Defining the molecular transcriptomic profile for predicting the clinical outcome of anthracycline resistant breast cancers. Defining metastases in relation with the primary tumor. BREAST-OMICS

This is an informational website about a research project realized in the National Program of RDI.—PN II-partnership:

Project description
Coordinator of the multidisciplinary consortium:
Project partners
Project course of events 

Project description

BREAST-OMICS subscribes to Health area 4.1.3. Investigation and intervention methods based on molecular and cellular medicine, genomics and proteomics.

This project is based on a highly complex partnership and seeks to develop the interdisciplinary sciences and high tech fields of genomics and proteomics in Romania, in the field of oncology. It would study tumor pathology and would use the newest technologies of genomics (microarray) and proteomics (protein-array) in breast cancer. The project will be carried on 36 months.
It is estimated that 1 out of 8 (13%) women will develop breast cancer during their life and 1 out of 33 will die from it.

New concepts that resulted from the microarray technology in the discovery of biomarkers have shown that the pattern of transcription or the proteins can lead to a correct and timely diagnosis. By evaluating a single biomarker, these modern technologies (microarray, protein array) are capable of providing data about the whole transcriptome or proteome associated with cancer. They are able to evaluate thousands or tens of thousands of transcription products or proteins associated with the genes of interest.

It is thus possible to study the differences between normal and tumor cells as well as the cellular mechanisms responsible for the development of cancer. The resistance to cytostatics is one of the major problems in the treatment of cancer and the study of pharmacogenomics, which analyzes the simultaneous expression of the entire cellular transcript, is the only tool capable of clarifying the function of the cell after a treatment with cytostatics.

The main aim of this project is to define of the molecular transcriptomic profile for the optimization of neoadjuvant anthracycline treatment in breast cancer.
The objectives of the study are: the identification of a molecular signal through microarray, the analysis of differential gene expressions during anthracycline treatment; the validation through tissue array studies of the principal proteins associated to the studied genes; the evaluation of proangiogenic molecules in the development of the tumor and during treatment; the identification of a molecular target for angiogenesis and immune system modulators that will help define metastases and the primary tumor.

Project time span: 14.09.2007— 30.09.2010
Funds: 2.000.0000 RON ( ? 500.000 EURO)
CNMP Project Code: 41-029/2007

Coordinator of the multidisciplinary consortium:
University of Medicine and Pharmacy „I. Hatieganu” Cluj Napoca (UMFCN).

Project partners:
The“ Ion Chiricuta” Cancer Institute – Comprehensive Cancer Center (IOCN),
The Institute of Public Health “Iuliu Moldovan„ Cluj-Napoca (ISPCN),
The „Solutions of Artificial Intelligence Applications” Association (SAIA)
S.C. Biona SRL.

Project management:

Assoc. prof. dr. Irimie Alexandru (UMF) - Project manager
Dr. Balacescu Ovidiu (IOCN) –Project responsible, IOCN partner
CSII Dr. Irimie Sorina (ISPCN) - Project responsible, ISPCN partner
Dr. Floares Alexandru (SAIA) – Project responsible, SAIA partner
Sef Lucrari Dr. Neagoe Ioana (Biona) - Project responsible, Biona partner

Conf Dr. Irimie Alexandru
The“ Ion Chiricuta” Cancer Institute – Comprehensive Cancer Center
Str. Republicii nr 34-36, Cluj Napoca, Cod 400015
Tel: 0264 - 450347, Fax: 0264 - 597692,

Project course of events:
14.09.2007 — project startup

Stage I (14.09.2007-15.12.2007)
The main objective of Stage I was to analyze the data available in the specialized literature regarding new diagnostic and treatment tendencies for breast tumors in stages IIIA and IIIB.

The UMF project manager presented an analysis of the data in the literature regarding the involvement of anthracycline treatment in breast cancer. The focus was set on the necessity to establish correctly all prognostic and predictive factors, in order to evaluate the evolution and response to therapy of patients with advanced locoregional breast cancer.

The IOCN partner presented the data regarding the usage of transcriptomic investigation techniques for anthracycline-resistant breast cancers. It is known that one of the most difficult problems in breast cancer management is predicting the response to chemotherapy. This was shown by several data from genetic studies that underline the fact that determining the genetic profile will become crucial to the therapeutic decision, in order to improve results and to offer better life quality to patients with breast cancer.

