Relationship between levels of Skp2 and P27 in breast carcinomas and possible role of Skp2 as targeted therapy

Estrogen receptor-negative breast carcinomas are more aggressive and are unresponsive to anti-estrogens. Thus, they clearly require new therapies targeted against specific genes and proteins actively engaged in the pathophysiology of cancer. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. We attempted to explore whether Skp2 may be a potential specific therapeutic target in the subset of aggressive breast carcinomas by investigating the possible relationship between expression of Skp2 and p27 proteins and estrogen receptor (ER). Immunohistochemical analysis of tumor tissues was employed to determine the expression of Skp2, p27, and ER proteins in 82 cases of primary breast carcinoma. Higher levels of Skp2 were detected more frequently in ER-negative tumors and tumors metastatic to the axillary lymph nodes. The expression of p27 was inverse with the histologic grade. Statistical analysis showed that the percentage of high Skp2 expressors was significantly greater in the group with low p27 expression than in the group with high p27 expression. The current study, together with the results from a previous study, demonstrated the existence of a subtype of high-grade, negative ER breast carcinomas with high Skp2 and low p27 levels. This implies that Skp2 may be a potential specific therapeutic target in a subset of aggressive breast carcinomas. Thus far, there is no specific therapy for the ER-negative and HER-2/neu resistant groups, which are among the subset of aggressive tumors.     

Insulin-like growth factor II (IGF-II) immunohistochemistry in breast cancer: relationship with the most important morphological and biochemical prognostic parameters

Recent in situ hybridization experiments have shown a high content of IGF-II mRNA in breast cancer stroma. The aim of this study was to examine the relationship between IGF-II protein expression and several prognostic parameters in 75 infiltrating ductal carcinomas (IDC) of the breast. Tissue sections were evaluated for proliferative activity, IGF-II protein, ER, PgR, p53, and p21 expression using immunohistochemical procedures. The degree of stromal proliferation was assessed. Menopausal status, axillary lymph node involvement and nuclear grade were known. Thirty-five patients (44.3%) were premenopausal and 47 (62.6%) had lymph node metastases. Marked stromal proliferation was found in 34 (45.3%) specimens and high nuclear grade in 20 (26.5%). Eighteen tumors (24%) showed no IGF-II immunostaining. In the positive cases, IGF-II was detected both in the tumor stroma and in the cytoplasm of epithelial cancer cells: a high IGF-II content was found in 12 specimens (16.0%), a low content in 14 (18.7%) and a moderate content in 31 (41.3%). Twenty-four tumors (32.0%) showed high proliferative activity. Both ER and PgR were expressed in the nucleus of cancer cells: 49 tumors (65.3%) were ER positive (ER+) and 34 (45.3%) PgR positive (PgR+). p21 protein was detected in 37 tumors (49.6%) and p53 in 12 (16%). IGF-II protein was not correlated with menopausal status, lymph node metastases, nuclear grade, proliferative activity, ER or p53. In contrast, IGF-II correlated strongly with stromal proliferation (p=0.008), PgR (p=0.03) and p21 (p=0.01). This study demonstrates that in IDC of the breast IGF-II protein is expressed in the epithelium and stroma of the majority of tumors and is correlated with stromal amount, PgR and p21 expression. These preliminary results indicate that IGF-II expression in breast cancer is connected with two important regulators of breast cancer growth and differentiation.     

Interactions between immunity,proliferation and molecular subtype in breast cancer prognosis

Background

Gene expression signatures indicative of tumor proliferative capacity and tumor-immune cell interactions have emerged as principal biology-driven predictors of breast cancer outcomes. How these signatures relate to one another in biological and prognostic contexts remains to be clarified.

Results

To investigate the relationship between proliferation and immune gene signatures, we analyzed an integrated dataset of 1,954 clinically annotated breast tumor expression profiles randomized into training and test sets to allow two-way discovery and validation of gene-survival associations. Hierarchical clustering revealed a large cluster of distant metastasis-free survival-associated genes with known immunological functions that further partitioned into three distinct immune metagenes likely reflecting B cells and/or plasma cells; T cells and natural killer cells; and monocytes and/or dendritic cells. A proliferation metagene allowed stratification of cases into proliferation tertiles. The prognostic strength of these metagenes was largely restricted to tumors within the highest proliferation tertile, though intrinsic subtype-specific differences were observed in the intermediate and low proliferation tertiles. In highly proliferative tumors, high tertile immune metagene expression equated with markedly reduced risk of metastasis whereas tumors with low tertile expression of any one of the three immune metagenes were associated with poor outcome despite higher expression of the other two metagenes.

