Comment on: Technical Issues Behind Molecular Monitoring in Chronic Myeloid Leukemia
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LETTER TO THE EDITOR
Comment on: Technical Issues Behind Molecular Monitoring in Chronic Myeloid Leukemia Mireille Crampe1 • Stephen E. Langabeer1
Ó Springer International Publishing Switzerland 2015
In a recent Commentary in Molecular Diagnosis & Therapy, Mattarucchi and colleagues raise several issues relevant to the quantitative PCR (qPCR) molecular monitoring of BCR-ABL1 transcripts in patients with chronic myeloid leukaemia (CML) that warrant further comment [1]. The BCR-ABL1 oncogene is the de facto genetic abnormality of all cases of CML with inhibition of the activity of the resultant protein the target of current tyrosine kinase inhibitor (TKI) therapies. As correctly stated, monitoring expression of BCR-ABL1 transcripts in CML is indeed a surrogate of disease response to therapy, yet it must also be acknowledged that DNA-based assays used to quantify the BCR-ABL1 copy number also follow this assumption. Quantification of BCR-ABL1 transcript expression by qPCR is currently by far the most widely adopted approach for molecular tracking, with consensus recommendations for defining deeper molecular responses recently published [2]. Pre-analytical variables can significantly affect RNAbased assays such as those for monitoring BCR-ABL1 transcripts and include red cell depletion, RNA extraction, complementary DNA (cDNA) synthesis, selection of qPCR primer/probe sets, use of appropriate internal controls and standards, and qPCR platform. Post-analytical variables such as evaluation of BCR-ABL1 and reference gene amplification and result reporting also require careful consideration. Many of these aspects have been comprehensively addressed previously, with consensus guidelines available [3, 4]. However, automating several or all aspects of the process would substantially reduce the impact of & Stephen E. Langabeer [email protected] 1
Cancer Molecular Diagnostics, Central Pathology Laboratory, St. James’s Hospital, Dublin 8, Ireland
these pre-analytical variables. An example of such an available platform is the cartridge-based XpertÒ BCRABL1 Monitor system, which is validated in several studies and is able to provide a feasible solution to many of the standardisation issues surrounding BCR-ABL1 monitoring [5–7]. Important parameters that require calculation when implementing a particular BCR-ABL1 monitoring method in the clinical laboratory include limit of quantification (LOQ) and limit of detection (LOD). A further requirement would be to determine the uncertainty of measurement (UOM), which provides information on the accuracy of measurement and is essential when monitoring the change in transcript levels, especially at low levels. UOM helps to establish the expected level of uncertainty associated with the measurement itself. For example, given a BCR-ABL1/ reference gene ratio of 2.4 % with a UOM of 1 %, the subsequent result should be either greater than 3.4 % or less than 1.4 % to be interpreted as a change in the BCRABL1 transcripts level. This can guide the clinician to differentiate between change
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