Piano di Attività
“Analysis of Hematologic Malignancies and Solid Tumors through SNPs arrayfor the prediction of genetic aberrations within target genes and throughGene Expression Profiling in order to explain the role of differential gene expressionin normal biological and disease processes.”
Background
Recent findings on the role of the genetic component in disease development and the success of drug treatment have opened new perspectives in the field of oncology for the implementation of personalized and tailored therapies with low toxicity and high efficacy for each patient.
Several evidences have shown that the tumor is a genetic disease, in fact characteristics and clinical-pathological responsiveness' of cancer cells to a specific therapeutic treatment is the result of genetic alterations acquired by tumor cells during their malignant transformation.
An accurate molecular characterization of tumors would thus allow the identification of molecular mechanisms underlying the malignant transformation and drug response as well as' the identification of new therapeutic targets delivering genetic biomarkers of disease and therapy useful for prognostic and therapeutic stratification of patients, the rationalization of pharmacological choice and the design of new treatments. In hematological malignancies field, already several authors have demonstrated the close association between prognosis and genetic subtype 1, 2 and highlighted important genetic biomarkers of drug response that are still widely used in the clinic 3.
The clinical value of molecular characterization of tumors has been demonstrated in solid tumors, highlighting how specific genotypes or alterations, in clinical practice, may allow therapies improving both in hematologic malignancies than in other malignancies 4. A significant evidence of how the study of cancer cells genetics may allow treatment with specific therapies is provided by the identification of the BCR-ABL fusion gene in chronic myeloid leukemia and the use of its specific inhibitor "imatinib", currently in use in the clinic with reasonable success 5
The close association between mutations in BCR-ABL gene and relapse 5, also underlines the clinic need to continuously identify alterations acquired by the chimeric gene to identify resistance cases and, therefore, therapeutic failure. Today, in the post-genomic era, an accurate genetic analysis of the genetic constitution of tumor cells is possible thanks to avant-garde technologies which, exploring the entire genome, allow genome-wide studies of the genetic heritage and cells-tissue transcriptome .
SNP-arrays are high-density oligonucleotides arrays that allow, on one hand, the identification of genetic polymorphisms and the realization of Genome-Wide Association Studies (GWAS), on the other hand, global analysis of cells karyotype making possible cytogenetic studies (CGH ) and Loss Of Heterozygosity (LOH) at high resolution. Cytogenetic analysis with SNP arrays has allowed the identification of chromosomal microaberrazioni (Copy Number Alteration, CNA) and LOH (also "copy-neutral" LOH, undetectable by other techniques commonly in use today) with high prognostic value and therapeutic in solid and haematological tumors.
The study of the genetic profile of SNPs arrays has allowed, for example, a better prognostic classification of adult and pediatric ALL, through the identification of CNA not known, otherwise undetectable with classical cytogenetics, and associated with the loss of genes involved in relevant cellular pathway, such as lymphoid development (IKZF1, PAX5, EBF1, VPREB1), the cell cycle regulation, (CDKN2A/CDKN2B, PTEN, BTG1, RB1), signaling (BTLA, CD200), pharmacological response (NR3C1), and the DNA mismatch repair (mTOR, HERC1, PRKCZ, and PIK3C2B) 6-10. Other authors have identified tumor-specific regions of LOH in pediatric patients with ALL associated with drug resistance or disease progression 11, 12.
Studies of Gene Expression Profiling consist of an assessment of the expression of the whole cellular transcriptome. Through comparative analysis, the study of gene expression profiling allows the identification of genetic and genes signature whose expression levels are associated with processes under study such as oncogenesis, tumor progression and therapeutic response. These studies, clarifying molecular mechanisms and highlighting all the elements potentially involved, open up new prospectives for a quick search of genetic biomarkers also previously unknown. The first studies of Gene Expression Profiling conducted in the field of pediatric ALL allowed a better classification and molecular genetic characterization of the different subtypes of ALL and AML identifying leukemic types with different gene expression profile for which has been then observed distinct therapeutic and prognostic course 13-21; secondly, they permit the identification of markers associated with tumor subtypes and prognosis 22-27 of patterns and genetic determinants of drug resistance, toxicity and therapeutic outcomes 28-32 as well as new targets for therapy 33-35 . Recent work has allowed a better molecular classification of lymphomas and cancers 36 still little known, such as colorectal cancer and gastrointestinal cancers, have been characterized offering new medical information useful for the disease treatment 37-38 .
In a recent work, for example, the analysis of the gene expression profile of cases of Gastrointestinal Stromal Tumors, GISTs, with and without mutation of KIT and PDGFRA has highlighted the receptor IGFR1 as a new target therapy for unmutated forms and therefore untreatable with conventional therapy 39.
In a recent survey of GISTs, the information of gene expression have been integrated with karyotype analysis performed by SNP arrays allowing the identification of genes affectively involved in chromosomal alterations associated with tumor 38. The integrated analysis has demonstrated the involvement of oncogenes and tumor suppressor genes known as KRAS in the amplification of chr 12p and KIF1B, PPM1A and deletion of NF2 in chr 1p, 14q and 22p respectively; in addition highlighted potential new tumor suppressor genes such as DAAM1, RTN1 and DACT1, involved in the deletion 14q23.1 highly frequent in cases of GISTs with KIT and PDGFRA mutation.
Rational
The treatment of oncological diseases is an issue of great importance for modern medicine. Although medical research has made satisfactory progress in the study of oncological diseases, still human tumors appear to be extremely complex and heterogeneous disease, with molecular structure little known and easily resistant or refractory to standard treatments. The innovative advances in molecular biology and nanotechnology now allow genome-wide study of gene expression and a high-resolution analysis of the karyotype allowing a more accurate characterization and, then, genetic and molecular classification of cancer. The results of these studies open new perspectives in clinical practice, for the identification of molecular biomarkers of disease and therapy as well as new drug targets toward which direct tumor-specific therapies, more effective and less toxic because drawn on the pathology molecular structure.
Objective
This project aims at a better characterization and molecular, clinical-prognostic classification of solid and haematological tumors in clinical practice to enable the optimization of existing therapeutic strategies through therapeutic and prognostic stratification of patients and the possibility of making therapeutic choices that are also innovative, based on a molecular structure of diseases, effectively targeted and effective. The scientific activity, conducted in collaboration with several research groups, will be carried on various solid tumors and human hemolymphopoietic neoplasm, of adults and children, through the implementation of high-resolution genome-wide expression profiling studies of gene expression and cell karyotype (studies of Copy Number) using the innovative microarray technology affymetrix.
Methods
The project will be done using the innovative technology of microarrays. In particular, gene expression studies will be combined to cytogenetic studies (studies of Copy Number) and Loss Of Heterozygosity (LOH) using the Affymetrix GeneChip.
The gene expression studies will be performed on GeneChip Human Genome U133 Plus 2.0 Arrays, high-density oligonucleotides chip containing probe sets for approximately 47000 transcripts whose 38500 known genes and on chips of the latest generation GeneChip Human Transcriptome Array 2.0 containing 44,699 known genes.
The copy number and LOH studies will be performed on Genome-Wide Human SNP Arrays 6.0 and Cytoscan HD arrays. The analysis of SNP arrays will be performed with Genotyping Console software and Chromosome Analysis Suite (CHAS) software.
Scientific training plan
The researcher will take care of the preparation of samples for hybridization and scanning of the chips for microarray analysis. He will also obtaining and analyzing data using statistical and bioinformatic tools dedicated for which he has already made training courses and study. In addition he will take care of the resolution of problems directly related to the proper performance of the microarray method.
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