Conclusions

These findings suggest that a productive interplay among multiple immune cell types at the tumor site promotes long-term anti-metastatic immunity in a proliferation-dependent manner. The emergence of a subset of effective immune responders among highly proliferative tumors has novel prognostic ramifications.     

Gene expression patterns in ovarian carcinomas

We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.     

Determination of stromal signatures in breast carcinoma

Many soft tissue tumors recapitulate features of normal connective tissue. We hypothesize that different types of fibroblastic tumors are representative of different populations of fibroblastic cells or different activation states of these cells. We examined two tumors with fibroblastic features, solitary fibrous tumor (SFT) and desmoid-type fibromatosis (DTF), by DNA microarray analysis and found that they have very different expression profiles, including significant differences in their patterns of expression of extracellular matrix genes and growth factors. Using immunohistochemistry and in situ hybridization on a tissue microarray, we found that genes specific for these two tumors have mutually specific expression in the stroma of nonneoplastic tissues. We defined a set of 786 gene spots whose pattern of expression distinguishes SFT from DTF. In an analysis of DNA microarray gene expression data from 295 previously published breast carcinomas, we found that expression of this gene set defined two groups of breast carcinomas with significant differences in overall survival. One of the groups had a favorable outcome and was defined by the expression of DTF genes. The other group of tumors had a poor prognosis and showed variable expression of genes enriched for SFT type. Our findings suggest that the host stromal response varies significantly among carcinomas and that gene expression patterns characteristic of soft tissue tumors can be used to discover new markers for normal connective tissue cells.     

Expression of the serine protease, matriptase, in breast ductal carcinoma of Chinese women: correlation with clinicopathological parameters

Matriptase is a serine protease expressed by cells of surface epithelial origin, including epithelial breast tumor cells. Matriptase cleaves and activates proteins implicated in the progression of cancer and represents a potential prognostic and therapeutic target. The aim of this study was to examine matriptase expression in breast tumors of Chinese women and to identify its clinicopathological correlations. Immunohistochemical analysis of matriptase was performed in tissue microarrays of 251 breast tumors including 30 fibroadenomas, 59 ductal carcinomas in situ (DCIS), 38 grade I invasive ductal carcinomas (IDC), 79 grade II IDC, and 45 grade III IDC. The matriptase scores were significantly higher in the tumors than their non-tumor counterparts (178+/-12 for fibroadenoma; 275+/-11 for DCIS; 299+/-10 for grade I IDC; 251+/-10 for grade II IDC; and 314+/-11 for grade III IDC). In cases of IDC, matriptase scores were significantly correlated with tumor staging and nodal staging. Our findings demonstrate that matriptase is over-expressed in breast ductal carcinoma of Chinese women. It therefore may be a good biomarker for diagnosis and treatment of malignant breast tumors.     

Google goes cancer: improving outcome prediction for cancer patients by network-based ranking of marker genes

Predicting the clinical outcome of cancer patients based on the expression of marker genes in their tumors has received increasing interest in the past decade. Accurate predictors of outcome and response to therapy could be used to personalize and thereby improve therapy. However, state of the art methods used so far often found marker genes with limited prediction accuracy, limited reproducibility, and unclear biological relevance. To address this problem, we developed a novel computational approach to identify genes prognostic for outcome that couples gene expression measurements from primary tumor samples with a network of known relationships between the genes. Our approach ranks genes according to their prognostic relevance using both expression and network information in a manner similar to Google's PageRank. We applied this method to gene expression profiles which we obtained from 30 patients with pancreatic cancer, and identified seven candidate marker genes prognostic for outcome. Compared to genes found with state of the art methods, such as Pearson correlation of gene expression with survival time, we improve the prediction accuracy by up to 7%. Accuracies were assessed using support vector machine classifiers and Monte Carlo cross-validation. We then validated the prognostic value of our seven candidate markers using immunohistochemistry on an independent set of 412 pancreatic cancer samples. Notably, signatures derived from our candidate markers were independently predictive of outcome and superior to established clinical prognostic factors such as grade, tumor size, and nodal status. As the amount of genomic data of individual tumors grows rapidly, our algorithm meets the need for powerful computational approaches that are key to exploit these data for personalized cancer therapies in clinical practice.     