The ISPCN partner introduced several facts from the specialized literature, concerning the involvement of life- and environment risk factors in the etiopathogenesis, evolution and prognosis of breast cancer. Breast cancer is a complex disease and the environment is considered to play an important part in its development; however, the extension of this part is not very clear. About 90% of the women who develop a breast cancer do not have cancer in their family history and, furthermore, there are studies on large groups of twins that have shown that most breast cancers cannot be explained in hereditary terms. Incidence discrepancies between countries suggest potential environment exposures; however, the relation between environment exposure and breast cancer is still very difficult to understand.

The SAIA partner brought forward several informations concerning the analysis techniques for transcriptomic data: pre-processing and identification of clusters that allow the tumor classification according to the levels of gene expression.
The Biona partner shortly described the work protocols and the legislative, communitary and international alignment regulations for processing human tissues as a support for biomedical research.

Stage II (16.12.2007-25.11.2008)
The activities of this stage were: establishing the patient groups according to the inclusion criteria specific to this study; planning the organization and standardization of analysis methods; developing a database of all patients; establishing a biobank; initiating the harvesting of biological samples (blood, tissues).

The UMF project manager
He introduced a review of the standardization of analysis methods according to the type of biological samples that were harvested.

The standardization of analysis methods was based on the usage of several types of analyses, including not only the classical method of extracting nucleic acids, but also new methods, based on commercial kits. The qualitative and quantitative evaluation of the extracted RNA was realized with the help of nanotechnology-based procedures. The devices that were used were: the NanoDrop Spectrophometer ND-100 and the Bioanalysor 2100 manufactured by Agilent.

Choosing these extraction methods was motivated by the qualitative and quantitative evaluations resulted from the two technologies. The chosen extraction methods (one for biopsies and the other for integral blood) use the principle of principle of selectively binding the RNA to a silica gel membrane and include the treatment with DNAse in their stages.

The IOCN partner presented the information regarding the infrastructure of the tumor bank, that is conceived and functioning according to European security regulations. Here, they present the harvesting protocols for biological samples and the consent protocols. Personal data, clinical information and the data concerning the harvested biological materials will be processed according to the „European Union Directive (Dir /95/46/EC) concerning the protection of individuals and the processing of their personal data” and also according to the state laws. All biological samples shall be processed in the same conditions, abiding by special, standardized protocols that are implemented currently by the Department of Functional Genomics. The safety of each patient in this study will be ensured and all harvested biological samples (tissues, blood) will be given an identification code. All tissue samples have been grouped according to the anatomo-pathologic diagnosis.

The ISPCN partner presented information about environmental factors that can be involved in the appearance and evolution of breast cancer. There is a series of factors, endogenous as well as exogenous, that increase the risk that this neoplasia affect the population. These factors include elements of genetic predisposition, hormonal status (age of menarche and duration of the menstrual period, endogenous hormonal status, pregnancies, breast feedings, usage of oral contraception, hormonal substitution therapy ), anthropometric features, lifestyle (ex. diet type and obesity, alcohol consumption or smoking), radiations, different environmental and occupational exposures.

This package of informations was included in the database, and the analysis of primary data was carried out according to the modules included in the evaluation questionnaire for breast cancer risk factors.

The SAIA partner displayed a series of data concerning the elaboration of selection criteria for the patients who will form the tested groups of this project, from the point of view of biomedical informatics. They selected methods for the most promising results, in order to identify clusters (subgroups or subpopulations) in patient populations or in order to classify patients, in situations that are almost equal to those in the study. They have identified all constraints introduced by these methods or, in other words, they identified potential selection criteria for patients or patient data; these criteria should be added to those established before, by different categories of doctors concerned with patient management. Thus, most methods aim at eliminating non-informative data and at processing the remaining information, so that the contents be maximized and then extracted.

