Comprehensive Validation
Comprehensive Validation
We developed and extensively validated a real-time PCR assay for the quantitation of bcr-abl to determine residual disease in patients with chronic myelogenous leukemia. This method quantitates the p210 and the p190 bcr-abl RNA fusion transcripts with results normalized to a housekeeping gene, using the 5'-exonuclease technique and the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). We parallel tested 372 clinical specimens and 50 peripheral blood samples from patients not known to have any myeloproliferative disorders. The results were 100% specific. Sensitivity studies showed that this method can detect bcr-abl in cell lines diluted to 0.0001% and can detect a single bcr-abl plasmid spiked into negative RNA. The between-run reproducibility showed a coefficient of variance (CV) of 12.3%, and within-run reproducibility showed a CV of 13.8%. This method can be used to reliably monitor the disease load in patients with bcr-abl-positive diseases.
The reciprocal translocation of the abl (Abelson murine leukemia) proto-oncogene on chromosome 9 to the bcr (breakpoint cluster region) gene on chromosome 22 creates a transcriptionally active, chimeric bcr-abl gene and gives rise to the Philadelphia chromosome. This gene encodes a 190- or 210-kd protein product, which is an active, deregulated, intracellular tyrosine kinase. By this mechanism, the fusion protein is thought to induce malignant transformation of hematopoietic stem cells.
The bcr-abl messenger RNA (mRNA) transcript is detectable by molecular methods in approximately 95% of cases of chronic myelogenous leukemia (CML), as well as some cases of acute lymphocytic leukemia (ALL; approximately 5% for children, 20% for adults). It also has been reported as present in some other hematologic disorders, albeit rarely. The bcr-abl gene and its mRNA transcript are tumor-specific markers. Before the availability of reliable and accurate quantitative polymerase chain reaction (PCR) methods, the clinical value of using bcr-abl mRNA PCR to monitor residual disease in CML was not established clearly. While a negative PCR result was consistently associated with remission, a positive result did not necessarily indicate progression or relapse. However, several reports more recently have shown that quantitative PCR may be more informative for minimal residual disease monitoring in these patients. These reports have shown that increasing values over time are indicative of progression or relapse.
This report describes the comprehensive validation for clinical use of a real time, 5'-exonuclease method for the quantitative detection of the p210 and p190 bcr-abl fusion transcripts, both normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPD).
Our method uses a 5'-exonuclease technique to generate fluorescence during the PCR process that is proportional to the starting quantity of template, which is monitored in real-time by using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Briefly, the principle of this method, called TaqMan (Applied Biosystems), is as follows: A sequence-specific probe is included in the PCR reaction, which is dual labeled with a reporter fluorophore at the 5'-end and a quencher fluorophore at the 3'-end. The probe anneals to the region of interest internal to the forward and reverse PCR primers. During the extension phase, the 5'
3' exonuclease activity of the Taq polymerase will cleave the reporter molecule. Its release is accompanied by a fluorescent signal, which the ABI 7700 instrument measures and records in real time.
The assay described herein quantitates the bcr-abl p210 and p190 transcripts, as well as a housekeeping gene, GAPD. We demonstrated that this assay is highly specific and that its sensitivity is such that it can reliably detect and quantitate 10 pg of bcr-abl RNA (0.0001%) in a background of negative RNA.
We developed and extensively validated a real-time PCR assay for the quantitation of bcr-abl to determine residual disease in patients with chronic myelogenous leukemia. This method quantitates the p210 and the p190 bcr-abl RNA fusion transcripts with results normalized to a housekeeping gene, using the 5'-exonuclease technique and the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). We parallel tested 372 clinical specimens and 50 peripheral blood samples from patients not known to have any myeloproliferative disorders. The results were 100% specific. Sensitivity studies showed that this method can detect bcr-abl in cell lines diluted to 0.0001% and can detect a single bcr-abl plasmid spiked into negative RNA. The between-run reproducibility showed a coefficient of variance (CV) of 12.3%, and within-run reproducibility showed a CV of 13.8%. This method can be used to reliably monitor the disease load in patients with bcr-abl-positive diseases.
The reciprocal translocation of the abl (Abelson murine leukemia) proto-oncogene on chromosome 9 to the bcr (breakpoint cluster region) gene on chromosome 22 creates a transcriptionally active, chimeric bcr-abl gene and gives rise to the Philadelphia chromosome. This gene encodes a 190- or 210-kd protein product, which is an active, deregulated, intracellular tyrosine kinase. By this mechanism, the fusion protein is thought to induce malignant transformation of hematopoietic stem cells.
The bcr-abl messenger RNA (mRNA) transcript is detectable by molecular methods in approximately 95% of cases of chronic myelogenous leukemia (CML), as well as some cases of acute lymphocytic leukemia (ALL; approximately 5% for children, 20% for adults). It also has been reported as present in some other hematologic disorders, albeit rarely. The bcr-abl gene and its mRNA transcript are tumor-specific markers. Before the availability of reliable and accurate quantitative polymerase chain reaction (PCR) methods, the clinical value of using bcr-abl mRNA PCR to monitor residual disease in CML was not established clearly. While a negative PCR result was consistently associated with remission, a positive result did not necessarily indicate progression or relapse. However, several reports more recently have shown that quantitative PCR may be more informative for minimal residual disease monitoring in these patients. These reports have shown that increasing values over time are indicative of progression or relapse.
This report describes the comprehensive validation for clinical use of a real time, 5'-exonuclease method for the quantitative detection of the p210 and p190 bcr-abl fusion transcripts, both normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPD).
Our method uses a 5'-exonuclease technique to generate fluorescence during the PCR process that is proportional to the starting quantity of template, which is monitored in real-time by using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Briefly, the principle of this method, called TaqMan (Applied Biosystems), is as follows: A sequence-specific probe is included in the PCR reaction, which is dual labeled with a reporter fluorophore at the 5'-end and a quencher fluorophore at the 3'-end. The probe anneals to the region of interest internal to the forward and reverse PCR primers. During the extension phase, the 5'
3' exonuclease activity of the Taq polymerase will cleave the reporter molecule. Its release is accompanied by a fluorescent signal, which the ABI 7700 instrument measures and records in real time.
The assay described herein quantitates the bcr-abl p210 and p190 transcripts, as well as a housekeeping gene, GAPD. We demonstrated that this assay is highly specific and that its sensitivity is such that it can reliably detect and quantitate 10 pg of bcr-abl RNA (0.0001%) in a background of negative RNA.
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