The study of the human genome laid the foundations for the search for a large number of molecular alterations related to different diseases. The Precision medicine allows us to know molecular alterations that can be detected and targeted for therapeutic purposes. There is little data in Argentina about the incidence and frequencies of alterations molecules associated with the most frequent tumors. Through the selection of a gene panel, analysis of the genetic information obtained analysis allows classifying tumors from a point of view therapeutic. On the other hand, through the same panel, markers of resistance to drugs that allow the incorporation of retreatment therapies. Together with the proposed panel, the ancestry of the patients will be evaluated to determine whether the frequencies of molecular alterations vary between the different ethnic origins of the country.
Platform Development A validation study of a new genetic panel of 52 genes will be carried out in tissue biopsies from adult subjects with the most prevalent solid tumors in Argentina. In a first phase, 200 retrospective samples of different tumors and matrices (e.g. paraffin tissue, fine needle biopsy and fresh tumor) to be able to analytically validate the proposed panel. Reproducibility tests will be carried out, repeatability and determination of the detection limit for each of the 52 markers studied. The choice of tumor types will be based on the frequency observed in the population from the Italian Hospital of Buenos Aires (HIBA), corroborated against agency data international cancer research (IARC-WHO). The distribution of samples of tumors (T) will be: 15% T digestive (colon), 15% T breast cancer, 15% T skin cancer (melanoma and basal cell carcinoma), 15% T lung cancer, 10% T prostate, 6% thyroid T, 6% ovarian T, 6% bladder/renal T and 12% rare T (nervous system central, musculoskeletal, head and neck, among others). In a second phase, once fine-tuned and analytically validated, the panel will be available to the treating doctor who may request the panel with prior consent informed of the subject. Tumor biopsies, both retrospective and prospective, are received and processed in the HIBA Pathological Anatomy Service by standardized methods that encompass embedding in paraffin (paraffin wad) and extraction of cellular genomic material (total cellular DNA -nuclear and mitochondrial- and RNA) by cutting 10 micron chips of thickness. Said paraffin material will be processed, in its entirety, with commercial kits of extraction of nucleic acids using Qiagen columns or equivalent. Gene Panel Sample Study: Proposed Massive Sequencing Method Thermo Fisher's S5 and Ion Torrent platforms will be used. The workflow comprises the amplification of specific regions and the generation of libraries to then be able to perform an emulsion PCR(Polymerase Chain Reaction). After a PCR enrichment step of emulsion, sequencing is carried out on chips of variable capacity, depending on the amount of data you wish to obtain. For the project, it is intended to process 32 samples in parallel with an Ion 540 chip. To comply with international standards, a validation phase will be carried out gene panel according to the guidelines recently published by the College American Association of Pathologists and the American Genetics Association: "Guidelines for Validation of Next Generation Sequencing based on Oncology Panel. A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologist", published in March 2017 6d The Oncomine Focus Assay panel It is a multimarker analysis that is carried out in massive sequencing equipment (S5) that evaluates 52 genes associated with treatment of solid tumors for which there is scientific evidence of its usefulness diagnostic, prognostic and/or predictive of therapeutic response The system analyzes DNA and RNA extracted from the tumor piece -biopsy- or blood, for example. The catalog of detectable alterations includes 35 hotspot genes, 19 Copy Number Variations (CNVs) and 23 gene fusions.
Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina