Digestive tumor bank protocol: from surgical specimen to the genomic studies of digestive cancer

Cancer is a complex polygenic and multifactorial disease, resulting from successive dynamic changes in the genome of somatic cells and from the accumulation of molecular alterations in both tumor cells and host cells. Genome amplifications, deletions, mutations, insertions, and rearrangements affect the expression of genes controlling regulatory processes such as proliferation, cell cycle dynamics, DNA repair, and apoptosis. These changes lead to genetic instability, initiation of tumor genesis, and progression to an increasingly malignant, invasive, and resistant phenotype. Host factors have a strong influence on the development and evolution of cancer, and there are many molecular heterotypic interactions between tumor cells and their normal neighbours. Apart from characterized genetic susceptibilities to hereditary cancers, and individual differences in immune defense, variations of yet unknown host background features definitely influence progression and disease outcome.
For the majority of cancers, including many malignancies of the gastrointestinal tract, our current means of diagnosis and treatment of the tumors are grossly insufficient.
In recent years the development of several gene expression profiling methods such as comparative genomic hybridization (CGH) (1), differential display (2), serial analysis of gene expression (SAGE) (3) and DNA arrays (4), associated with the sequencing of the human genome, have provided an opportunity to monitor and investigate the complete cascade of molecular events leading to tumor development and progression.
DNA arrays allow quantitative and simultaneous measurement of the mRNA expression levels of thousands of genes in a biological sample. The technology is based on the hybridization of a complex probe, derived from tissue RNA, to large sets of DNA fragments that represent target genes arrayed on a solid support.
DNA arrays applications include comparative genomic analyses for the identification of chromosomal imbalances (Matrix-CGH), SNP analyses for the detection of mutations and genetic polymorphisms, proteome analyses using peptide and antibody arrays and gene expression profiling for the simultaneous assessment of transcript levels of up to several tens of thousands of genes in a single experiment., making them ideal tools to study the complex network of transcriptional changes that are associated with the malignant transformation of normal cells.
The mRNA hybridized to the complementary DNA used by DNA arrays is extracted from fresh normal and tumoral tissues, so the first step in genomic research is the long-term tissue bank.
DNA arrays technology is rapidly spreading worldwide and has the potential to significant change the therapeutic approach to patient affected with tumor.
Given the central role played by surgeons in the current management of patients with solid cancer, it is of paramount importance for them to know the principles that characterize this laboratory tool to critically appreciate the results originating from this biotechnology. (5)
The clinical objective is a more accurate cancer classification system, using the comprehensive gene expression profiles of tumors to identify new distinct molecular subgroups within clinically and morphologically similar groups of tumors.
Post-genome era discovery processes, in order to deliver high quality and consistent results that should be expected with actually available technologies, must be based upon the combination of two main components: (1) high quality, carefully selected biological material associated with diagnostic and clinical data from large scale patient cohorts and (2) state of art technology platforms including genomics, proteomics and bioinformatics platforms.
SCIENTIFIC PARTNERSHIP BETWEEN FUNDENI CLINICAL INSTITUTE AND RNTECH COMPANY
In order to improve the scientific and medical activities, the Fundeni Clinical Institute (FCI) has been developed a scientific collaboration agreement for research with RNtech, a biotech developing company in the field of cancer diagnostic and treatment.
This agreement is a premiere in the romanian scientifically medical society, being an ambitious project of molecular biology and clinical studies based on a high skilled team both in research and clinical fields.
The scientific collaboration agreement, signed in August 2002 and modified in June 2004 has the main object to undertake in common the creation of a center of biological resources (Biobank).
This partnership made possible an exclusive access to high standard biological materials and data combined with state of art technology.
The following issues arise from this agreement:
1. Center of biological resources and the collection of biological materials - (blood samples, urinary samples, normal and tumoral tissues from the operative specimen)
The center of biological resources (Biobank) achieves biological materials from patients hospitalized in FCI with cancer along with clinical and pathological information and imposes some ethical, scientific and technical standards:
· The patient's individual and written consent is taken after an individual explanation of the research objective on these biological products made by the physician;
· The data obtained from biological products and clinical data are covered by the medical secret;
· All information concerning the patient becomes anonymous and confidential, only the partners know about the patient's identity and RNtech will be in possession of a numeric code for every patient;
· The high-quality biological samples are retrieving from patients diagnosed with a digestive cancer, mainly, but other tumoral pathologies are also accepted, and they are associated with complete clinical files. The samples are prepared by the cryogenic method (under liquid nitrogen) at -170ºC
· Tissues samples coming from the surgical resected specimen in the setting of the surgical interventions with curative intention are harvested without interfering with pathological diagnostic;
· The blood and urinary samples will be collected only with the physician's consent and if the patient has given his/her agreement before the surgical intervention.
2. Standardized protocol of working for the biological collection - Biobank
The FCI (by the Departments of Surgery and Pathology) and RNtech established a standardized protocol regarding the process of harvesting, identification, coding and storage of the biologic products. From this protocol every department involved will write it's normalized and standardized operative procedures and the institute will look after their application.
A. Identification of the operational procedures in the protocol of the Biobank Department of Surgery:
1. Patient selections for the protocol;
2. Explanation of the project and obtaining the written individual informed consent;
3. Obtain the clinical information;
4. Harvesting biological fluids (blood and urine);
5. Transportation of the blood and urinary samples in specific conditions;
6. Taking the documents and biological fluids;
7. Preparation for the surgical intervention the cases included in the protocol;
8. Surgical intervention for the resection of the operative specimen;
Department of Pathology:
1. Transportation and receipt of the operative specimen;
2. Preparation of the specimen and registration of the case introduced in the protocol;
3. Harvesting and freezing the tissues chosen by the pathologist from the operative specimen;
4. Transportation of the frozen tissues specimen in liquid nitrogen;
5. Preparation of the operative specimen for the pathological examination;
6. Treatment with paraffin of the samples from pathological examination;
7. Preparation of the histological blades for the microscopic examination;
8. Pathological diagnosis after the microscopic exam and refilling of the standardized histological file;
9. Taking photos for every type of sample examined;
10. Protocol of storage of the histological cassettes with paraffin;
11. Protocol of storage of the histological blades;
12. Protocol of storage of the pictures digitalized of the samples after the microscopic exam;
13. Protocol of storage of the information on the histological control in the notebooks of laboratory and in the histological files.
Department of Biochemistry and Rntech Laboratory (Fig 1, 2):
1. Receipt of the blood and urinary samples and first registration of the samples received;
2. Preparation of the serum / urine for the storage and registrations of the cases included in the protocol;
3. Receipt of the samples tissue on liquid nitrogen, temporary storage, registration of the cases included in the protocol;
4. Preparation of tissues for the definitive storage and storage of the information gotten;
B. Standardized documents utilized in the protocol of the Biobank
For security reasons regarding patient data the files contain a code of identification for each patient included in RNtech database. Only the Departments of Surgery and Biochemistry from FCI could make the relationship between the code and patient identification. This files respect the international terminology of the domain (ethical, medical).
Tree types of files are used:
· Standardized written individual informed consent file.
· Standardized clinical data file.
· Histological quality control file.
C. Storage of the information's and biological products:
Department of Surgery:
· Storage of clinical files
· Storage of the photos of resected specimen in a database
Department of Biochemistry:
· Storage of the standardized files - written consent, copy of clinical and histological files (in non anonymous profile)
· Storage of the annulated files
· Storage of the information regarding identity and location of the patients.
Department of Pathology:
· Storage of paraffin blocks
· Storage of the histological blades
· Storage of the digitalized histological photos
· Storage of histological files
RNtech Laboratory:
· Storage of biological fluids
· Storage of tissues specimens (Fig. 3)

