Comparison of competitive-nested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients.

Guo JQ, Lin H, Kantarjian H, Talpaz M, Champlin R, Andreeff M, Glassman A, Arlinghaus RB.

Department of Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

Real-time RT-PCR has great advantages for estimating transcript levels in a variety of situations. These include relative rapid assay times (hours), reliability and ease of performing replicate analyses.

In contrast, competitive PCR is a very labor-intensive procedure requiring a few days to generate useful data. We compared the same samples from CML patients by both methods.

Importantly, we used the Bcr-Abl junction plasmid DNA, which is used as a competitor in the .manual competitive PCR assay, to generate a standard curve for the real-time assay. This permitted reporting the real-time data as the number of BCR-ABL transcripts per microg of total RNA, which is the same format used for the competitive PCR assay. In this study, a
total of 435 peripheral blood and marrow samples from 285 CML patients were analyzed by RT-PCR; these patients were undergoing therapy by STI-571, interferon, and bone marrow transplantation treatment. Most samples also had assay values for the Philadelphia chromosome (Ph), FISH and Western blotting for the Bcr-Abl oncoprotein.

Our findings indicated that the real-time assay was less sensitive than the manual competitive RT-real-time RT-PCR, except for levels of BCR-ABL below 200 transcripts per microg of RNA. In both patient and cell line experiments, dividing the BCR-ABL transcripts by the total ABL transcripts virtually eliminated the difference between real-time BCR-ABL transcript values and quantitative competitive BCR-ABL transcript values, indicating that both BCR-ABL and ABL transcripts were underestimated by the real-time assay.

In addition, the increased sensitivity of the nested, competitive RT-PCR was readily apparent in patients with minimal residual disease, which by the real-time were negative in the majority of patients but were positive by nested,
competitive RT-PCR in 44.6% (n = 29) of samples analyzed (n = 65). These findings indicate that real-time RT-PCR, when normalized for the total ABL transcripts, can be used to monitor CML patients during therapy, but we suggest that nested, competitive RT-PCR be used to determine BCR-ABL/ABL transcript ratios at low transcript values or especially when real-time analyses are negative.

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[4883] Cytogenetics May Be More Sensitive Than FISH in Monitoring a
Subset of CML Patients Treated with Gleevec.

Kathleen E. Richkind, Patricia Mowery-Rushton, Joyce Murata-Collins,

Christian Lytle. Cancer Laboratory, Genzyme Genetics, Santa Fe, NM, USA

Fluorescence in situ hybridization analysis (FISH) of interphase cells has been recommended as the technique of choice for monitoring treated CML patients. However, we have observed an unexpectedly high number of treated CML patients who were cytogenetically positive at low levels for t(9;22) , but were negative for a bcr/abl rearrangement by interphase FISH.

This finding raises issues regarding the actual sensitivities of CC and FISH analyses in the monitoring of patients with CML, and may indicate a clinically significant subset of patients in whom a low level of mitotically active Ph+ cells persist despite apparent remission as indicated by FISH analysis.

Twenty-eight patients diagnosed with CML and treated either with interferon (2 patients) or Gleevec (STI571) (26 patients) were followed by both CC analysis of 20-30 metaphase cells and interphase FISH studies with BCR/ABL ES probe set (Vysis) during the course of their treatment. All patients at one or more time points in their treatment had a positive CC result with 1 Ph+ cell observed in 20-30 cells examined. Four patients had multiple analyses with 1/20 cells positive for t(9;22). Eleven patients had correspondingly positive FISH results (per cent of interphase cells with bcr/abl fusion = 2%-7.5%), while 17 patients had negative FISH results (per
cent of interphase cells with bcr/abl fusion = 0% - 1%). Of the 11 patients who had concordant positive CC/FISH results, five have had subsequent followup studies - four patients have achieved cytogenetic remission and one patient has relapsed. Nine of the 17 patients with discordant CC/FISH results had subsequent followup studies; four of these patients have cytogenetically persistent disease or relapse, while five achieved cytogenetic remission.

Follow up periods after ascertainment of the study which detected 1/20 Ph+ cell ranged from 2 months to 4 years (average=6mos). Use of classical cytogenetics (CC) analysis in monitoring treated CML patients, with examination of 20-30 metaphase cells, results in a theoretically less sensitive test than FISH for a bcr/abl rearrangement, which routinely involves examination of 200-300 cells.

Our results suggest that the sensitivity of CC is increased beyond theoretical expectations due to enrichment of the target population by selection for mitotically active cells, which may represent a subset of cells resistant to the anti-proliferative effects of Gleevec.

Contrary to current guidelines, it may be important to perform both FISH and cytogenetic studies in the monitoring of treated CML patients, because interphase FISH analysis may have a higher false negative rate than expected at low levels of disease.

CML Prisms--Literature for Members ofasian CML Support Group

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