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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-10302009-090817 |
| Date | 30 October 2009 |
| Creators | van Deventer, Jacob Jacobus |
| Contributors | Prof VJ Louw, Prof CD Viljoen |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-10302009-090817/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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