SummaryMolecular testing for the BCR-ABL1 fusion gene by real time quantitative polymerase chain reaction (RT-qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT-qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society.

SummaryMolecular genetic assays for the detection of the JAK2 V617F (c.1849G>T) and other pathogenetic mutations within JAK2 exon 12 and MPL exon 10 are part of the routine diagnostic workup for patients presenting with erythrocytosis, thrombocytosis or otherwise suspected to have a myeloproliferative neoplasm. A wide choice of techniques are available for the detection of these mutations, leading to potential difficulties for clinical laboratories in deciding upon the most appropriate assay, which can lead to problems with inter-laboratory standardization. Here, we discuss the most important issues for a clinical diagnostic laboratory in choosing a technique, particularly for detection of the JAK2 V617F mutation at diagnosis. The JAK2 V617F detection assay should be both specific and sensitive enough to detect a mutant allele burden as low as 1-3%. Indeed, the use of sensitive assays increases the detection rate of the JAK2 V617F mutation within myeloproliferative neoplasms. Given their diagnostic relevance, it is also beneficial and relatively straightforward to screen JAK2 V617F negative patients for JAK2 exon 12 mutations (in the case of erythrocytosis) or MPL exon 10 mutations (thrombocytosis or myelofibrosis) using appropriate assays. Molecular results should be considered in the context of clinical findings and other haematological or laboratory results.

Both ITD and D835 mutations of the fms-like tyrosine kinase (FLT3) gene cause constitutive activation of the receptor, in the absence of ligand. We have examined a cohort of 91 patients, AML (80) and MDS (11), to determine the prevalence of these mutations and any correlations between the two mutations and disease prognosis. FLT3/ITD (ITD+) or D835 mutations (D835+) were not detected in MDS patients examined. However, 10% (8/80) and 7.5% (6/80) of AML patients were ITD+ and D835+, respectively. ITD+ patients have a higher rate of relapse than patients with wild-type (WT) FLT3. Median overall survival was 4.6 months (range 0.6-36.2) for ITD+ and 19.85 months (range 0.2-197.5) for WT patients (P=0.0066), and disease-free survival (DFS) was also worse for ITD+ patients than FLT3/WT patients (P=0.047). FLT3/ITD is also a significant prognostic marker for overall survival (OS) and DFS in patients in the standard karyotype group (P=0.0040, 0.0365, respectively). ITD is more prevalent in patients in the standard karyotype category (7/41, 17.1%) as compared to patients in the poor-risk category (1/32, 3.1%). Similar to ITD, D835 mutations were found to be more frequent in patients with standard-risk rather than poor-risk cytogenetic category. WBC count (mean 63.8 x 10(9)/l) was significantly higher in ITD+ patients than patients with D835 mutations (mean 34.8 x 10(9)/l) and WT patients (mean 26.4 x 10(9)/l) (P=0.004). D835 mutants did not appear to have a worse median OS or DFS compared with the WT group. We conclude that FLT3/ITD mutations may be an important prognostic marker in AML, especially in the standard/good risk karyotype groups, where it may allow risk-directed therapy.

No abstract