D. Scientific committee reunion and activity report - a weekly standardized activity report is sent to the scientific committee in order to evaluate the activity of the team
3. Ethical, scientific and technical responsibilities of the staff involved
Ethical responsibilities: the setting up of the specific conventions to insure the legal and functional prelevation of the biologic materials, while following the ethical principles implemented in the European Community and the United States; FCI accepts the constitution of a file for accreditation of this collection by the competent organisms and assumes the ethical responsibility.
Technical and scientific responsibilities: creation of a technological platform for biological products collection, establish a standardized protocol of procedures, maintaining a high-quality control system.
BIOBANK RESULTS AND FUTURE PERSPECTIVES
From August 2002 till July 2006 1296 patients were included in Biobank. Data is summarized in table 1.
Until now 4 studies are running:
1. The gene expression profile of the mucosa from normal colon, adenomas and colonic adenocarcinoma with studying of sequential deregulations of the genes (up and down regulation) from normal colonic mucosa to adenocarcinoma.
2. Study of the gene expression profile and biomarkers correlated with clinical and pathological parameters in ductal pancreatic adenocarcinoma (GENOPACT).
3. Genic profiles induced by transcriptional suppression of ETS1 in pancreatic cancer
4. Expression of caveolins 1, 2 and 3 in pancreatic cancer as molecular targets in diagnosis and therapy.
In these studies after identification of a gene or (much common) a set of genes using a DNA array- based experiment, confirmation of this results with accuracy is commonly effectuate with quantitative real - time PCR. For further validation of the expression of proteins coded by investigated genes, standard immunohistochemistry or Western blot techniques will be assessed.
As further applications because translational gene expression regulation and posttranslational protein modifications are also of crucial importance in determining cell function, DNA array should be complemented with the latest new technologies such as tissue micro array (6) and proteomics (7, 8).
Using these powerful research techniques we hope that our studies will describe a part of the molecular portrait of the biological phenomena underlying tumor development and progression.

References
1. Pinkel, D., Segraves, R., Sudar, D. - High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nat. Genet., 1998, 20:207.
2. Broude, N.E. - Differential display in the time of micro-arrays. Expert Rev. Mol. Diagn., 2002, 2:209.
3. Velculescu, V.E., Zhang, L., Volgelstein, B. - Serial analysis of gene expression. Science, 1995, 270:484.
4. Brown, P.O., Botstein, D. - Exploring the new world of the genome with DNA microarrays. Nat. Genet., 1999, 21:S33.
5. Mocellin, S., Provenzano, M., Rossi, C.R. - DNA array-based gene profiling: From surgical specimen to the molecular portrait of cancer. Annals of Surgery, 2005,1:16.
6. Kallioniemi, O.P., Wagner, U., Kononen, J. - Tissue microarray technology for high-throughput molecular profiling if cancer. Hum. Mol. Genet., 2001, 10:657.
7. Le Naour, F. - Contribution of proteomics to tumor immunology. Proteomics, 2001, 1:1295.
8. Lawrie, L., Fothergill, J., Murray, G. - Spot the differences: proteomics in cancer research. Lancet Oncol., 2001, 2:270.

• Timeviewed: 4928 times