Highly Sensitive Multiplex RT-PCR System for the Detection of all Common BCR-ABL Transcripts Associated with Different Leukemias

ABSTRACT

After the recent characterization of leukemia-specific DNA rearrangements, molecular methods have become primary tools for the diagnosis and monitoring of many hematological malignancies, due to their superior sensitivity and accuracy over other conventional methods. The BCR-ABL fusion transcripts resulting from the t[9; 22] translocation are distinct hallmarks of Philadelphia chromosome positive [Ph[+]] leukemias. There are clear associations between different isoforms of the BCR-ABL fusion protein and specific phenotypes of these leukemias. Each isoform also has a significant prognostic value and can be a critical indicator of the clinical outcome. In this study we have adopted, with modifications, a highly sensitive and specific method to screen simultaneously for the most frequent BCR-ABL fusion transcripts, namely, p210 [b3a2/b2a2], p190 [ela2] and p230 [e19a2]. A multiplex reverse-transcriptase polymerase chain reaction [RT-PCR] protocol with nested primer strategy for each of the above fusion transcripts was carefully optimized. RNA integrity, cDNA synthesis and PCR amplification were checked using internal control primers for the normal untranslocated BCR gene. Over 100 clinical samples were collected from hospitals in Amman between 2003 and 2005. This system was applied successfully on 100 clinical samples previously diagnosed as chronic myelogenous leukemia [CML] and acute lymphocytic leukemia [ALL]. RNA extracts from established leukemic cell lines carrying the translocations of interest were used as external positive controls. Representative PCR products were sequenced to verify the specificity of the amplification system. Our multiplexed nested RT-PCR assay provides a sensitive, accurate, time-saving and cost-effective diagnostic tool for the diagnosis and monitoring of patients with Ph [+] leukemias