Abstract
Imatinib induces a complete cytogenetic response in the majority of patients and a reduction of BCR-ABL to 0.1% of the level at diagnosis is associated with longer progression free survival. However, the methods of BCR-ABL detection differ from study to study. Mostly, quantitative real-time PCR assay (RQ-PCR) has been used however, the control gene and expression of results have not been standardized to date. In Japan, only Amp-CML, a commercial kit composed of TMA (transcription mediated amplification) and HPA (hybridization protection assay) has been approved by the government. BCR-ABL was expressed by copy number per microgram RNA. The range of detection is between 50 to 2500 copies per assay. If 0.05 ug of RNA is used for the assay, the detection ranges between 1000 to 50000 copies per microgram RNA. However, the accuracy and sensitivity of Amp-CML in comparison with RQ-PCR are not well known. Thus we compared Amp-CML with RQ-PCR using a large number of patients enrolled in the Japan Adult Leukemia Study Group (JALSG).
In the JALSG clinical trial, imatinib was used as initial therapy for 171 patients with chronic myelogenous leukemia. We measured levels of BCR-ABL transcripts in the blood of all patients in this trial before and 1, 2, 3, 6 and 12 months after the treatment using RQ-PCR and Amp-CML. In both assays the results expressed the copy numbers of BCR-ABL per microgram of RNA. The median number of BCR-ABL transcripts detected by RQ-PCR was 98,964 at diagnosis and 63,442, 33182, 3342, 331 and 98 at 0, 1, 2, 3, 6, 12 months, respectively, after treatment. The median number of BCR-ABL transcripts detected by Amp-CML was >50000 at diagnosis and >50000, 20284, 2180, <1000 and <1000 at 0, 1, 2, 3, 6, 12 months, respectively, of treatment. These results suggested that results of Amp-CML and RQ-PCR were well correlated (R=0.67), although the range of Amp-CML was restricted. In the next step, we attempted to improve the sensitivity by increasing the amount of RNA used for Amp-CML. Using 0.5 ug of RNA, the range of detection by Amp-CML is between 100 and 5000 copies, and 50 additional samples were analyzed. In this range, BCR-ABL copy number was also well correlated. (R=0.968) Using 2ug of RNA, we are investigating more sensitive methods expecting range of detection by Amp-CML is between 25 and 1250 copies. Tentatively 13 samples which showed 10–100 copy/ug by RQ-PCR was reevaluated by both methods. Ratio of Amp-AML to RQ-PCR is 1.4 in median and range is 0.3-2.1. Thus Amp-CML may have accuracy even the very small range. Thus, Amp-CML can be used for the monitoring BCR-ABL transcripts during treatment with imatinib.
Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR
