Hematopathology
Contributions
Type: Publication Only
Background
In India treatment of Chronic Myeloid Leukemia (CML) is supported through an International Patient Assistance Program in which imatinib mesylate is provided to economically disadvantaged patients free of cost. As a result most of these patients opt for treatment and need regular molecular monitoring. However, commercial kits for BCR-ABL monitoring are cost prohibitive for our patients. It is also currently recommended to test for BCR-ABL copy numbers in triplicates to ensure making confident calls at low levels placing further economic strain in resource constrained settings especially if all quality control measures are to be followed. In that context it is required to develop cost effective BCRABL monitoring technologies. Globally, a majority of centres use the EAC protocol for CML, an assay that suffers from background noise. We addressed these problems by modifying the legacy real time PCR (qPCR) process. We cloned a duplex qPCR compatible plasmid that enables us to multiplex BCRABL and ABL reaction in a single well using FAM- black hole quencher1 (BHQ1) and HEX-BHQ1 probes modified with locked nucleic acids (LNA).
Aims
To develop a custom duplex PCR compatible plasmid and develop and standardize a LNA probe based assay for BCR-ABL and ABL.
Methods
A 1600bp amplicon from a newly diagnosed CML (E13A2) patient into a pJET1.2/blunt cloning vector. This was linearized and using Avogadro’s number an estimate of copy numbers were arrived at and a standard curve was created. Accuracy of dilutions were confirmed by using Ipsogen’s standards. Using Modified EAC protocol: BCR-ABL probe was truncated to 16 nucleic acids labelled with FAM and BHQ1 with three LNA modifications whereas the ABL probe was 19 nucleic acids (HEX and BHQ1) with 4 LNA modifications. Calibration to the WHO calibrator was done using a secondary standard (Asuragen Inc) to derive an international scale conversion factor. Assay precision was monitored using two levels of controls in the duplex assay (median NCN 0.1% and 0.05%). We compared the normalized copy number (NCN) derived from the duplex assay with the simplex assay in 87 samples of CML (NCN ranging from 0.002 to 108.5, median 0.14%).
Results
The plasmid was successfully cloned and confirmed by sanger sequencing. The duplex assay did not lead to loss of MMR in any patient also we did not face any issues with false positivity. We found a good correlation between the simplex and duplex assay (r2=0.97). In all runs, slope was between -3.2 to -3.6, R2>0.98. The international scale conversion factor was 0.79. Over 60 runs the BCRABL assay has a CV of 38.9% and 48.1% for high and low precision controls.
Summary
This assay has enabled us to maintain a high level of quality control (runs in triplicates, precision, no template as well as no reverse transcription controls) yet process 22 samples per run. As a result the cost of the assay is low. We would be happy to share this plasmid to other users from resource constrained setting such as ours.
To the best of our knowledge, this is first paper to use LNA probes for CML minimal residual disease (MRD) monitoring. The use of a duplex PCR enables us to significantly decrease the cost of the assay ($5/patient) yet maintain a high standard of quality control.
Keyword(s): Chronic myeloid leukemia, MRD, Real time PCR
Type: Publication Only
Background
In India treatment of Chronic Myeloid Leukemia (CML) is supported through an International Patient Assistance Program in which imatinib mesylate is provided to economically disadvantaged patients free of cost. As a result most of these patients opt for treatment and need regular molecular monitoring. However, commercial kits for BCR-ABL monitoring are cost prohibitive for our patients. It is also currently recommended to test for BCR-ABL copy numbers in triplicates to ensure making confident calls at low levels placing further economic strain in resource constrained settings especially if all quality control measures are to be followed. In that context it is required to develop cost effective BCRABL monitoring technologies. Globally, a majority of centres use the EAC protocol for CML, an assay that suffers from background noise. We addressed these problems by modifying the legacy real time PCR (qPCR) process. We cloned a duplex qPCR compatible plasmid that enables us to multiplex BCRABL and ABL reaction in a single well using FAM- black hole quencher1 (BHQ1) and HEX-BHQ1 probes modified with locked nucleic acids (LNA).
Aims
To develop a custom duplex PCR compatible plasmid and develop and standardize a LNA probe based assay for BCR-ABL and ABL.
Methods
A 1600bp amplicon from a newly diagnosed CML (E13A2) patient into a pJET1.2/blunt cloning vector. This was linearized and using Avogadro’s number an estimate of copy numbers were arrived at and a standard curve was created. Accuracy of dilutions were confirmed by using Ipsogen’s standards. Using Modified EAC protocol: BCR-ABL probe was truncated to 16 nucleic acids labelled with FAM and BHQ1 with three LNA modifications whereas the ABL probe was 19 nucleic acids (HEX and BHQ1) with 4 LNA modifications. Calibration to the WHO calibrator was done using a secondary standard (Asuragen Inc) to derive an international scale conversion factor. Assay precision was monitored using two levels of controls in the duplex assay (median NCN 0.1% and 0.05%). We compared the normalized copy number (NCN) derived from the duplex assay with the simplex assay in 87 samples of CML (NCN ranging from 0.002 to 108.5, median 0.14%).
Results
The plasmid was successfully cloned and confirmed by sanger sequencing. The duplex assay did not lead to loss of MMR in any patient also we did not face any issues with false positivity. We found a good correlation between the simplex and duplex assay (r2=0.97). In all runs, slope was between -3.2 to -3.6, R2>0.98. The international scale conversion factor was 0.79. Over 60 runs the BCRABL assay has a CV of 38.9% and 48.1% for high and low precision controls.
Summary
This assay has enabled us to maintain a high level of quality control (runs in triplicates, precision, no template as well as no reverse transcription controls) yet process 22 samples per run. As a result the cost of the assay is low. We would be happy to share this plasmid to other users from resource constrained setting such as ours.
To the best of our knowledge, this is first paper to use LNA probes for CML minimal residual disease (MRD) monitoring. The use of a duplex PCR enables us to significantly decrease the cost of the assay ($5/patient) yet maintain a high standard of quality control.
Keyword(s): Chronic myeloid leukemia, MRD, Real time PCR