Georgia Papachristopoulou

Quantitative expression analysis and study of the novel human kallikrein-related peptidase 14 gene (KLK14) in malignant and benign breast tissues

Human kallikrein-related peptidase 14 gene (KLK14) is regulated by androgens and progestins. This gene is expressed in the central nervous system and endocrine tissues such as the breast, prostate and ovary. The differential KLK14 mRNA expression levels are related to several human neoplasias, among them breast cancer. The aim of this study was to analyse the KLK14 expression in breast tissues and to investigate its differential diagnostic and prognostic value in the mammary carcinomas. For this purpose, we isolated total RNA from 70 malignant and 33 benign specimens. After testing RNA quality, we synthesised cDNA by reverse transcription and applied a highly sensitive quantitative real-time PCR (qRT-PCR) method for KLK14 mRNA quantification using the SYBR Green® chemistry. HPRT1 was used as a reference gene and the BT20 breast cancer cell line as a calibrator. Relative quantification analysis was performed using the comparative CT method 2-ΔΔCT. KLK14 expression was detected in both types of breast tumours. However, a statistically significant increase of the KLK14 mRNA level was observed in the malignant, compared to the benign tumour samples (p<0.001), highlighting its value in discriminating these breast lesions. Elevated KLK14 expression profiles were associated with higher tumour grade (p=0.043) and size (p=0.007) in cancerous samples. Furthermore, KLK14 mRNA expression showed negative correlation in a statistically significant manner with estrogen receptor status (p=0.024). In accordance with logistic regression models (p=0.012) and receiver-operating-characteristics analysis (p<0.001), KLK14 gene expression could be evaluated as a putative independent diagnostic biomarker in breast tumour biopsies.

Kallikrein-related peptidase 6 (KLK6) expression in the progression of colon adenoma to carcinoma.

Abstract KLK6 is a secreted trypsin-like serine protease. KLK6 mRNA expression and its association with colon cancer (CC) progression was studied using quantitative real-time PCR. We examined the expression of KLK6 in 232 colon tissues (cancerous, non-cancerous, and adenomatous). We proved that KLK6 expression in CC behaves as a continuous variable, as its expression correlates significantly with increasing tumor stage (p=0.004) and histological grade (p=0.007). Interestingly, the expression of KLK6 in adenomas was significantly higher than that in the cancerous or non-cancerous tissues examined (p<0.001). Cox proportional hazard regression model using univariate analysis revealed that positive KLK6 expression is a significant factor for disease-free survival (DFS) (p=0.017) and overall survival (OS) (p=0.002) of patients. Kaplan-Meier survival curves demonstrated that KLK6-negative expression is significantly associated with longer DFS (p=0.009) and OS (p=0.001). ROC analysis showed that KLK6 expression has significant discriminatory power in distinguishing cancerous from non-cancerous colon tissues (p<0.001), or cancerous from adenoma tissues (p=0.001), or adenoma from non-cancerous colon tissues (p<0.001). Additionally, strong KLK6 immunostaining was seen in the cancer cells of selected CC sections, as well as in glandular cells and inflammatory cells of adenomas. In conclusion, KLK6 may represent a potential unfavorable prognostic biomarker for CC.

Unravelling a p73-regulated network: The role of a novel p73-dependent target, MIR3158, in cancer cell migration and invasiveness

The transcription factor p73 is homologous to the well-known tumor-suppressor p53. The p73-regulated networks are of significant clinical interest, because they may substitute for impaired p53-regulated networks which are commonly perturbed in cancer. Herein, we aimed to characterize a p73-regulated network that mediates cell migration and restores anti-oncogenic responses in p53-mutant cancer cells. In this study, we demonstrate that p73 regulates a network underlying cell migration, which consists of MIR34A/MIR3158/vimentin/β-catenin/lef1. The p73 isoforms transactivate the miRNA components (MIR34A/MIR3158) of this network, which in turn, downregulate their EMT-related mRNA co-targets (vimentin/β-catenin/lef1) to decrease cell-migration. Modulation of this network, by increasing the level of the novel p73-dependent target MIR3158, was found to induce anti-oncogenic/anti-invasive responses in p53-mutant cancer cells. Taken together, a p73-regulated, MIR3158-containing, network restores anti-invasive phenotypes in p53-mutant cancer cells; this property could be exploited towards the development of anticancer therapeutics.

Identification of a STAT5 target gene, Dpf3, provides novel insights in chronic lymphocytic leukemia.

