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Rational Selection of Pcr-Based Platforms for Pharmacogenomic Testing

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Rational Selection of Pcr-Based Platforms for Pharmacogenomic Testing WCRJ 2014; 1 (4): e391 RATIONAL SELECTION OF PCR-BASED PLATFORMS FOR PHARMACOGENOMIC TESTING A. DE MONACO 1, A. D’ORTA 2, C. FIERRO 3, M. DI PAOLO 4, L. CILENTI 5, R. DI FRANCIA 1 1Hematology-Oncology and Stem Cell Transplantation Unit, National Cancer Institute, Naples, Italy. 2DD Clinic, Caserta, Italy. 3UOS Hematology and Cellular Immunology, Azienda dei Colli Monaldi Hospital, Naples, Italy. 4CETAC Research Center, Caserta, Italy. 5Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, USA. ABSTRACT: Background : Genotyping is crucial to the identification of genetic markers underlying development of neoplastic diseases and individual variations in responses to specific drugs. Cost- and time-effective technologies able to accurately identify genetic polymorphisms will dramatically affect routine diagnostics processes and future therapeutic developments. However, such methods need to fulfill the principles of analytical validation to determine their suitability to assess nucleotide polymorphisms in target genes. Approach: This article reviews the recent developments of technologies for genotyping of sin - gle nucleotide polymorphisms (SNPs). For the appropriate choice of any method, several criteria must be considered: i) known or unknown genetic variations in a given cancer gene; ii) needs of testing within pharmacogenomics studies; iii) diffusion and availability of large platforms and re - quired equipments; iv) suitability of tests for routine diagnostics; v) capacity of methods to offer a specific and sensitive detection of mutant alleles within great excess of wild-type alleles in a given sample; vi) suitability for high-throughput implementation. Content: This review is intended to provide the reader with a better understanding of the various technologies for pharmacogenomics testing in the routine clinical laboratory. A brief overview is given on the available technologies for detection of known mutations together with a precise description of the homogeneous technologies and platforms currently employed in genotyping analysis . Based on the criteria proposed here, potential users may evaluate advantage and limitations of the different analytical platforms and possibly identify the most appropriate one according to spe - cific operative settings and diagnostic needs. KEY WORDS: Genotyping methods, Analytical validations, Molecular diagnostics . Abbreviations: Single nucleotide polymorphisms (SNPs) ; Matrix-Assisted Laser Desorption/Ionization time of flight Single-strand Conformational Polymorphism (SSCP) ; Allele (MALDI TOF) ; Fluorescent Resonance Energy Transfer Specific Amplification (ASA) ; Amplification Refractory Mu - (FRET) ; Locked nucleic acid (LNA) ; Oligonucleotide liga - tation System (ARMS) ; Restriction fragment length poly - tion assay (OLA) ; Rolling Circle Amplification (RCA) ; High morphism analysis (RFLP) ; Peptide Nucleic Acid (PNA) ; Resolution Melting (HRM) . Corresponding Author : A. De Monaco, MD; e-mail: [email protected] 1 RATIONAL SELECTION OF PCR-BASED PLATFORMS FOR PHARMACOGENOMIC TESTING INTRODUCTION fectiveness and practicability. These methods are usually amenable to automation and high-through - Recent research demonstrates that certain genetic put processing with 96-well plates, the current in - polymorphisms are linked to significant variations dustry standard, but can be further implemented by among individuals in the response, in terms of ac - 384-well plate capabilities. Maximum automation tivity and toxicity, to a given drug 1. To confirm the can be achieved by fully integrated systems with ro - candidate genetic markers emerging from such botic processing of 96- or 384-well plates through - studies, there is a commensurate need for pharma - out the stages of DNA extraction, PCR set-up, cogenomic laboratories to design and validate tar - amplification, detection, and data interpretation 6. geted genotyping assays capable of rapidly identify Many of these strategies are now commercially the individual Single Nucleotide Polymorphism available and this field is characterized by intense (SNP) of interest within confirmatory clinical stud - competition mixed to many examples of produc - ies and in the routine clinical practice. In recent tive cooperation and cross licensing 7. No single years, a number of increasingly complex tech - genotyping platform stands out as ideal and it is nologies have been applied to the qualitative and likely that many of the different technologies de - semi-quantitative detection of polymorphisms and scribed in this article will be employed in com - mutations in DNA (for simplification, we shall bined studies