The CIN4 Chromosomal Instability qPCR Classifier Defines Tumor Aneuploidy and Stratifies Outcome in Grade 2 Breast Cancer

Purpose

Quantifying chromosomal instability (CIN) has both prognostic and predictive clinical utility in breast cancer. In order to establish a robust and clinically applicable gene expression-based measure of CIN, we assessed the ability of four qPCR quantified genes selected from the 70-gene Chromosomal Instability (CIN70) expression signature to stratify outcome in patients with grade 2 breast cancer.

Methods

AURKA, FOXM1, TOP2A and TPX2 (CIN4), were selected from the CIN70 signature due to their high level of correlation with histological grade and mean CIN70 signature expression in silico. We assessed the ability of CIN4 to stratify outcome in an independent cohort of patients diagnosed between 1999 and 2002. 185 formalin-fixed, paraffin-embedded (FFPE) samples were included in the qPCR measurement of CIN4 expression. In parallel, ploidy status of tumors was assessed by flow cytometry. We investigated whether the categorical CIN4 score derived from the CIN4 signature was correlated with recurrence-free survival (RFS) and ploidy status in this cohort.

Results

We observed a significant association of tumor proliferation, defined by Ki67 and mitotic index (MI), with both CIN4 expression and aneuploidy. The CIN4 score stratified grade 2 carcinomas into good and poor prognostic cohorts (mean RFS: 83.8±4.9 and 69.4±8.2 months, respectively, p = 0.016) and its predictive power was confirmed by multivariate analysis outperforming MI and Ki67 expression.

Conclusions

The first clinically applicable qPCR derived measure of tumor aneuploidy from FFPE tissue, stratifies grade 2 tumors into good and poor prognosis groups.     

Expression of Polycomb Targets Predicts Breast Cancer Prognosis

Global changes in the epigenome are increasingly being appreciated as key events in cancer progression. The pathogenic role of enhancer of zeste homolog 2 (EZH2) has been connected to its histone 3 lysine 27 (H3K27) methyltransferase activity and gene repression; however, little is known about relationship of changes in expression of EZH2 target genes to cancer characteristics and patient prognosis. Here we show that through expression analysis of genomic regions with H3K27 trimethylation (H3K27me3) and EZH2 binding, breast cancer patients can be stratified into good and poor prognostic groups independent of known cancer gene signatures. The EZH2-bound regions were downregulated in tumors characterized by aggressive behavior, high expression of cell cycle genes, and low expression of developmental and cell adhesion genes. Depletion of EZH2 in breast cancer cells significantly increased expression of the top altered genes, decreased proliferation, and improved cell adhesion, indicating a critical role played by EZH2 in determining the cancer phenotype.     

Analysis of the candidate tumor suppressor Ris-1 in primary human breast carcinomas

Frequent chromosome 3 losses have been described in several tumors types, which strongly suggest the presence of one or several tumor suppressor genes. Recently, a novel candidate tumor suppressor gene termed Ris-1 (for Ras-induced senescence 1) has been identified at chromosomal position 3p21.3. Ris-1 has been proposed to participate in anti-tumor responses that resemble cellular senescence and that are elicited by oncogenes such as Ras. To analyze the role of Ris-1 as a putative tumor suppressor gene in human breast cancer, we have performed a real-time quantitative analysis of its mRNA expression in 60 patients. Moreover, we carried out a first approach to evaluate the most common inactivation mechanism that can affect expression levels of tumor suppressor genes (mutation, promoter hypermethylation and allelic losses). Furthermore, a correlation study between expression as well as inactivating mechanisms of Ris-1 and several clinico-pathological parameters of the tumors was designed, with the objective of appraising the prognostic value of Ris-1 status. Decreased expression of Ris-1 was observed in 23% of the cases and overexpressed Ris-1 was detected in 15% of the primary breast tumors. Our data showed high frequency of LOH (30%) at one of the markers used. Nevertheless, a polymorphism related with the expression levels was described. Statistically significant correlations were found between decreased Ris-1 expression and negative progesterone receptors, as well as between overexpressing Ris-1 tumors and high histological grade. Despite all these data, we conclude that the suggested role of Ris-1 as tumor suppressor gene is not evident, at least in breast cancer. Future and larger series studies in different tumor types are necessary to clarify Ris-1 function in human cancer.     

