Cervicall-arsen-array

This is an informational website about a research project -CERVICALL-ARSEN-ARRAY, granted by Executive Unit for financing education Higher, research Development and innovation (UEFISCDI).

http://www.iocn.ro/en/Research/The-projects-web-pages/Cervicallarsenarray.html

a. The title of the project:
“Analysis of microRNA biomarkers as predictive values for cervical cancer treatment, in relationship with environmental exposure to arsenic, phthalates and ETS.”
b. Acronym: Cervicall-arsen-array
c. Project summary:
Cervicall-arsen-array is an inter and multidisciplinary research project under the requirements of the program PN-II-PT-PCCA-2011-3. The activities under this project will be conducted during a period of 48 months.
The project is classified under the 4th. HEALTH, thematic area 4.1.3 Methods of investigation and intervention based on molecular and cellular medicine, genomics and proteomics. The research domain is cervical cancer and the project is aiming to identify new molecular targets to evaluate the treatment efficiency of advanced stages (IIB-IIIB) of cervical cancer by combining the noncoding RNA (miRNA) functional genomic data with toxic exposure (arsenic, phtalathes and cotinine).
The main goal of the Project is to elaborate a prediction model for therapy response (pharmacogenomics) of patients with advanced cervical cancer (stage IIB-IIIB) based on tissue and serum genomic signature (miRNA-microarray) and to investigate if toxic exposure to mixture (arsenic, phthalates and cotinine as a ETS biomarker) could influence the treatment response.

d. Project objectives

General objective: elaborate a prediction model for therapy response in patients with advanced cervical cancer


Specific objectives:
O1. Identification of specific miRNA signature in response to concurrent chemo radiotherapy in patients with stage IIB-IIIB cervical cancer as predictive molecules to treatment outcome;
O2. Explore a possible correlation of miRNA signatures in tumor biopsies with the ones from serum samples in order to develop a noninvasive prediction test;
O3. Investigate if and how the exposure to arsenic, phthalates and cotinine mixtures of the patients with cervical cancer could influence the treatment response


e. Consortium components:

Coordinator of the multidisciplinary consortium:
The Oncology Institute “Prof Dr Ion Chiricuta” Cluj-Napoca (IOCN), named CO; Project Director: Dr. Ovidiu Balacescu

Project partners:
P1-The University of Medicine and Pharmacy Cluj-Napoca (UMFCN), named P1, Manager Partner P1: Prof Dr. Ioana Neagoe
P2- Environmental Health Center Cluj-Napoca (CMS), named P2, Manager Partner P2: Dr. Iuliana Neamtiu Adina
P3- Babes Bolyai University Cluj-Napoca (UBB), named P3, Manager Partner P3: Dr. Roba Carmen Andreea

f. Contracting Authority:
Executive Unit for financing education Higher, research Development and innovation (UEFISCDI).

Project code: 96/2012


g. Project time span: (54 month) 02.07.2012— 05.12.2016


h. The activities and responsibilities of the participants:
Coordinator – IOCN ♦ defining the including criteria ♦ standardizes the biobank storage methods ♦ processing the biological samples ♦ management of database ♦ extraction of nucleic acids (DNA, RNA) ♦ synthesis of microarray probes ♦ microarray and bioinformatics analysis ♦ data validation by qRT-PCR♦ tissue and serum data correlation ♦ integrating clinical and molecular data ♦ management the clinical data of the patients up to five years. ♦ preparing the final report of the study ♦ dissemination and exploitation of results.

Partner 1 – UMF CN ♦ defining the patients groups necessary for genomics studies♦ management of database date ♦ quality control for nucleic acids ♦ HPV genotyping♦ synthesis of microarray probes ♦ in silico studies to identify the optimal conditions, primers and probes for qRT-PCR assay ♦preparing the final report of the study ♦ dissemination and exploitation of results.
Partner 2 – CMS ♦ developing the conceptual model for assessing human exposure to phthalates, arsenic and contaminants contained in cigarette smoke ♦ piloting integrated conceptual model for assessing exposure to mixtures of pollutants ♦ development the model on the establishment of measurable indicators of the process of assessing the exposure to phthalates, arsenic and contaminants contained in cigarette smoke
♦ validation of the experimental model by specific measurements of biological products and integrated risk analysis ♦ development and implementation of bio-toxicology model. ♦ preparing the final report of the study ♦ dissemination and exploitation of results.
Partner 3 – UBB ♦ analysis of biological samples with the determination of phthalates and their metabolites ♦ quality control of chemical analysis of phthalates and their metabolites ♦ preparing the final report of the study ♦ dissemination and exploitation of results.

i. The budget of the project

Currency project:

3.000.0000 RON state budget; 250.000 RON co financing



j. Contact details of the project:

Contact:

Dr. Ovidiu Balacescu

The Oncology Institute „Prof Dr. Ion Chiricuta” Cluj-Napoca, Romania
Department of Functional Genomics, Proteomics and Experimental Pathology


34-36 Republicii str,
Cluj-Napoca
Code 400015
Cluj
Romania

Tel: 0264-590638
Email: obalacescu@yahoo.com; ovidiubalacescu@iocn.ro

k. Partial results:

Phase I (02.07.2012-5.12.2012)

– Standardization, validation and application of biological samples collection and processing protocols; The collection and processing of biological samples (tissue, blood, urine, nails, cervical lavage) were established according to the project objectives; special kits that contain all the necessary consumables for sample harvesting were prepared and used. The biological samples were processed specifically, in similar conditions based on standardized protocols.