STAT5 controls essential cellular functions and is encoded by two genes, Stat5a and Stat5b. To provide insight to the mechanisms linking hematologic malignancy to STAT5 activation/regulation of target genes, we identified STAT5 target genes and focused on Dpf3 gene, which encodes for an epigenetic factor. Dpf3 expression was induced upon IL-3 stimulation in Ba/F3 cells, while strong binding of both STAT5a and STAT5b was detected in its promoter. Reduced expression of Dpf3 was detected in Ba/F3 cells with Stat5a and Stat5b knock-down, suggesting that this gene is positively regulated by STAT5, upon IL-3 stimulation. Furthermore, this gene was significantly up-regulated in CLL patients, where DPF3 gene/protein up-regulation and strong STAT5 binding to the DPF3 promoter, correlated with increased STAT5 activation, mainly in non-malignant myeloid cells (granulocytes). Our findings provide insights in the STAT5 dependent transcriptional regulation of Dpf3, and demonstrate for the first time increased STAT5 activation in granulocytes of CLL patients. Novel routes of investigation are opened to facilitate the understanding of the role of STAT5 activation in the communication between non-malignant myeloid and malignant B-cells, and the functions of STAT5 target genes networks in CLL biology.

Human kallikrein-related peptidase 12 (KLK12) splice variants discriminate benign from cancerous breast tumors

OBJECTIVES As kallikrein-related peptidase 12 (KLK12) has been implicated in the cancer progression and alternative splicing plays significant role in this disease, the aim of this study was to examine the expression profile and the clinical impact of the KLK12 splice variants in breast cancer. DESIGN AND METHODS Total RNA was isolated and reverse transcripted from 141 tissues. Afterwards, quantitative real-time PCR were conducted, followed by the performance of the comparative CT (2-ΔΔCT) method for relative quantification, whilst their correlation with the clinicopathological features of breast malignancies were assessed by statistical analysis. RESULTS Both KLK12sv1/2 and KLK12sv3 showed higher expression in non-cancerous than in cancerous samples. KLKsv1/2 (P = 0.001) upregulated and KLK12sv3 (P < 0.001) downregulated in the malignant compared to the benign tumors and their discriminative ability was verified by ROC curve analysis. Moreover, KLK12sv3 was associated with grade (P = 0.012) and hormonal receptor status (P = 0.001). Furthermore, Kaplan-Meier and Cox regression analyses showed that patients with positive KLK12sv1/2 and KLK12sv3 levels presented a significantly longer disease-free survival (P = 0.014 and P = 0.013, respectively) and overall survival (P = 0.062 and P = 0.004, respectively). CONCLUSIONS Our results demonstrate the discriminative value of KLK12sv1/2 and KLK12sv3 between benign and malignant breast tumors as well as their potential favorable prognostic significance in breast adenocarcinoma.

A comprehensive clinicopathological evaluation of the differential expression of microRNA-331 in breast tumors and its diagnostic significance

OBJECTIVE MicroRNA-331 (miR-331) has shown regulatory activity against several genes whose expression has been claimed to be deregulated in breast tumors, including that of epidermal growth factor receptor 2 (HER2). Herein, the clinical value of miR-331 expression was investigated by analyzing its levels in breast benign and malignant tumors. METHODS The expression levels of miR-331 were quantified via real-time PCR in 130 malignant and 66 benign breast tissue specimens collected after surgical resection of primary tumors. The generated data were analyzed by applying several statistical tests in order to examine the relationship of miR-331 expression with various established clinicopathological features and survival data of patients. RESULTS Our data showed that miR-331 was overexpressed in malignant breast tumors compared to their benign counterparts both overall (P = 0.026) and individually when the subgroups of fibroadenoma and invasive ductal carcinoma were analyzed with each other (P = 0.001). ROC curve analysis confirmed the diagnostic value of these variations, providing an AUC value equal to 0.597 (P = 0.026) and 0.663 (P = 0.001), respectively. Furthermore, miR-331 levels were elevated (P = 0.026) in ductal cancerous specimens compared to the lobular ones but failed to correlate with other clinicopathological features or survival data of the breast cancer patients. CONCLUSIONS Our results provide evidence that miR-331 levels might provide valuable information regarding the differential diagnosis of benign and malignant breast tumors but present no prognostic value for breast cancer.