Loss of circadian clock gene expression is associated with tumor progression in breast cancer

Several studies suggest a link between circadian rhythm disturbances and tumorigenesis. However, the association between circadian clock genes and prognosis in breast cancer has not been systematically studied. Therefore, we examined the expression of 17 clock components in tumors from 766 node-negative breast cancer patients that were untreated in both neoadjuvant and adjuvant settings. In addition, their association with metastasis-free survival (MFS) and correlation to clinicopathological parameters were investigated. Aiming to estimate functionality of the clockwork, we studied clock gene expression relationships by correlation analysis. Higher expression of several clock genes (e.g., CLOCK, PER1, PER2, PER3, CRY2, NPAS2 and RORC) was found to be associated with longer MFS in univariate Cox regression analyses (HR<1 and FDR-adjusted P < 0.05). Stratification according to molecular subtype revealed prognostic relevance for PER1, PER3, CRY2 and NFIL3 in the ER+/HER2- subgroup, CLOCK and NPAS2 in the ER-/HER2- subtype, and ARNTL2 in HER2+ breast cancer. In the multivariate Cox model, only PER3 (HR = 0.66; P = 0.016) and RORC (HR = 0.42; P = 0.003) were found to be associated with survival outcome independent of established clinicopathological parameters. Pairwise correlations between functionally-related clock genes (e.g., PER2-PER3 and CRY2-PER3) were stronger in ER+, HER2- and low-grade carcinomas; whereas, weaker correlation coefficients were observed in ER- and HER2+ tumors, high-grade tumors and tumors that progressed to metastatic disease. In conclusion, loss of clock genes is associated with worse prognosis in breast cancer. Coordinated co-expression of clock genes, indicative of a functional circadian clock, is maintained in ER+, HER2-, low grade and non-metastasizing tumors but is compromised in more aggressive carcinomas.     

Expression of checkpoint kinase 2 in breast carcinomas: correlation with key regulators of tumor cell proliferation, angiogenesis, and survival

Checkpoint kinase 2 (Chk2) is a cell-cycle-checkpoint kinase that may act as a tumor suppressor gene due to its important role in DNA damage signaling and cell cycle regulation. The role of Chk2 expression in mammary tumorigenesis, however, is still poorly understood. This study was designed to assess the relationship between the expression of Chk2 and well-established prognostic factors, including disease-free-survival and overall survival; and several regulators of cell proliferation and invasiveness in breast carcinomas, including oncogenes, tumor suppressor genes, apoptosis-related proteins, and angiogenesis-related markers. Immunohistochemistry with 27 primary antibodies was performed in 100 formalin-fixed paraffin-embedded samples of not otherwise specified invasive ductal carcinomas. Clinical data were retrieved from medical files. In normal mammary parenchyma adjacent to the tumors Chk2 stained the nuclei of epithelial cells. Downexpression of Chk2 protein was observed in 23 carcinomas and correlated with advanced disease. Among the regulators of tumor cell proliferation and invasiveness analyzed, the downexpression of Chk2 correlated only with reduced expression of p27 and telomerase. There was no difference between the overall survival and disease-free survival rates according to Chk2 status. In conclusion, Chk2 correlated with reduced expression of h-TERT and p27, but not with angiogenic factors. Chk2 expression also did not interfere in the outcome of the patients.     