– It was founded the biological samples Biobank related to the research project; the biological samples were stored in liquid nitrogen and/or in -80 freezers according to the international protocols for functional genomic studies


– Were defined the inclusion and exclusion criteria for advanced cervical cancer patient selection; the criteria were established based on histopathological, clinical and therapeutic scheme data, according to the proposed study design

– Was developed a conceptual model for assessing human exposure to phthalates, arsenic and ETS (cotinine); the conceptual model was established based on human dosimetry which allows for the determination of several indicators for arsenic, phthalates and ETS, called biomarkers. In the arsenic case, the measured biomarker is arsenic levels in blood, urine and nails, in phthalates case the biomarkers are the phthalates and urine metabolites of these and for environmental cigarette smoke exposure, the biomarker is the cotinine levels in urine.


Phase II (06.12.2012-05.12.2013)

- New biological samples were collected from the patients entered in the study. During the biological samples harvesting and their processing activities was consolidated the biobank and the database by integration of new patients based on inclusion criteria

- Biological samples (blood, tissue, cervical lavage) were processed according with standardized protocols; the biological samples were processed according to each sample type and stored in the biobank

- Cervical lavage samples were processed to identify HPV genotyping (37 subtypes); the HPV genotyping analysis includes the evaluation of 37 HPV subtypes, of which 13 are of high risk. The obtained results showed a significant percent of patients positive for HPV 16, but also the presence of other high risk subtypes such as HPV 18 and 33 and correlations between the 16, 18, 52, 33, 35 and 58 high risk HPV subtypes.

- Was validated the experimental model, by measuring the specific biological products and integrated risk analysis (pilot study); The pilot study that evaluates the exposure influence was generated based on patient selection (case-control study), collection of mixture exposure data (cigarette smoke, arsenic and phthalates) during an interview, biological samples harvesting and the biochemical analysis of the biological samples to determine cotinine and arsenic levels. The study results showed that cotinine levels in urine, as biomarker for smoke exposure is significantly correlated with active smoking (p=0.037).

- Was conducted the pilot study regarding the genomic analysis (miRNA) on tumor tissue samples. During this pilot study, was performed a microarray evaluation of miRNA expression on FFPE samples in a group of advanced cervical cancer patients with different response to therapy (complete response vs. non response). Our group has previously identified and validated (mRNA and proteins) the activation of BRCA/FA pathway as resistance mechanism to therapy in cervical cancer. The current study showed a number of 7 specific miRNAs differentially expressed between the two groups. 3 of these miRNAs have been identified to transcriptionally regulate some genes involved in DNA repair mechanisms.

Phase III (06.12.2013-05.12.2014)

- New biological samples were included in the biobank; new biological samples were harvested and processed to consolidate the biobank and the database by introducing new patients into the study

- The pilot study was extended to validate the experimental model of exposure to arsenic, phthalates and cotinine. A total of 51 patients were analyzed to evaluate the mixture exposure. The measured arsenic in urine had values around 0.35 to 202.5 µg/l and in nails around 0.21 – 86.34 µg/l. The cotinine levels in urine were around 2.4-395.41 µg/g creatinine, with a median value for creatinine of 37.5 µg/g creatinine. The regression model analysis of the variables of phthalates metabolites did not show any significant correlation between the exposure and urine biomarkers.

- the miRNA study on tissue samples has been extended and the miRNA expression was correlated with the treatment response; the pilot study was extended on 16 FFPE tissue samples from patients that were chemo-radio treated with known 6 months therapy response. 11 miRNAs were identified to be differentially expressed between the two groups. The functional analysis of these 11 miRNAs showed that these are involved in regulation of processes like cellular interaction, cellular growth and proliferation, cellular motility and apoptosis.

- A study to assess the alteration of miRNA in the whole blood samples of patients with cervical cancer was conducted; During the partial hemogenomics study (microarray study on whole blood) were analyzed 22 blood samples harvested from cervical cancer patients with known therapy response at 6 months. Microarray analysis revealed 125 miRNAs that are differentially expressed between the two groups. The IPA functional analysis grouped these miRNAs in proliferation and development processes, cell cycle, cellular survival and death and cellular signaling and interaction.


- A pilot study to investigate specific miRNAs in cervical tissue by qRT-PCR was conducted. A number of 12 mutitargeting (3 mRNAs minimum targets) miRNAs were evaluated based on the miRNA-mRNA interaction analysis. Of these, 7 miRNAs were considered negative for amplification (35 cycles threshold), 4 miRNAs were positive for both group of patients being underexpressed in the non-responsive patients, levels that are correlated with increased levels of targeted mRNA in the BRCA/FA pathway. One miRNA was overexpressed in the non-responsive group of patients.