Expression Analysis of miR-29b in Malignant and Benign Breast Tumors: A Promising Prognostic Biomarker for Invasive Ductal Carcinoma With a Possible Histotype-Related Expression Status

&NA; The transcript levels of miR‐29b, which has been claimed to be involved in breast cancer molecular pathology, were evaluated with regard to their clinical utility by analyzing miR‐29b expression in 177 breast tissue samples of either benign or malignant characterization. Our data showed that miR‐29b might constitute a promising prognostic marker for invasive ductal carcinoma. Background: Aberrations in microRNA levels seem to provide valuable information regarding breast cancer prognosis and therapy. In this study, we sought to analyze miR‐29b expression in breast tumors and thus explore its clinical value. Materials and Methods: One hundred twenty‐one malignant and 56 benign breast tissue specimens were collected and subjected to extraction of total RNA, which was polyadenylated and reverse transcribed to cDNA. Subsequently, a highly sensitive quantitative real‐time polymerase chain reaction protocol was developed and miR‐29b levels, estimated via the comparative CT method, were finally subjected to comprehensive statistical analysis. Results: MiR‐29b levels did not differ between the analyzed benign and malignant breast tissue specimens, but were found to be significantly (P = .010) decreased in invasive ductal adenocarcinomas compared with their lobular counterparts, albeit receiver operating characteristics curve analysis did not verify the latter correlation. Additionally, miR‐29b expression was elevated in samples with positive estrogen receptor status (P = .021) in the overall population, whereas it was negatively correlated (P = .035) with primary tumor staging in the ductal subset and increased in poorly‐differentiated tumors of lobular origin (P = .041). Furthermore, Kaplan–Meier and Cox regression analyses showed that patients with ductal carcinoma and elevated miR‐29b levels had a significantly longer disease‐free survival (P = .010) and a lower risk to relapse (hazard ratio = 0.35, 95% confidence interval, 0.15‐0.81; P = .014). Conclusion: Our results provide evidence that miR‐29b levels constitute a promising biomarker of favorable prognosis for patients with invasive ductal breast carcinoma and imply that its expression status might be affected by the histological origin of breast malignancy.

Sa1944 Immunohistochemical Localisation of Kallikrein-Related Protease 14 (KLK14) in Colon Tissues During Cancer Development: Expression and Clinical Evaluation

variability in the sequence quality, reads generated, or basic coverage metrics. To assess the fidelity of variant calls in the EUS FNA exome data, results were compared to mutations previously detected by a 50 gene cancer panel. Results: The sequencing study was successful with a sufficient/expected number of reads per sample generated (Table 1). The mean percent duplicate reads per sample was 12.9% (range 4.8-38.6%). Exome coverage varied between samples; 50-85% of the exome was covered at 50x, and 20-50% of the exome was covered at 100x. The 50 gene panel comparator set comprised of 87 mutations across 12 samples. Comparison revealed that 86 of 87 mutations reported by the panel were also detected by exome sequencing. Conclusion: This is the first report to demonstrate the success of EUS FNA exome-sequencing. There was sufficient coverage in all samples and the variant identification between the 50 gene panel and the exome sequencing was highly concordant. The findings reveal the potential clinical utility of this method when applied to routine clinical cytology specimens to identify potential therapeutic targets which could be readily applied in a clinical research trial and eventually direct clinical care. Table 1

Sa1693 Cathepsin-B in Colon Cancer: Gene Expression and Clinical Evaluation

INTRODUCTION. Colon cancer (CC) remains the third leading cause of worldwide cancerrelated death in men and women. Currently, available prognostic and/or predictive markers for colon cancer lack specificity and sensitivity. Developing new biomarker for early detection, accurate diagnosis and therapeutic treatment for CC is of great importance in improving the clinical outcome of the disease. Cathepsin B (CB), a lysosomal cysteine protease, is expressed constitutively in lysosomes, however its expression and localization change in cancer. High levels of expression of CB at both gene and protein levels have been observed in different types of cancer. AIM. The aim of this study was to analyze the CB expression in different stages of CC progression and to evaluate its clinical relevance. MATERIALS & METHODS. We examined for first time, using quantitative real time PCR, the expression of CB in 185 colonic tissue specimens from 130 patients; 50 were pairs of cancerous-normal tissues, 17 were cancerous tissues and 63 were adenomas for 5 of which normal paired mucosa were also available. RESULTS. We proved that CB was up regulated in the cancer specimens in comparison to their normal pairedmucosa (p<0.001), as well as in the adenomas in comparison to normal tissue (p<0.001). CB expression was found to be associated with histological grade (p=0.037). Cox proportional hazard regression model using univariate and multivariate analysis revealed that high status CB expression is a significant factor for disease-free survival(DFS) (p=0.037 and 0.0038, respectively) and overall survival (OS)(p= 0.003 and p=0.0037, respectively) of patients. Receiver-operating characteristic (ROC) analysis of our results showed that CB has discriminatory value between CC and adenomas tissues (area under the curve [AUC]=0.711). Kaplan-Meier survival curves demonstrated that CB expression of low status is significantly associated with longer DFS (p=0.023) as well as OS (p=0.002). CONCLUSION. Present results suggest that CB gene expression may represent a useful marker of unfavorable prognosis for CC patients with discriminatory power between CC and adenoma patients.