A Novel Nanofluidics-Based Single-Platform Molecular Testing Approach for Chronic Myelogeneous Leukemia (CML)
Wesley O Greaves, Shalini Verma, Keyur P Patel, Rajesh Singh, Neelima G Reddy, Raquel Giffard, Sema Hai, Hui Yao, Li Shen, L Jeffrey Medeiros, Rajyalakshmi Luthra. The University of Texas MD Anderson Cancer Center, Houston, TX
Background: Reverse transcriptase-based quantitative PCR (RT-qPCR) and fluorescence in situ hybridization (FISH) are widely used for molecular monitoring of patients with CML. Only FISH is routinely used to detect extra copies of the Ph chromosome, a common harbinger of progression. Additionally, most clinical RT-qPCR approaches currently used to detect BCR-ABL1 target only common fusion transcripts: e13a2 (b2a2), e14a2 (b3a2) and e1a2. These methods do not detect rare variants, such as e19a2 and those involving exon 3 of ABL. We describe a novel single-platform, nanofluidics-based qPCR design that enables comprehensive simultaneous detection of common and rare BCR-ABL1fusion transcripts and assessment of Ph chromosome copy number.
Design: We used the 48 x 48 Dynamic Array Integrated Fluidic Circuit (IFC) system (Fluidigm®, San Francisco, CA) and Taqman probe-based qPCR to simultaneously detect 8 distinct variant transcripts of BCR-ABL1: e1a2, b2a2, b3a2, e19a2, e1a3, b2a3, b3a3 and e19a3. K562, KBM7 and B15 cell lines and custom plasmids were used as positive controls. Previously tested RNA from peripheral blood and bone marrow samples of 36 CML patients with known BCR-ABL1 transcripts and archival DNA from 2 patients with extra copies of Ph chromosome by FISH were analyzed to validate the design. ABL1 was used as an internal control and plasmids with known amounts of target sequences were utilized to generate standard curves. Quantification of transcripts was compared with RT-qPCR results generated using ABI 7900 (Applied Biosystems Inc., Foster City, CA) for each sample. Receiver operating curve analysis was used to identify the cutoff values for IFC. Statistical analysis was performed using Pearson correlation coefficient. The 2-ΔΔCt method was used for Ph chromosome copy number analysis, with RNAse P as an endogenous reference and normal DNA as a diploid control.
Results: There was 100% concordance between IFC and ABI 7900 qPCR for identification of BCR-ABL1 transcripts in all plasmids and patient samples tested, with a correlation coefficient of 0.83 for transcript quantification. We confirmed additional copies of Ph chromosome in K-562 cells and patient samples.
Conclusions: Nanofluidics technology enables cost-effective, comprehensive, single-platform qPCR testing of BCR-ABL1 in patients with CML, including assessment of rare transcript variants. Ph chromosome copy number also can be assessed using a novel qPCR approach and can be potentially integrated into a nanofluidics-based platform to accurately predict early disease progression.
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 215, Wednesday Morning