aimed to find disease genes and mainly refer to point mutations, though in general, novel drug targets. small deletions or insertions can be as efficiently 2 detected by the methods described here) . NEEDS TO DETECT GENETIC VARIATIONS Traditional techniques for SNP genotyping de - IN CANCER CHEMOTHERAPY tection by Single-Strand Conformational Poly - morphism (SSCP) and Heteroduplex analysis have now been largely replaced by high-throughput Pharmacogenomic approaches have been applied to methods including “in silico” discovery platforms. many existing chemotherapeutic agents in an effort These latter methods generate much more data and to identify relevant inherited variations that may bet - are easier to automate. ter predict patient response to treatment and toxic - A recent breakthrough in high-throughput strate - ity 8. Genetic variations which can alter the amino gies is represented by DNA chip technology, which acid sequence of the encoded protein, include nu - allows the combined detection and identification of cleotide repeats, insertions, deletions, translocations mutations 3. However, for many applications appro - and SNPs. Genetic polymorphisms in drug metab - priate chips will be only available in the forthcom - olizing enzymes like Cytochrome P450 family, drug ing years. Thus, conventional screening methods for transporters like Multidrug Resistance-1, and other point mutations and small deletions will most prob - molecular targets have been actively explored with ably keep their place in the diagnostic laboratory for regard to functional changes in phenotype (altered a reasonable amount of time. Costs, however, are expression levels and/or activity of the encoded pro - projected to be high and assay performance and re - teins) and their contribution to variable drug re - sults interpretation will remain strictly dependent on sponse 9,10 . Clinically relevant examples of genetic the availability of highly qualified and well-trained defects highlighting the relevance of cancer phar - personnel. We will highlight some of the most pop - macogenomics in optimizing cancer chemotherapy ular homogeneous technologies that are currently by improving its efficacy and safety are given in used in specialized laboratory, making the transition Table 1 . A new generation of anticancer drugs has from the research setting to the clinical laboratory been recently designed with high specificity toward and discuss key aspects in their validation for geno - tumour cells, providing a broader therapeutic win - typing in pharmacogenomics 4. dow with less toxicity as compared to conventional Homogeneous methods are essentially “single- chemotherapy; these drugs represent a new and tube” assays in which all of the processes required promising approach to targeted cancer therapy 11 . for target amplification and detection occur in a New agents are designed to interfere with a spe - single “closed-tube ” reaction (except for Pyrose - cific molecular target, usually a protein with a crit - quencing), without a solid phase. Combining the ical role in tumour growth or progression (i.e. a thermal cycling system with the signal detection tyrosine kinases). There are multiple types of other system allows the on-line monitoring of the PCR targeted therapies already clinically available, in - amplification process 5. In this review post PCR cluding monoclonal antibodies, antisense in - agarose gel-based detection methods, will be con - hibitors, proteasome inhibitors, enzyme-activity sidered as homogeneous. modifiers and immuno-modulatory drugs. Obvi - The advantages of homogeneous methods in - ously, any of these new agents may exert a selec - clude reduced risk of cross-contamination, time-ef - tive pressure on tumour cells that elaborate 2 RATIONAL SELECTION OF PCR-BASED PLATFORMS FOR PHARMACOGENOMIC TESTING TABLE 1. MOST COMMON GENETIC ABNORMALITIES IN CANCER GENES AND THEIR EFFECT IN CHEMOTHERAPY OUTCOMES. GENE Polymorphism Molecular Drug Effect on (nucleotide effect therapy translation) Cytochrome Various nucleotide Decreased enzyme Cyclophosfamide Inter-individual variability P450 family translation activity Etoposide in Pharmacokinetics Paclitaxel TPMT2, 3A, 3C Various Rapid degradation 6-MP Hematopoietic toxicity Polymorphism Thioguanine UGT1A 28 TA repeats in Low expression Irinotecan Neutropenia toxicity 5’ promoter MDR1 (C3435T) Low expression Various Drug resistance TYMS 3 tandem repeats High expression 5-FU, Methotrexate Drug resistance DHFR (T91C) Increase enzyme Methotrexate Drug resistance activity MTHFR (C677T) Decreased enzyme Methotrexate Toxicity activity c-KIT (T1982C) Constitutive signal Imatinib Desensitizes activity (T81421A) activation in GIST c-KIT D816V Imatinib Good response in Semaxinib t(8;21)-positive AML EGFR L858R Gefitinib Good response in NSCLC Erlotinib ABL
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