Serial monitoring of T315I BCR-ABL mutation by Invader assay combined with RT-PCR

Abstract

We recently developed an Invader assay combined with reverse transcriptase polymerase-chain-reaction in order to quantify T315I bcr-abl transcripts. Using this assay, we serially monitored T315I bcr-abl transcripts in chronic myeloid leukemia (CML) patients whose bcr-abl transcripts were still detectable at 6 months after starting imatinib therapy. Although, we continued to monitor bcr-abl transcripts in 14 CML patients (13 chronic phases and 1 accelerated phase) for up to 12 months, there were no patients who were apparently resistant to imatinib due to the T315I mutation. In contrast, in a case of Philadelphia chromosome-positive acute lymphoid leukemia being treated with chemotherapy including imatinib, we monitored both wild-type and T315I bcr-abl transcripts, and found increased levels of T315I transcripts during relapse (0% at the time of diagnosis and 54.8% at relapse). Thus, our new approach could be a useful tool to study the kinetics of mutant clones and the pharmacokinetics of drug resistance with regard to the T315I mutation.

Induced pluripotent stem cells generated from diabetic patients with mitochondrial DNA A3243G mutation

“…The first 10 and 12 positions in the primary probes represent the 5′ flap of Invader reaction. The cleavage enzyme, fluorescence resonance energy transfer probe, signal probe and Invader oligo were added to the microplates, including diluted plasmids that included the primary probe/ Invader oligo binding region, after which the Invader assay was carried out as previously described [21]. The plates were incubated at 63°C in the fluorescence microplate reader (FluoDia-T70; Otsuka Electronics, Osaka, Japan).…”

“…The plates were incubated at 63°C in the fluorescence microplate reader (FluoDia-T70; Otsuka Electronics, Osaka, Japan). Fluorescence values for carboxyfluorescein (3243A; wavelength/ bandwidth: excitation 485/20 nm; emission 530/25 nm) and Redmond red (3243G; excitation 560/20 nm; emission 620/ 40 nm) were measured every 2 min for a period of 4 h. To detect A3243G heteroplasmy, we calculated the copy number of 3243A and 3243G with a standard curve using quantitative Invader assay as described [21,22]. The A3243G ratio was based on the ratio of the all-copy (3243A and 3243G) to the 3243A copy.…”

Influence of CYP3A5 and drug transporter polymorphisms on imatinib trough concentration and clinical response among patients with chronic phase chronic myeloid leukemia

“…The samples used for IM response evaluation were obtained from patients on the same day as samples used for measurement of plasma trough levels. In patients without a CCyR, mutation analysis of the bcr-abl fusion transcript was performed using the Invader assay, 31 which detects 25 mutations, including M244V, L248VR, G250E, Q252H, Q252R, Y253F, Y253H, E255K, E255V, E279K, F311L, T315A, T315I, F317L, M351T, F359IV, V379I, L387M, H396P, H396R, S417Y, E459K and F486S.…”

Successful prior treatment with dasatinib followed by stem cell transplantation in a patient with CML in blastic crisis with a BCR–ABL mutation

“…The best response to imatinib was a complete cytogenetic response. However, the patient developed severe thrombocytopenia requiring blood transfusion, and cytogenetic analysis revealed Ph in 15/21 metaphases with M244V mutation of the BCR-ABL kinase domain [3]. In October 2006, the patient was started on salvage therapy for imatinibresistant CML [4]; at that time, the corrected BCR-ABL to ABL ratio was 141.25 and after treatment, the M244V-positive clone levels became undetectable.…”