Phase IV (06.12.2014-05.12.2015)

- New biological samples were included in the biobank; new biological samples were harvested and processed to consolidate the biobank and the database by introducing new patients into the study
- Biological samples (blood, tissue, cervical lavage) were processed according with standardized protocols; the biological samples were processed according to each sample type and stored in the biobank
- During this phase was performed an integrated study based on specific measurements in biological samples and exposure data and the preparation of the experimental model validation for the integrated analysis of mixture exposure and development of dedicated biotoxic model at sample level. The relationship of linear regression between the treatment response and mixture exposure (cigarette smoke, arsenic and phthalates) quantified through exposure biomarkers measured in urine (cotinine, creatinine, phthalates metabolites) did not reveal a statistical significant correlation between the urine biomarkers and the variable that measures the treatment response.
- The study of miRNAs expression on tissue samples consisted in a screening microarray analysis (Agilent 8x60k) on 32 FFPE tissue samples. 4 miRNAs were identified to be of interest as possible modulators of BRCA1, BRCA2, FANCD2 and RAD51 genes, which are involved in the intrinsic resistance of cervical cancer. The miRNA validation will be performed during next phase.
- The pilot study of miRNA screening in serum samples by microarray analysis consisted in the analysis of 24 serum samples collected before therapy from patients with cervical cancer. The quality analysis of the microarray slides showed poor hybridization efficiency, possibly due to the presence of inhibitors in the serum samples or the reduced number of differentially expressed miRNA between samples. One miRNA was identified to be differentially expressed between the two groups and correlated with intrinsic resistance, the data validation will be performed in the following phase.

Phase IV (06.12.2014-05.12.2015)
- New biological samples (post-therapy) were included in the biobank and processed according with standardized protocols
- During this phase was completed the integrated study based on specific measurements for the integrated analysis of mixture exposure (cigarette smoke, arsenic and phthalates) quantified through exposure biomarkers measured in urine (cotinine, creatinine, phthalates metabolites) and development of the biotoxicologic model at sample level;
- Microarray analyzes have identified sets of miARN considered with multi-target potential on FA/BRCA pathway. This data can be exploited in future studies that aim to explore and validate the functional role of these miARN for therapeutic purposes.
- At serum level was identified a small number of miARN, their role in predicting response to therapy should be confirmed by future studies.


l. Final results of the project :

- A prediction model for therapy response in patients with advanced cervical cancer (stage IIB-IIIB) based on tissue and serum genomics signature (miRNA) related to a pharmacogenomics and PBPK prediction prototype.


m. Publications:

Chapter book (international):
1. O Tudoran, O Balacescu, I. Berindan--‐Neagoe I., Molecular Pathways Of Angiogenesis In Cervical Cancer. Molecular Pathways of Angiogenesis in Cervical Cancer (pp. 311-326) In Angiogenesis: insights from a systematic overview; Ed. Santulli G., Nova Science Publishers, 2013.
https://www.novapublishers.com/catalog/product_info.php?products_id=39794

Publications:
1. Tudoran OM, Soritau O, Balacescu L, Pop L, Meurice G, Visan S, Lindberg S, Eniu A, Langel U, Balacescu O, Berindan-Neagoe I. PDGF beta targeting in cervical cancer cells suggest a fine-tuning of compensatory signalling pathways to sustain tumourigenic stimulation. J Cell Mol Med. 2015 Feb;19(2):371-82. (ISI)

2. Balacescu O, Balacescu L, Tudoran O, Todor N, Rus M, Buiga R, Susman S, Fetica B, Pop L, Maja L, Visan S, Ordeanu C, Berindan-Neagoe I, Nagy V. Gene expression profiling reveals activation of the FA/BRCA pathway in advanced squamous cervical cancer with intrinsic resistance and therapy failure. BMC Cancer. 2014 Apr 8;14:246. (ISI)

3. Neamtiu I, Dumitrascu I, Roba C. Chemical Analysis of Urinary Cotinine in Subjects Exposed to A Mixture of Contaminants. Indian Journal of Applied Research. 2014 (3). 56-58 (BDI).

4. Neamtiu I, Dumitrascu I, Roba C. Exposure to Arsenic and Environmental Tobacco Smoke in A Hospitalized Population Group Diagnosed with Cervical Cancer -Preliminary Results From A Pilot Study. IJSR. 2014, 3(3); 71-73 (BDI).

5. Neamtiu IA, Bloom MS, Dumitrascu I, Roba CA, Pop C, Ordeanu C, Balacescu O, Gurzau ES. Impact of exposure to tobacco smoke, arsenic, and phthalates on locally advanced cervical cancer treatment-preliminary results. PeerJ. 2016 Sep 8;4:e2448. doi: 10.7717/peerj.2448.