The application of real-time quantitative PCR in the diagnostics of chronic myeloid leukaemia

[摘要] English: CML is a cancer of the white blood cells and it effects on average one individualin every 100,000. Since it was first described in 1845 by John Hughes Bennettand the subsequent discovery of the Philadelphia chromosome by NowelI andHungerford in 1960, this hematopoietic malignancy has received much attentionin terms of scientific study. Elucidating the pathogenic pathway has lead to thedevelopment of targeted therapy. In 2001 imatinib mesylate was introduced asfirst line therapy for CML. The success of imatinb was illustrated during theIRIS trial by Real-time quantification of BCR-ABL mRNA.BCR-ABL expression levels are correlated to disease stage and progression.BCR-ABL mRNA quantification is therefore the most accurate and sensitiveprognostic marker to monitor CML patients. Hence, Real-time PCR for BCRABLhas been introduced in many international laboratories to allow foraccurate and reliable monitoring to improve and manage patient treatment.Standardization became problematic due to the ease of method developmentand robustness for Real-time quantification of BCR-ABL mRNA by differentlaboratories. As a result a plethora of methods for Real-time quantification ofBCR-ABL mRNA have been published. This is especially problematic forlaboratories with limited means undertaking to develop and implement such amethod. Since there are no standardized guidelines, in-house development isrequired. Furthermore, availability of commercial copy number standards forcontrol and target genes makes it difficult to implement anyone method fromthe literature especially since there is criticism for the genes where standardsare commercially available.From a thorough analysis of the literature, problem areas considering RNAextraction, the choice of priming for cDNA synthesis, primers and probes forReal-time PCR as well as a specific control gene together with copy numberstandards and reference material were clearly defined. Based on thisinformation, best laboratory practice regarding common methodology fromliterature was established. Only recently through an initiative known as EuropeAgainst Cancer (EAC) has there been a concerted effort to facilitate regionalstandardization of Real-time quantification of BCR-ABL mRNA.During this study a modified EAC method for Real-time quantification of BCRABLmRNA was developed and validated with the emphasis to improvereproducibility. Instead of ABL or BCR, GUS was used as control gene basedon recommendations from literature. Based on statistical analysis it wasconcluded that the modifications did not bias the percentage BCR-ABL result.It cannot be emphasised enough that standardization for Real-time monitoringof BCR-ABL is most crucial as it will ultimately facilitate molecular laboratoriesto develop this diagnostic with much greater ease. In order for standardizationto be realized, copy number standards as well as reference material for qualitycontrol purposes needs to become more readily available. In addition to that,specific guidelines for assay criteria such as appropriate Ct values and analysisof data must also be developed. By streamlining Real-time quantification ofBCR-ABL the treatment and monitoring of CML patients can be improved on aglobal scale.

[发布机构] University of the Free State