The Biona partner exposed a short description of the protocols for harvesting samples of breast cancer, biopsies from tumors, by freezing or other alternative preservation techniques; the identification of tumor banks from European networks that have access to informational databases; building relationships with the above-mentioned and standardizing the methods of including the samples harvested from the breast tumor pathology in these banks.
During this stage, we organized an ESMO course related to the topic of our project: Breast Cancer Management: A European Perspective „ Cluj-Napoca, October 2-5, 2008.


Stage III (26.11.2008-15.09.2009)
During this stage, our aim was both to harvest new biological samples for the consolidation of our biobank, as well as to process the samples in order to prepare the study in genomics and proteomics.

The UMF project manager
He built a database of all clinical and paraclinical data available for each patient; this data will be used both for the analysis of biological samples, and for the division in groups and subgroups. According to this information, patients will be split in groups; these will be useful in the pharmacogenomic analyses by micro array, in order to identify the prediction and evolution markers of advanced locoregional breast cancer after the anthracyclin therapy. Our database contains 82 patients until now; 30 of them will participate in the study, according to the inclusion-exclusion criteria we have presented in the previous report. The grouping of patients was done according to age, histological type, hormonal status (presence/absence of estrogen/progesterone receptors and HER2). Another considered criterion was the number of treatment cycles provided, as well a the treatment response rate. Until now, 3 patients have responded completely to treatment.

The IOCN partner continued the activity of including patients in the biobank, according to the previously established selection criteria. The biological samples (blood, tissue) were harvested and conserved differently, according to their nature, respecting the harvesting protocol. Processing the biological samples of blood and tissue (RNA extraction) and the synthesis of micro array probes (complementary DNA) will be done in special locations, with 2nd class manipulation hoods for biological samples. The biological samples we have introduced in the biobank have been processed in similar conditions, according to the previously established protocols. The extracted RNA samples have been evaluated qualitatively and quantitatively and an optimal extraction method has been chosen for each type of biological sample.
Until now, we have included 82 patients in the pharmacogenomic study. We have harvested blood (for the RNA extraction) and serum, as well as tissue samples (punction biopsy, incisional biopsy and post-treatment exeresis elements) from 69 patients before therapy and from 20 patients after the anthracycline therapy.

The SAIA partner meets the clinical needs by creating a series of comprehensive analytical instruments that can study complex pathologies like cancer; these needs have lead to the creation of technologies like genomics and proteomics, that evolved quickly and that are capable to provide complex informational databases, necessary for clinical cancer research. In order to prepare studies in genomics and proteomics, it is necessary to establish the pre-processing and cluster identification algorithms. Applying the algorithms: 1) K-means, 2) Fuzzy C-means, 3) hierarchy clustering or 4) Mixture of Gaussian is considered appropriate for the issues caused by measuring errors as well as by missing data, in order to extract patterns from protein arrays. Considering the amplitude and complexity of the Breast-Omics project, we consider that applying the CRISP-DM method is appropriate for the extraction of information (patterns) from the Breast-Omics data set, but also for their processing into knowledge.

The BIONA partner
In this stage of the project, he defines the institutional framework in which tumor banks can develop, function and be standardized (especially those that harvest breast tissue from cancer patients), in accordance with minimal quality standards, which are obligatory for the creation of databases and for sample harvesting. In this respect, we present the protocols that certify that all conditions of long-term preservation have been respected, with subsequent applicability and controlled access to informational data.


1. Alexandru Irimie, Ovidiu Balacescu, Claudia Burz, Emil Puscas, Loredana Balacescu, Oana Tudoran, Meda Rus, Bogdan Fetica, Carmen Lisencu, Redis Roxana, Patriciu Achimas, Ioana Berindan-Neagoe. Prognostic significance of serum level of different growth factors and their correlation with estrogen receptors in patients with locally advanced breast cancer. Romanian Biotechnological Letters, 2011, (16), 5, 6523-6534.

2. Sorina Irimie, Mariana Vlad, Ileana Maria Mirestean, Ovidiu Balacescu, Carmen Lisencu, Emil Puscas, Bogdan Fetica, Patriciu Achimas, Loredana Balacescu, Ioana Berindan-Neagoe, Alexandru Irimie. Risk Profile in a Sample of Patients with Breast Cancer from the Public Health Perspective. Applied Medical Informatics, 2010, 27(4), 21-30.