Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR

Abstract

We sought to establish a rapid and reliable RT-PCR approach for detection and quantification of BCR-ABL fusion transcripts using the LightCycler technology. This device combines rapid thermocycling with online detection of PCR product formation and is based on the fluorescence resonance energy transfer (FRET) between two adjacent hybridization probes carrying donor and acceptor fluorophores. A pair of probes was designed that was complementary to ABL exon 3, thus enabling detection of all known BCR-ABL variants and also normal ABL as an internal control. Conditions were established to amplify less than 10 target molecules/reaction and to detect one CML cell in 10 5 cells from healthy donors. To determine the utility of the assay, we quantified BCR-ABL and ABL transcripts in 254 samples (222 peripheral blood, 32 bone marrow) from 120 patients with CML after therapy with IFN-␣ (n ‫؍‬ 219), allogeneic BMT (n ‫؍‬ 17), chemotherapy (n ‫؍‬ 11), or at diagnosis (n ‫؍‬ 7). The level of residual disease in the 245 BCR-ABL positive specimens was expressed as the ratio of BCR-ABL/ABL. This ratio was compared to results obtained by three established methods from contemporaneous specimens. A highly significant correlation was seen between the BCR-ABL/ABL ratios determined by the LightCycler and (1) the BCR-ABL/ABL ratios obtained by nested competitive RT-PCR (n ‫؍‬ 201, r ‫؍‬ 0.90, P Ͻ 0.0001); (2) the proportion of Philadelphia chromosome positive metaphases determined by cytogenetics (n ‫؍‬ 81, P Ͻ 0.0001); and (3) the BCR ratio determined by Southern blot analysis (n ‫؍‬ 122, P Ͻ 0.0001). We conclude that real-time PCR with hybridization probes is a reliable and sensitive method to monitor CML patients after therapy. The major advantages of the methodology are (1) amplification and product analysis are performed in the same reaction vessel, avoiding the risk of contamination; (2) the results are standardized by the quantification of housekeeping genes; and (3) the complete PCR analysis takes less than 60 min.

LH receptor gene expression in cumulus cells in women entering an ART program

“…Such a low number of copies cannot be identified with RT-PCR. Of note, the presence of cDNA was verified in all samples using G6PD as a reporter gene [8], even in samples where LHR splice variants were not detected through RT-PCR.…”

Early molecular and cytogenetic response is predictive for long-term progression-free and overall survival in chronic myeloid leukemia (CML)

“…The expressions of BCR-ABL and total ABL were determined by quantitative reverse transcriptase polymerase chain reaction from peripheral blood samples 14 and standardized according to the international scale (BCR-ABL IS ). [18][19][20] Only patients expressing typical BCR-ABL transcripts (b2a2, b3a2 or both b2a2 and b3a2) and monitored in standardized labs were considered. Patients with atypical transcripts (e1a2, n ¼ 3; b2a3, n ¼ 3; b3a3, n ¼ 3; b2a3 and b3a3, n ¼ 2; e19a2, n ¼ 10), unknown transcripts or monitored in nonstandardized labs (n ¼ 52) were excluded from the analysis.…”

Dynamics of the emergence of dasatinib and nilotinib resistance in imatinib-resistant CML patients

“…7 BCR-ABL and beta-glucuronidase (GUS) transcripts were quantified as described previously. 8 Importantly, for the current study, mutations were considered to be significant only if detected by two independent methods (D-HPLC/DS, ARMS or L-PCR). Findings from only one method were excluded from further analysis.…”