BCRABL transcript detection by quantitative real-time PCR : are correlated results possible from homebrew assays?
BACKGROUND
Quantitative real-time PCR has become the predominant molecular technique to monitor BCRABL levels in response to treatment in Ph(+) leukemia patients. However, without some form of standardized methodology between laboratories, the correlation of results is difficult.


METHODS
Using TaqMan-based assays, parallel quantitative real-time PCR analysis was performed on 70 clinical specimens at Vanderbilt University Medical Center and Virginia Commonwealth University. While the same positive control cell line (K562) and quality control gene (BCR) were used, the RNA isolation technique, cDNA synthesis, BCR control cell line, and PCR primer and probe sequences were different.


RESULTS
The detection of BCRABL-positive results spanned a dynamic range from 10(0) to 10(5)/100,000 cells. Forty-three samples were negative at both facilities. A Spearman rank correlation analysis was performed for the 22 BCRABL-positive paired results. The correlation coefficient, r(s), was 0.9435 (p < 0.00001), suggesting a strong correlation of the results. One discordant result was obtained for consecutive samples from one patient with a low BCRABL copy number as a result of a minimal RNA yield at one laboratory.


CONCLUSIONS
These results suggest that quantitative real-time PCR assays for BCRABL detection can be comparable between laboratories despite significant differences in methodologies if the same positive control cell line and quality control gene are used. It is imperative that some level of assay standardization be adopted between laboratories, not only for patients who are monitored at different facilities, but also for larger investigative studies in which hematologic, cytogenetic and molecular responses are to be compared.
Fossey
Sallyanne C
SC
Molecular Genetics Laboratory, Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Ferreira-Gonzalez
Andrea
A
Garrett
Carleton T
CT
Dumur
Catherine I
CI
Vnencak-Jones
Cindy L
CL
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001549:Benzamides; D001853:Bone Marrow / Q000378:metabolism / Q000473:pathology; D016026:Bone Marrow Transplantation; D016313:Genes, abl; D006801:Humans; D000068877:Imatinib Mesylate; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000473:pathology / Q000628:therapy; D007951:Leukemia, Myeloid / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D010879:Piperazines / Q000627:therapeutic use; D016133:Polymerase Chain Reaction; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D011743:Pyrimidines / Q000627:therapeutic use; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis*; D012680:Sensitivity and Specificity
D000970:Antineoplastic Agents; D001549:Benzamides; D010879:Piperazines; D011743:Pyrimidines; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D000068877:Imatinib Mesylate