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 individual in every 100,000. Since it was first described in 1845 by John Hughes Bennett and the subsequent discovery of the Philadelphia chromosome by Nowell and Hungerford in 1960, this hematopoietic malignancy has received much attention in terms of scientific study. Elucidating the pathogenic pathway has lead to the development of targeted therapy. In 2001 imatinib mesylate was introduced as first line therapy for CML. The success of imatinb was illustrated during the IRIS 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 sensitive prognostic marker to monitor CML patients. Hence, Real-time PCR for BCRABL has been introduced in many international laboratories to allow for accurate and reliable monitoring to improve and manage patient treatment. Standardization became problematic due to the ease of method development and robustness for Real-time quantification of BCR-ABL mRNA by different laboratories. As a result a plethora of methods for Real-time quantification of BCR-ABL mRNA have been published. This is especially problematic for laboratories with limited means undertaking to develop and implement such a method. Since there are no standardized guidelines, in-house development is required. Furthermore, availability of commercial copy number standards for control and target genes makes it difficult to implement any one method from the literature especially since there is criticism for the genes where standards are commercially available. From a thorough analysis of the literature, problem areas considering RNA extraction, the choice of priming for cDNA synthesis, primers and probes for Real-time PCR as well as a specific control gene together with copy number standards and reference material were clearly defined. Based on this information, best laboratory practice regarding common methodology from literature was established. Only recently through an initiative known as Europe Against Cancer (EAC) has there been a concerted effort to facilitate regional standardization of Real-time quantification of BCR-ABL mRNA. During this study a modified EAC method for Real-time quantification of BCRABL mRNA was developed and validated with the emphasis to improve reproducibility. Instead of ABL or BCR, GUS was used as control gene based on recommendations from literature. Based on statistical analysis it was concluded that the modifications did not bias the percentage BCR-ABL result. It cannot be emphasised enough that standardization for Real-time monitoring of BCR-ABL is most crucial as it will ultimately facilitate molecular laboratories to develop this diagnostic with much greater ease. In order for standardization to be realized, copy number standards as well as reference material for quality control purposes needs to become more readily available. In addition to that, specific guidelines for assay criteria such as appropriate Ct values and analysis of data must also be developed. By streamlining Real-time quantification of BCR-ABL the treatment and monitoring of CML patients can be improved on a global scale.

[发布机构] University of the Free State