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Chronic myelogenous leukaemia (CML) [OMIM #608232] represents about 15-20% of all cases of adult leukaemia and acute lymphoblastic leukaemia (ALL) [OMIM #601626] accounts for approximately 80 per cent of all childhood leukaemia cases.
Nearly all cases of CML and a minority of cases of ALL are caused by a t(9;22) (q34;q11) chromosome translocation – known as the Philadelphia chromosome – which fuses 2 genes: BCR and ABL. The BCR-ABL fusion acts as an oncogene and promotes genomic instability.
The advent of effective chemotherapy for CML in the late 1990s immediately demonstrated the need for accurate measurement of the amount of the abnormal clone remaining in the patient. This became known as the measurement of minimal residual disease (MRD) and quickly became the most useful measurement in monitoring the effectiveness of treatment in individual patients.
Measurement of MRD is routinely performed by reverse-transcription real-time PCR (RT-PCR) by quantifying levels of BCR-ABL mRNA transcripts in peripheral blood and bone marrow samples. The technique can determine accurately the response to treatment and is particularly valuable for patients who have achieved complete chromosomal remission. Despite efforts to establish standardised protocols for BCR-ABL fusion transcript quantitation there is still substantial variation in the way in which RT-PCR for BCR-ABL is carried out and how results are reported in different laboratories world-wide.
The amount of BCR-ABL translocation is usually measured by comparison with the level of a normal control gene transcript. However, this is the limit of conformity and several different control genes, assay methods and reporting strategies are in use by different groups around the world.
There was a clear need for standardisation of measurement and at the same time the relationship between BCR-ABL levels and clinical outcome needed to be established. The IRIS trial established a standard baseline for measurement – (100% BCR-ABL on the ‘international scale’) and a major molecular response (good response to therapy) was defined as a 3-log reduction in the amount of BCR-ABL – 0.1% BCR-ABL on the international scale.
However, the samples used to define these values were quickly exhausted and traceability relied on the internal QC data of one laboratory in Adelaide. Other laboratories wishing to align their results with the international scale could do so by exchanging samples with the Adelaide laboratory and by this process conversion factors were established for several laboratories.
Subsequently one more laboratory in Mannheim has become able to derive conversion factors and although this system works well, it is very laborious and consequently only open to a limited number of laboratories at any given time.
The collaborative validation study has assigned BCR-ABL / BCR; BCR-ABL / ABL; BCR-ABL /GUSB values for four different freeze-dried cellular materials, each containing different amounts of BCR-ABL. The materials consist of 4 different dilutions of K562 cells (Philadelphia chromosome positive) in HL60 cells (Philadelphia chromosome negative). Alignment of these materials with the pre-existing international scale is vital to allow consistent and comparable quantitative data to be obtained in all countries. The values have been assigned by a small number of expert laboratories who have been able to show that their conversion factor – which allows them to convert their local results to the international scale – is stable over time.
Mean µg RNA yield after extraction
BCR/ABL 1 08/192
BCR/ABL 2 08/194
BCR/ABL 3 08/196
BCR/ABL 4 08/198
BCR-ABL / ABL
BCR-ABL / BCR
The panel comprises four different dilutions of K562 cells (Philadelphia chromosome positive) in HL60 cells (Philadelphia chromosome negative). Samples are presented as approximately 1.5x10E6 cells freeze-dried genomic DNA in glass ampoules. The collaborative validation study data indicates that these materials are suitable for use in RNA extraction and subsequent Q-PCR assays to quantitate the BCR-ABL fusion gene transcript.
Dr Ross Hawkins - Principal ScientistDr Jennifer Boyle – Principal ScientistDr Pia Sanzone – Senior ScientistMr Malcolm Hawkins - Senior Scientist Dr Leandro Lo-Cascio - Senior ScientistMr Miltiades Stylianou – ScientistMr Noble Ossai - Scientist
BCR-ABL fusion gene quantitation09/138: WHO International Genetic Reference Panel for the quantitation of BCR-ABL translocationOther genomic reference materials