Background and aim: Molecular mechanism of translocation and outcome in variant chronic myeloid leukaemia (vCML) has been a topic of debate. While several cytogenetic studies suggest a low response to Imatinib Mesylate, others demonstrate a similar disease course in both classical and vCML. Besides, many studies comprehensively also link tyrosine kinase domain (TKD) mutations with aggressive clinical outcome. Thus, we aim to study the molecular mechanism of translocation, identify the third partner chromosomes and comment on the disease course and clinical outcome. Method:We cytogenetically characterised 25 vCML cases to determine the third partner chromosome, mechanism of translocation and prognostic outcome. We also compared vCML cases with and without TKD mutation to most appropriately outline the clinical consequence and ascertain the potent cause of unresponsiveness to treatment.Results: Third partner chromosome in variant translocation was defined by conventional and molecular cytogenetics. Although in our study most cases showed inadequate clinical response attributable to TKD mutation rather than variant translocation, we observed an inferior outcome in cases involving chromosome 5 as the third partner. Conclusion:Thus, we conclude that characterising and reporting new cases of variant translocations, involving various different chromosomes as third partner (with different breakpoints) by cytogenetics, will lead to a better understanding of the disease. To the best of our knowledge, this kind of delineate study has not been applied to precisely comment on the prospects of cytogenetically characterised vCML. K E Y W O R D S tyrosine kinase domain mutation, variant chronic myeloid leukaemia 1 INTRODUCTION Chronic myeloid leukaemia (CML) is the malignant transformation and abnormal proliferation of myeloid progenitor cells. An untreated CML is a bi or tri-phasic disease. European Leukaemia Net (ELN) 2009 characterizes chronic phase CML (CML-CP) by splenomegaly, increased neutrophils and <10% blasts; accelerated phase (CML-AP) by 10-19% blasts and thrombocytopenia unrelated to therapy and blast crises (CML-BC) by increased blasts in blood or bone marrow, clusters of blasts and extramedullary blast involvement. 1 Most patients are diagnosed in CML-CP which usually has an insidious onset. CML accounts for 15% of all leukaemias in adults and shows a slight male

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World Health Organization states the importance of conventional cytogenetics and FISH in hematological malignancy for accurate diagnosis, treatment and monitoring response to therapy. Most FISH probes, however, are Analyte- Specific reagents (not FDA approved) and thus an elaborate validation procedure prior to diagnostic use becomes essential. This study focuses on validating FISH probes by assessing the analyte- sensitivity, specificity, accuracy, precision and determining normal reference ranges (cut-offs). Eight probes from two different manufacturers each were validated using cytogenetically normal peripheral blood (negative controls) and leukemia positive bone marrow samples (positive controls) to determine the most suitable probe for use in a diagnostic set-up. Both the controls were cytogenetically defined before initiating the validation procedure. Alongside this, the probe constructs were studied to understand signal co-localization, size and intensity. Accuracy was determined by metaphase FISH, precision by standard deviation or inter-observer variability and analyte specificity and sensitivity using standard formulae. The cut-off or the normal reference range was derived by BETAINV function in Microsoft Excel. Based on performance characteristics and qualitative data most relevant probes were suggested for diagnostic use. Although validation procedures may differ between test centres, it should be a mandate pre-clinical practice. A validated FISH probe surges dependability on generated reports and this study presents the most rudimentary yet essential parameters in a FISH probe validation.