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

Abstract

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.

Afrikaans: CML is ʼn maligniteit van die witbloedselle en dit affekteer gemiddeld een individu per 100,000. Vandat dit die eerste keer in 1845 beskryf is deur John Hughes Bennett en die daaropvolgende ontdekking van die Philadelphia chromosoom deur Nowell en Hungerford in 1960, geniet hierdie hematopoïetiese maligniteit veel aandag in terme van wetenskaplike studie. Ontbloting van die patogeniese weg het gelei tot die onwikkeling van teikengerigte terapie. In 2001 is imatinib mesylaat bekendgestel as die nuwe eerste linie vir die bestryding van CML. Die sukses van imatinib was duidelik gedurende die IRIS proewe toe BCR-ABL mRNA vlakke met behulp van kwantitatiewe PKR gekwantifiseer is. BCR-ABL uitdrukkingsvlakke word gekorreleer met siekte toestand en ontwikkeling. BCR-ABL mRNA kwantifisering is die akkuraatste en sensitiefste prognostiese merker vir die monitering van CML pasiënte. Gevolglik is “Realtime PCR” vir BCR-ABL kwantifisering deur vele internasionale laboratoriums bekend gestel vir akkurate en betroubare monitering wat sal help met die verbetering en bestuur van behandeling vir pasiënte. Standaardisering het problematies geword a.g.v. die gemak waarmee die “Real-time” tegniek deur verskillend laboratoriums ontwikkel kon word. Die uiteinde hiervan was dat ʼn aansienlike hoeveelheid metodes vir die kwantifisering van BCR-ABL uitdrukkingsvlakke gepubliseer is. Dit het juis ʼn probleem geword vir laboratoriums met beperkte hulpbronne om hierdie metode te onwikkel en te implementer aangesien daar geen standaard riglyne beskikbaar is nie. Dit het gelei tot menige intern-ontwikkelde metodes aangesien ʼn tekort aan kommersieel verkrygbare kopiegetal standaarde vir kontrole- sowel as teiken geen dit onmoontlik maak om ʼn spesifieke metode, soos gepubliseer in die literatuur, te implementer. ʼn Deeglike literatuurstudie het probleem areas soos RNA ekstraksie, die keuse van peilstukke vir cDNA sintese, peilstukke en peilers vir “Real-time PCR” sowel as ʼn spesifieke kontrole geen tesame met kopie getal standaarde en verwysingsmateriaal, duidelik gedefinieer. Na aanleiding van hierdie inligting is die beste laboratorium gebruik n.a.v. algemene metodiek van die literatuur vasgestel. Daar is onlangs ʼn inisiatief aangewend bekend as “Europe Against Cancer (EAC)” om streeks standaardisasie vir “Real-time” kwantifisering van BCR-ABL mRNA te fasiliteer. ʼn Tekort aan kommersiële kopie getal standaarde en verwysingsmateriaal staan steeds sentraal tot die frustrasie van ander pogings om die kwessie van metodiek standaardisering op te los. Gedurende hierdie studie is ʼn gemodifiseerde EAC metode vir “Real-time” kwantifisering van BCR-ABL mRNA ontwikkel en geverifieer met beklemtoning om reprodusering te verbeter. Eerder as om ABL of BCR as kontrole gene te gebruik, is GUS vir hierdie doeleinde geïmplementeer met verwysing na aanbevelings van verskeie publikasies. Statistiese analise het getoon dat hierdie veranderinge nie enige resultate vir BCR-ABL kwantifisering onregmatig beïnvloed het nie. Die belangrikheid van metodiek standaardisering vir “Real-time” kwantifisering van BCR-ABL mRNA kan nie genoeg benadruk word nie aangesien dit uiteindelik die implementering van die metode deur verskeie molekulêre laboratoriums sal vergemaklik. Hiervoor sal kopie getal standaarde asook verwysingsmateriaal vir kwaliteitskontrole meer geredelik kommersieel beskikbaar moet wees. Verder sal spesifieke riglyne vir reaksie kriteria soos aanvaarbare Ct waardes en data analise ook ontwikkel moet word. Deur “Realtime” kwantifisering van BCR-ABL mRNA te standaardiseer, sal die behandeling en monitering van CML pasiënte op ʼn globale skaal verbeter kan word.