Syndecan-1 and Syndecan-4 Are Independent Indicators in Breast Carcinoma

Syndecan proteoglycans may be key regulators of tumor invasion and metastasis because this four-member family of transmembrane receptors regulates cell adhesion, proliferation, and differentiation. Their expression can also serve as prognostic markers. In breast carcinomas, syndecan-1 overexpression correlates with poor prognosis and aggressive phenotype. Syndecan-4 is expressed in most breast carcinoma cell lines, but its role in malignancy is unclear. A possible relationship between syndecan-1 and syndecan-4 expression and established prognostic factors in breast carcinomas was examined. Duplicate samples of 114 benign and malignant breast disease cases were stained for the two syndecans. Clinicopathological information was available for all cases. Syndecan-1 was detected in 72.8% of cases, with significant association between its expression and histological tumor type (p<0.05) and high grade tumors (p<0.05). Syndecan-4 was expressed in 66.7% of cases; expression correlated significantly with positive estrogen (p<0.01) and progesterone (p<0.01) receptor status. Independent expression of the two syndecans was noted from an analysis of single and double positive cases. There was a statistical relationship between syndecan-1 presence in high-grade tumors and absence of syndecan-4, whereas syndecan-4 presence in cases positive for estrogen and progesterone receptor associated with syndecan-1 absence. These syndecans may, therefore, have distinct roles in regulating breast carcinoma cell behavior.     

Gene expression profiling reveals two separate mechanisms regulating apoptosis in rectal carcinomas in vivo

The level of apoptosis in rectal carcinomas of patients treated by surgery only predicts local failure; patients with intrinsically high-apoptotic tumors develop less local recurrences than patients with low levels of apoptosis. To identify genes involved in this intrinsic apoptotic process in vivo, 47 rectal tumors with known apoptotic phenotype (24 low- and 23 high-apoptotic) were analyzed by oligonucleotide microarray technology. We identified several genes differentially expressed between low- and high-apoptotic tumors. Unsupervised clustering of the tumors based on expression levels of these genes separated the low-apoptotic from the high-apoptotic tumors, indicating a gene expression-dependent regulation. In addition, this clustering revealed two subgroups of high-apoptotic tumors. One high-apoptotic subgroup showed subtle differences in mRNA and protein expression of the known apoptotic regulators BAX, cIAP2 and ARC compared to the low-apoptotic tumors. The other subgroup of high-apoptotic tumors showed high expression of immune-related genes; predominantly HLA class II and chemokines, but also HLA class I and interferon-inducible genes were highly expressed. Immunohistochemistry revealed HLA-DR expression in epithelial tumor cells in 70% of these high-apoptotic tumors. The expression data suggest that high levels of apoptosis in rectal carcinoma patients can be the result of either slightly altered expression of known pro- and anti-apoptotic genes or high expression of immune-related genes.     

Correlations of Differentially Expressed Gap Junction Connexins Cx26, Cx30, Cx32, Cx43 and Cx46 with Breast Cancer Progression and Prognosis

Background and Aims

Connexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers.

Materials and Methods

Meta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models.

Results

The expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other’s prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively.

Conclusion

Differential expression of Cx43 and Cx30 may serve as potential positive and negative prognostic markers, respectively, for a clinically relevant stratification of breast cancers.     

The investigation of DNA repair polymorphisms with histopathological characteristics and hormone receptors in a group of Brazilian women with breast cancer

The association of tumor differentiation and estrogen receptor expression with the prognosis of breast cancer has been well established. Nevertheless, little is yet reported about the association of morphological characteristics of the tumor, estrogen receptor status and polymorphisms in low penetrance genes. The aim of the present study was to investigate a possible association between DNA repair gene polymorphisms (XRCC1, XPD, XRCC3, and RAD51) with histological type, grade and hormone receptor expression in a series of breast cancers. A cross-sectional study was carried out to evaluate 94 women with breast carcinoma, who had already been selected and included in a study on the association of DNA repair gene polymorphisms. For immunohistochemistry, formalin-fixed, paraffin-embedded tissue samples from breast tumors were consecutively retrieved from the histopathology files of our institution. DNA obtained from blood samples of the same patients was investigated for the presence of the following polymorphisms: Arg-399Gln located in the XRCC1 gene; 135C/G located in the RAD51 gene; Lys751Gln located in the XPD gene and Thr241Met located in the XRCC3 gene. Polymorphisms were considered to be independent variables and hormone receptor expression and the morphological characteristics of the tumors comprised the dependent variables. No statistically significant association was found between gene polymorphisms and hormone receptor status. The association between XRCC1-Arg399Gln polymorphism and ductal carcinoma was statistically significant (P = 0.02). The association of the XPD-Lys751Gln polymorphism with histological grade was also tatistically significant (p = 0.05). In conclusion, the XRCC1 genotype was found to be associated with ductal carcinoma histotypes and XPD genotype with low histological grade, which is the most frequent pattern of sporadic breast carcinomas.