This is an informational website about a research project realized in the National Program of RDI.—PN II
This study is granted by The National University Research Council Bucuresti, (IDEI grant number 397/2007).

Project description
Duration of project
Total funds
Research Team
Direct implication of PhD students
The objectives and activities and the degree of realisation
Project Director
Results obtained in the project
Published articles
Perfecting PhD students

Project description
Prostate cancer is the second leading cause of adult male death from cancer in the developed countries. The molecular genetics analysis of prostate cancer revealed the basic mechanisms involved in prostate carcinogenesis are intricate and require a comprehensive investigation. Identification of new biomarkers for detection and diagnosis of prostate cancer and targets for the design of drug treatments could give insights of the biochemical and molecular pathways involved in normal cell modification into cancer cells and describe a real progress of the prostate cancer treatment. Good clinical procedure for the definition, transcriptomics characterization and identification of molecular biomarkers which are studying prostate cancer due to occurance of the genomics (microarrays) and proteomics (two-dimensional gel electrophoresis, MALDI/TOF, SELDI/TOF) technology have the ability to provide meta data for comprehensive data bases essential in clinical trials and translational research. The overall aim of this project is to determine new molecules involved in prostate cancer (molecular biomarkers) with expression fold directly involved in prostate cancer, noninvasive growth and proliferation as well as in identification of new predictive biomarkers xin the blood serum and urine. The main aim of this study is high-throughput genomics analysis based on the microarrays reaction (whole Human Genome) to obtain several transcriptomics signatures (Clusters of differentiation) in benign vs. malignant prostate tissue. Secondary project’s objectives are to investigate and validate the genes of interest by high-throughput techniques in blood serum and urine specimens (quantitative PCR, 2DE proteomics). We also attend to correlate gene expression level of the interest genes from the tissue, blood serum and urine involved in the transformation of normal prostate cells in malignant as well as the classification of the molecular biomarkers importance in noninvasive screening.

Duration of project: 01.10.2007 – 30.09.2010
Total funds: 1.000.000 RON
Amount financed: 771272.25 Lei
Project ID—CNCSIS: 314/2007

Research Team:
Dr. Ovidiu Balacescu- Project Director

Assistent Prof. Dr. Coman Ioan
Associate Prof. Dr. Neagoe Ioana
Dr. Popescu Sorin
Dr. Ramona Suharoschi, PhD student
Dr. Nicu Crisan, PhD student
Dr. Bogdan Feciche, PhD student

Project Director :

Dr. Ovidiu Balacescu- Project Director

Cancer Institute “I. Chiricuta” Cluj-Napoca
Experimental Pathology Department, Gh. Bilascu Str. nr 34-36, Cluj-Napoca, Cod 400015
Tel: 0264-590638, Fax: 0264-590638, Email:

Direct implication of PhD students:

Young researchers (PhD students) are involved in all stages of the projects function of their speciality:
• Samples collection
– Dr. Nicolae Crisan (PhD student)
– Dr. Bogdan Feciche (PhD student)
• Serum extraction from blood
– Dr. Nicolae Crisan (PhD student)
– Dr. Bogdan Feciche (PhD student)
• RNA extraction from tissues and blood
– Dr Ramona Suharoschi (PhD student)
• Synthesis and evaluation of microarray probes
– Dr Ramona Suharoschi (PhD student)
• PCR array study on RNA extracted from blood
– Dr Ramona Suharoschi (PhD student)
– Dr. Nicolae Crisan (PhD student)
• Microarray study on the tissue samples
– Suharoschi Ramona Dr (PhD student)
• Study by electrophoresis proteomics
– Suharoschi Ramona Dr (PhD student)
• Writing scientific publications
– Dr Ramon Suharoschi (PhD student)
– Dr. Nicolae Crisan (PhD student)
– Dr. Bogdan Feciche (PhD student)
• Patent research
– Dr. Nicolae Crisan (PhD student)

The objectives and activities and the degree of realisation:

2007 (the objectives and the activities were realised in totality)
Ob- Biobank founding: tissue, blood, (RNA serum) urine;
A1- Selection the patients function of PSA value and DRE
A2- Grouping the patients function the stage of the disease (benign vs. malign)
A3- Biological samples collection (tissue, blood and urine)

2008- (The objectives and the activities were realised in totality)
Ob - Bio bank consolidation, processing the samples and preparing the functional genomics study;
A1- New biological samples collection tissue, blood, urine,
A2- RNA extraction from tissues; quality and quantity evaluation by nanotechnologies,
A3 – cDNA synthesis
A4 – synthesis of microarray probes (cRNA-Cy3)

2009 (the objectives and the activities adapted function of budget were realised in totality)
Ob- Realize the functional genomics study and results validation;
A1- realizes the microarray reaction, preliminary evaluation, bioinformatics study;
A2- results validation (genes of interest from blood and tissue) using qRT-PCR.

