Contributions
Abstract: PB1810
Type: Publication Only
Background
Chronic myeloid leukemia (CML) is a myeloproliferative, clonal and acquired hematological disease that is included within myeloproliferative neoplasms (WHO 2016). Its main characteristic is the presence (95% of the cases) of a small chromosome denominated Philadelphia chromosome, coming from the reciprocal translocation between the chromosomes 9 and 22. Depend where the break-point occurs, different isoforms of the fusion gene BCR-ABL may appear. For the diagnosis of CML, detection of BCR-ABL rearrangement is crucial; and molecular biology techniques, such as RT-PCR, may be the only data at that point, but most current RT-PCR methods for detecting BCR–ABL are designed and optimized for detecting the majority forms (e14a2 and e13a2) without distinguishing between them. Characterization of the transcript is not necessary for the diagnosis but permits follow-up at the molecular level and differentiate between different BCR-ABL isoforms at the time of the CML diagnosis could be taken into account in future studies to investigate its role into the prognosis.
Aims
To develop a new multiplex RT-PCR method coupled to fragment analysis by capillary electrophoresis to identify different BCR-ABL isoforms: e13a3, e19a2, e14a3, e6a2, e1a3, e13a2, e14a2, e1a2 and e8a1.
Methods
34 CML patients BCR-ABL positive by qRT-PCR and 1 negative control for BRC-ABL fusion gene were included in this study. Three hundred nanograms of total RNA from leucocytes were used for retro-transcription (SuperScript ® IV). Subsequently, Multiplex PCR reactions were assessed using primers described by Burmeister in 2008 [ABL-3 primer labeled with carboxyferrescein (FAM)]. G6PD was chosen as endogenous gene control using G6PD-F labeled with hexachloro-fluorescein phosphoramidite (HEX). Capillary electrophoresis of the multiplex RT-PCR reaction was done in an ABI3130XL analyzer, using ILS600 as marker.
Results
BCR-ABL fusion RNAs were detected in all patients (34/34), on the other hand we did not detect BCR-ABL on the negative control. The main isoform identified was e14a2 (detected in 22 out of 34 patients, 64.7%). Twelve patients were positive for e13a2 BCR-ABL isoform (35.3%). Interestingly we identified 7 patients (20.5%) with co-expression of e14a2 and e13a2 isoforms, being in all these cases the e14a2 isoform mainly expressed.
Conclusion
RT-PCR combined with capillary electrophoresis is revealed as a sensitive technique for the detection of different isoforms of BCR-ABL and may be included as a BCR-ABL first screening. Quantification with qRT-PCR might only be done in positive samples. Unfortunately we could not detect any isoform besides the majority ones, due to the size of our cohort. Finally, our study validates previous studies on the main BCR-ABL isoforms (e14a2 and e13a2) percentage detected in CML patients.
Session topic: 7. Chronic myeloid leukemia - Biology
Keyword(s): Diagnosis, Chronic myeloid leukemia, BCR-ABL
Abstract: PB1810
Type: Publication Only
Background
Chronic myeloid leukemia (CML) is a myeloproliferative, clonal and acquired hematological disease that is included within myeloproliferative neoplasms (WHO 2016). Its main characteristic is the presence (95% of the cases) of a small chromosome denominated Philadelphia chromosome, coming from the reciprocal translocation between the chromosomes 9 and 22. Depend where the break-point occurs, different isoforms of the fusion gene BCR-ABL may appear. For the diagnosis of CML, detection of BCR-ABL rearrangement is crucial; and molecular biology techniques, such as RT-PCR, may be the only data at that point, but most current RT-PCR methods for detecting BCR–ABL are designed and optimized for detecting the majority forms (e14a2 and e13a2) without distinguishing between them. Characterization of the transcript is not necessary for the diagnosis but permits follow-up at the molecular level and differentiate between different BCR-ABL isoforms at the time of the CML diagnosis could be taken into account in future studies to investigate its role into the prognosis.
Aims
To develop a new multiplex RT-PCR method coupled to fragment analysis by capillary electrophoresis to identify different BCR-ABL isoforms: e13a3, e19a2, e14a3, e6a2, e1a3, e13a2, e14a2, e1a2 and e8a1.
Methods
34 CML patients BCR-ABL positive by qRT-PCR and 1 negative control for BRC-ABL fusion gene were included in this study. Three hundred nanograms of total RNA from leucocytes were used for retro-transcription (SuperScript ® IV). Subsequently, Multiplex PCR reactions were assessed using primers described by Burmeister in 2008 [ABL-3 primer labeled with carboxyferrescein (FAM)]. G6PD was chosen as endogenous gene control using G6PD-F labeled with hexachloro-fluorescein phosphoramidite (HEX). Capillary electrophoresis of the multiplex RT-PCR reaction was done in an ABI3130XL analyzer, using ILS600 as marker.
Results
BCR-ABL fusion RNAs were detected in all patients (34/34), on the other hand we did not detect BCR-ABL on the negative control. The main isoform identified was e14a2 (detected in 22 out of 34 patients, 64.7%). Twelve patients were positive for e13a2 BCR-ABL isoform (35.3%). Interestingly we identified 7 patients (20.5%) with co-expression of e14a2 and e13a2 isoforms, being in all these cases the e14a2 isoform mainly expressed.
Conclusion
RT-PCR combined with capillary electrophoresis is revealed as a sensitive technique for the detection of different isoforms of BCR-ABL and may be included as a BCR-ABL first screening. Quantification with qRT-PCR might only be done in positive samples. Unfortunately we could not detect any isoform besides the majority ones, due to the size of our cohort. Finally, our study validates previous studies on the main BCR-ABL isoforms (e14a2 and e13a2) percentage detected in CML patients.
Session topic: 7. Chronic myeloid leukemia - Biology
Keyword(s): Diagnosis, Chronic myeloid leukemia, BCR-ABL