The introduction of tyrosine kinase inhibitors (TKIs), starting with imatinib and followed by second and third generation TKIs, has significantly changed the clinical management of patients with chronic myeloid leukemia (CML). Despite their unprecedented clinical success, a proportion of patients fail to achieve complete cytogenetic remission by 12 months of treatment (primary resistance) while others experience progressive resistance after an initial response (secondary resistance). BCR‐ABL1 kinase domain (KD) mutations have been detected in a proportion of patients at the time of treatment failure, and therefore their identification and monitoring plays an important role in therapeutic decisions particularly when switching TKIs. When monitoring KD mutations in a clinical laboratory, the choice of method should take into account turnaround time, cost, sensitivity, specificity, and ability to accurately quantify the size of the mutant clone. In this article, we describe in a “manual” style the methods most widely used in our laboratory to monitor KD mutations in patients with CML including direct sequencing, D‐HPLC, and pyrosequencing. Advantages, disadvantages, interpretation of results, and their clinical applications are reviewed for each method. Am. J. Hematol., 2012. © 2011 Wiley Periodicals, Inc.

MethodsThe IWHS cohort includes 41,836 women who, in January 1986, were randomly sampled from the Iowa State driver's list and responded to an initial questionnaire. This questionnaire queried numerous characteristics, including height to the nearest inch.As detailed previously [1], the IWHS data were linked to Centers for Medicare Services (CMS) enrollment and health care utilization data from 1986 through 2004. Medicare provides payment for all or part of health care for most US residents aged 65 and older [24], and data stemming from the payment of Medicare bills is now used widely as a population-based data source for clinical occurrences [24]. Of IWHS cohort members surviving to 65 years, 98% (40,377 of 40,997) met our CMS inclusion criteria and were therefore followed for incident VTE events [1].IWHS participants were considered to have had a VTE if any of the following VTE ICD-9 codes occurred on their Medicare MedPAR (hospitalization) discharge diagnosis records: 415.13, 451.13, 451.2, 451.81, 451.9, 453.0, 453.1, 453.2, 453.3, 453.43, 453.8, 453.9 [1]. Provoked VTE was classified on the basis of ICD codes indicating malignancy at the time of discharge, surgery in the three months prior to VTE diagnosis, hospitalization of 4 days or trauma in the two months prior to the VTE, or a serious medical disease at the time of hospital discharge. All remaining cases were classified as unprovoked [1].A recurrent event was defined as one that occurred subsequent to, but independently of, the incident event. We had no specific information about independence of events. Therefore, in order to distinguish index VTE events from recurrent VTE events, we required a 6-month lag period between the discharge date of the initial event and the admission date of the subsequent event [1].For this analysis of height and VTE recurrence, we included only women who experienced an incident event. Cox proportional hazards regression was used to evaluate the relation between height and VTE recurrence. As in our previous IWHS manuscript, which reported the association between height and risk of incident VTE

Molecular monitoring of minimal residual disease (MRD) using quantitative real-time PCR (qPCR) to measure BCR-ABL1 transcript levels has become an essential tool in management of patients with chronic myeloid leukaemia (CML). The assay shows superior specificity and sensitivity compared with conventional morphology, cytogenetic, and fluorescent in situ hybridization. In an attempt to increase sample throughput and assay accuracy, we have developed a duplex assay that measures both BCR-ABL1 target and endogenous control transcripts in the same reaction with no loss of sensitivity compared with the conventional simplex test.The BCR-ABL1 fusion gene is a consequence of the t(9;22)(q34;q11) chromosomal translocation (resulting in the Philadelphia chromosome, Ph), a primary leukemogenic event in CML. Transcriptional control is dysregulated and the subsequent chimeric protein contains a constitutively activated tyrosine kinase with a multitude of downstream targets involved in cellular proliferation, avoidance of apoptosis and genomic instability [1]. The BCR-ABL1 fusion gene is also found in some cases of acute lymphoblastic leukemia (Ph1ALL) and in rare instances of acute myeloid leukemia.Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become the standard tool for molecular assessment of residual disease in several hematological malignancies. As a result of the Europe against Cancer (EAC) initiative, optimized dual-labeled hydrolysis (TaqMan) probe and primer sets were developed for a wide range of leukemia-associated fusion genes, including BCR-ABL1 [2]. Endogenous control genes, used to normalize the target gene expression, were evaluated, and ABL1 was judged to be the most appropriate [3]. Particular effort has been expended recently to optimize and standardize the quantification of BCR-ABL1 transcripts especially following to the worldwide use of first-and second-generation tyrosine kinase inhibitor-based therapies [1][2][3][4][5][6].Because of a high throughput of MRD samples, both in-house and referral, a routine assay was initially developed for use in our laboratory that quantitated BCR-ABL1 and ABL1 transcripts in separate qPCR runs (simplex tests). This assay was based on the EAC protocol but utilized different ABL1 primers and probes, hence referred to as the ''Hammersmith'' set, because of the presence of a G6PD sequence insert (part of an earlier competitive PCR assay) in the ABL1 exon 3 [7].With the advent of a range of optimised, multiplex-capable reagent master mixes, the opportunity arose to streamline the simplex assay (Sx), which historically tested ABL1 and BCR-ABL1 in separate plate, into a single plate where both ABL1 and BCR-ABL1 reactions were carried out in a single well (hence referred to as duplex assay; Dx). This approach offers several advantages, including lower cost, time saving while providing higher throughput and increased precision due to the removal of inter-run variations. We took considerable care to ensure that gained benefits were not at the expense ...

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