated image acquisition and analysis system, scored IHC signal. Tissues were scored 0 to 3 (0=negative, 3=strong positive). ANOVA p-value of .01 or lower was considered significant for expression comparisons. Regression analysis was used for correlating Estrogen Receptor ER/Progesterone Receptor PR status with MRN expression patterns. MRE11, RAD50, and NBS1 were downregulated using siRNA. Protein expression levels were quantified by western blot. Boyden Chamber assay was used to measure cellular invasion through laminin-rich ECM, lrECM (Matrigel). Results: In non-invasive S1 cells, 3DlrECM culturing resulted in higher levels of MRN than in 2D. In pre-invasive S3-C and invasive T4-2 cells, 3DlrECM no longer upregulated MRN. Consequently, in 3DlrECM cultures non-invasive S1 cells had a higher expression of MRE11, RAD50 and NBS1 compared to invasive T4-2 cells. IHC on tissue sections showed that, on average, normal tissues have higher levels of MRN expression than DCIS and DCIS have higher levels of expression than IDC. Multiple MRN expression patterns were observed in subsets of patients when benign, DCIS, and IDC transitions were compared. Decreases in MRN expression correlated with ER/PR negativity: “single decrease” in benign to DCIS to IDC progression was associated significantly with ER negative status for MRE11 and RAD50, not NBS1. MRE11, RAD50, and NBS1 siRNAs downregulated their protein levels in invasive T4-2 cells, resulting in a significant increase in cell invasiveness through ECM. Discussion: First, we described a new role for ECM signaling in up-regulating the DNA repair protein complex MRN in non-tumorigenic cells, which gets misregulated in pre-invasive and invasive cells resulting in a progressive decrease in MRN levels in culture and in tissues. Reciprocally, we showed that downregulation of MRN upregulates invasion through ECM, suggesting it is a negative regulator of invasion in addition to its role as a gatekeeper of genome stability. Finally, different patterns of MRN expression changes in progression associated with ER/PR status suggest MRN levels as a candidate biomarker of patient-specific clinical outcomes. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-17.

Tumor histology in lymph vessels and lymph nodes for the accurate prediction of outcome among breast cancer patients treated with neoadjuvant chemotherapy

The present study investigated fibrotic foci (FFs), the grading system for lymph vessel tumor emboli (LVTEs), and the histological characteristics of nodal metastatic tumors that were significantly associated with the outcomes of 115 patients with invasive ductal carcinoma (IDC) who had received neoadjuvant chemotherapy. We compared the outcome predictive power of FFs, the grading system for LVTEs, and the histological characteristics of metastatic tumors in lymph nodes with the well-known clinicopathological characteristics of tumor recurrence and tumor-related death in multivariate analyses. The presence of FFs, as assessed by a biopsy performed before neoadjuvant chemotherapy, significantly increased the hazard rates (HRs) for tumor-related death in all the cases and in cases with nodal metastasis. The grading system for LVTEs, which was assessed using surgical specimens obtained after neoadjuvant chemotherapy, was significantly associated with increasing hazard rates (HRs) for tumor recurrence and tumor-related death in all the cases and in cases with nodal metastasis. Moderate to severe stroma in nodal metastatic tumors and five or more mitotic figures in nodal metastatic tumors were significantly associated with elevated HRs for tumor recurrence and tumor-related death among all the cases. These results indicated that FFs, the grading system for LVTEs, and the histological characteristics of tumor cells in lymph nodes play important roles in predicting the tumor progression of IDCs of the breast in patients treated with neoadjuvant chemotherapy.

Expression of Indian Hedgehog signaling molecules in breast cancer

To investigate the clinicopathological significance and expression pattern of Hedgehog (Hh) signaling molecules in breast normal glands and invasive ductal carcinoma. A total of 142 cases, including 21 of normal breast and 121 of invasive ductal carcinoma of the breast, were immunohistochemically analyzed for Ihh, Ptch, Smo, Gli-1, Gli-2, and Gli-3 protein expression. All of Hh signaling molecules were greatly enhanced in invasive ductal carcinoma compared with the normal breast epithelia. The expressions of Ihh, Smo, and Gli-2 were increased in PR negative cases, and the expressions of Ihh, Ptch, and Gli-1/2/3 were statistically correlated with increased proliferating index of Ki-67 in invasive ductal carcinoma. Ihh and Gli-1/2/3 expressions were correlated with node metastasis. Additionally, the protein expressions of Ihh, Ptch, and Gli-2 were correlated with the clinical stage of breast cancer. Hedgehog signaling molecules play an important role in the progression of invasive ductal carcinoma of breast.

Primary tumour–vessel tumour–nodal tumour classification for patients with invasive ductal carcinoma of the breast

There are many studies that show biological differences between invasive ductal carcinoma (IDC) with and without nodal metastasis, but no prognostic classification taking into consideration any biological differences between them is currently available. We previously investigated the histological characteristics that play an important role in tumour progression of IDCs according to their nodal status, and a new prognostic histological classification, the primary tumour–vessel tumour–nodal tumour (PVN) classification, was devised based on the histological characteristics of IDCs with and without nodal metastasis. Multivariate analyses using the Cox proportional hazard regression models were used to compare the ability of the PVN classification to predict tumour recurrence and death in 393 IDC patients based on the following histological classifications: (1) the pTNM classification, (2) the Nottingham Prognostic Index, (3) the modified Nottingham Prognostic Index, and (4) the histologic grade. In IDCs without nodal metastasis, only the PVN classification significantly increased the hazard rates (HRs) of tumour recurrence and death (P<0.05), independent of the hormone receptor status. Similarly, in IDCs with nodal metastases, only the PVN classification significantly increased the HRs of tumour recurrence and death (P<0.05), independent of the hormone receptor status. We conclude that the PVN prognostic histological classification is the best classification available for IDC of the breast.