2010 (depending on the objectives and activities tailored to the budget were made fully diminished)
Ob- Making proteomics study using electrophoresis analysis, correlation of functional genomics data with those of proteomics
A1-Extraction of proteins from the same biological samples (tissue, serum) from which to extract RNA used for functional genomics studies
A2 - Correlation levels of gene expression (messenger RNA) with poteinele related to serum and tissue levels
A3-Correlation serum protein levels of tumor biomarkers value of DRE and PSA values

Results obtained in the project:
Bio bank of biological samples in prostate field (tissue, blood, urine)
In the bio bank were included samples from over 70 patients who have been diagnosed with prostate adenocarcinoma. Also, the biobank include blood and serum samples from 10 healthy volunteer, as well as normal tissue fragments from prostatectomy sample of the 7th pacients.
Biological samples have been handling in respect with the functional genomics standards. RNA extraction from tissue has been done by classical method with phenol isopropanol-ethanol followed by purification onto the spin-column (Rneasy mini kit) (Qiagen). The RNA extraction was performed from the blood samples collected directly in the PaxGene tubes using PaxGene kit (Sigma). All the samples were treated with DNaza in order to avoid DNA contamination. The RNA extraction from urine samples has been done with mini RNA Isolation I KitTM. Bioanalyser 2100 (Agilent) and Nanodrop ND-1000 were used for quantitative and qualitative measurements as Q/C of all RNA extraction. The advantage that a nanotechnology is that it uses a small amount of genetic material and obtains accurate and reproducible results. We synthesized microarrays probes labeled with Cy3 using LILAK kit (Agilent).
The serum biomarkers with non-invasive predictive value are related with angiogenesis, one of the most important pathways involved in tumor progression. Thus we study serum probes of 40th patients grouped as prostate pathology: prostate adenocarcinoma (n=17), BPH (benign prostatic hyperplasia) (n=11), chronic prostatitis (n=6), and a control group of healthy people (n=6). We assessed 8 angiogenic markers (PDGF-BB, VEGF, FGF-b, ANG, KGF, TIMP-1, ICAM-1, ANG-2) using FAST Quant® array technique. The results show a difference statistical significance of the expression of KGF, ANG2, PDGF-BB and TIMP-1 for the selected pathology.

Microarray data from tissue revealed genes involved in molecular mechanisms as: angiogenesis, transcription, cell cycle regulation, apoptosis, kinase activity. Because of budget diminution have been chosen 4 genes, involved in angiogenesis, for validation on qRT-PCR (VEGFA, TIMP1, PDGFA, TNF).

In the blood we evaluate 84 genes involved in angiogenesis mechanism. For this study we used 12 samples, 7 tumor samples and 5 controls (normal tissue). The evaluation of mechanism of angiogenesis was made using the The Human Angiogenesis RT² Profiler™ PCR Array, and Roche technology (Light Cycler 480). The bioinformatic analysis was made using RT² Profiler™ PCR Array Data Analysis, using ΔΔCt method, based fold-change calculations with normalisation for all the genes. Between PCa and HBP were not found significant statistically difference, maybe because of the small number of samples. On the other hand, between PCa and control were obtained 37 genes different expressed (5 were overexpressed and 32 underexpressed). The threshold was  ±1.5.

Published  articles:

1. Bălăcescu L, Bălăcescu O, Crişan N, Fetica B, Petruţ B, Bungărdean C, Rus M, Tudoran O, Meurice G, Irimie A, Dragoş N, Berindan-Neagoe I. Identifying molecular features for prostate cancer with Gleason 7 based on microarray gene expression profiles. Rom J Morphol Embryol. 2011;52(4):1195-202.

2. Balacescu O, Neagoe I, Balacescu L, Crisan N, Feciche B, Tudoran O, Coman I, Irimie A. Angiogenesis serum protein quantification for Prostate patholgy (BDI). Curr Urol 2008; 2: 181-187 (ISSN 1661-7649). Karger group.

3. Floares, A. Balacescu, O. Floares, C. Balacescu, L. Popa, T. Vermesan, O. Mining knowledge and data to discover intelligent molecular biomarkers: Prostate cancer i-Biomarkers; 10.1109/SOFA.2010.5565613 2010: pag 113 – 118.

National patent submitted to OSIM (Regsistration nr (OSIM): A/10030/2010. Noninvasive method for prostate cancer detection based on a set of genes involved in the mechanism of angiogenesis.
Holders: Balacescu Ovidiu, Neagoe Ioana, Balacescu Loredana

Perfecting PhD students:

In the second phase of the project were done training courses of 3 weeks each for PhD students: Nicolae Crisan (1-21 July 2008) and Feciche Bogdan (22 June – 14 July 2008) at the European Oncologic Institute of Milano, Department of Urology.
Also during this project phase we recorded the completion of a doctoral thesis of the third PhD student:
Suharoschi Ramona: in vitro and in silico Prostate Cancer Research. PhD in Medical Science, order of MECT no. 439/12.03.2008.
Nicolae Crisan: Improving diagnosis and therapy of prostate adenocarcinoma in the context of the occurrence of urinary tumor markers and minimally invasive interventions. December 11, 2010 public presentation
Doctoral students have gained experience in both developing research projects, and in writing scientific papers and patents making.