Acute myeloid leukemias with t(8;21) or inv(16)/t(16;16) are commonly referred to as Core-Binding Factor AML and represent 5-8% of all AMLs . These genomic rearrangements are characterized by disruption of the CBF and CBF genes respectively. Both genes encode a subunit of core-binding factor complex, an important transcriptional regulator of hematopoiesis involved in myeloid differentiation. CBF AML are considered a favorable AML risk group based on high remission rate and survival probabilities. However, the presence of the cKIT D816V mutation has been reported to harbor an unfavorable prognostic implication in CBF-AML. The proto-oncogene cKIT encodes a receptor for SCF. It belongs to the type-III receptor of the tyrosine kinase subfamily, that is characterized by five extracellular immunoglobulin-like domains, a single transmembrane helix, a cytoplasmic juxtamembrane domain, and a kinase domain. The cKIT D816V mutation is found in 20–25% of patients with t(8;21) and in about 30% with inv(16)/t(16;16). cKIT D816V causes the indipendent-ligand activation of the tyrosine kinase receptor cKIT, and the activation of downstream pathways supporting cell proliferation and survival. CBF AML patients exhibiting D816V mutation in cKIT gene, are classified in the intermediate risk category, since they present an high incidence of relapse and a low overall survival. Moreover the replacement of the aspartic acid residue with valine at codon 816 causes resistence to Imatinib, the tyrosine kinase inhibitor usually used in the treatment of CBF AML to suppress the overexpressed cKIT. In this case, patients are treated with TKIs of second generation, such as Dasatinib or Nilotinib. So, the single point mutation D816V in cKIT gene, represents a critical prognostic factor for CBF AML patients and an important indicator to define the appropriate treatment. Several PCR-based methods are developed for the detection of D816V mutation, such as PCR followed by direct DNA sequencing, enzymatic digestion with HinfI enzyme (RFLP), or dHPLC. However, most of the PCR-based methods result expensive, time-consuming and labor-intensive. At the moment Sanger sequencing is still the gold-standard for D816V detection, even if it presents a low sensitivity (10–15%). The aim of this work is to improve the detection of cKIT D816V mutation in CBF AML patients by the development of rapid, user-friendly and cost-effective assay based on Allele-Specifc Loop mediated isothermal AMPlification (AS-LAMP) technology. The AS-LAMP assay is based on LAMP technology that amplifies target DNA at costant temperature thanks to the employment of the DNA polymerase with strand-displacement activity. AS-LAMP assay is able to detect the single point mutation thanks to the cooperation of two elements: a loop primer specific for the mutated cKIT sequence, and a blocker that binds only the wild type gene. While the blocker suppresses the aspecific amplification of wild type alleles, the LB primer recognizes the mutated codon and allows its amplification. The AS-LAMP assay includes also the amplification of the housekeeping ABL gene as internal control, that is informative to exclude false-negative results due to the failure of DNA extraction procedure. The target and the IC are detected in real-time in 500 nm and 530 nm channels respectively, thanks the use of two labeled probes specifically binding cKIT or ABL genes. The AS-LAMP assay was optimized on synthetic plasmid containing the cKIT sequence and was successfully validated on negative and positive clinical samples, confirming in all cases the results obtained with PCR-based methods. In conclusion the AS-LAMP assay is able to detect the D816V mutation with high sensitivity and specificity in only 40 minutes and it is easy-to-use. For these reasons the AS-LAMP assay represents a powerful tool for molecular diagnostics applications in clinical routine.
|Data di pubblicazione:||26-feb-2016|
|Titolo:||Development of a Novel Molecular Assay Based on Allele-Specific Loop Mediated Isothermal Amplification for Rapid Detection of cKIT D816V Point Mutation in Core-Binding Factor Acute Myeloid Leukemia Patients|
|Settore Scientifico Disciplinare:||BIO/11 - BIOLOGIA MOLECOLARE|
|Corso di dottorato:||BIOTECNOLOGIE INDUSTRIALI - 15R|
|Citazione:||(2016). Development of a Novel Molecular Assay Based on Allele-Specific Loop Mediated Isothermal Amplification for Rapid Detection of cKIT D816V Point Mutation in Core-Binding Factor Acute Myeloid Leukemia Patients. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).|
|Parole Chiave (Inglese):||cKIT D816V, AS-LAMP, CBF AML|
|Appare nelle tipologie:||07 - Tesi di dottorato Bicocca post 2009|