Contributions
Abstract: PB1740
Type: Publication Only
Background
“Acute promyelocytic leukemia (APL) with PML-RARA” (WHO, 2016) is characterized by the presence of the aforementioned fusion gene, classically secondary to the t(15;17)(q24.1;q21.2) translocation, although cases of variant or cryptic translocations have been described.
The presence of the translocation and its fusion product can be identified by conventional karyotyping (CK), interphase FISH analysis using adequate probes, and molecular biology (MolB) through polymerase chain reaction (PCR), three techniques with different sensitivities, advantages, disadvantages and limitations, that have been well described in the literature.
Aims
We aim to compare the outcome of the simultaneous application of the three techniques in a real-world setting.
Methods
We reviewed all peripheral blood (PB) or bone marrow aspirate (BM) samples that were analysed in our lab over a five-year period (from January 1st 2013 to February 14th 2018) for a suspected diagnosis of APL at presentation or relapse, and that were submitted for near simultaneous testing using at least two of the three techniques. Samples that were submitted for analysis but that were not processed due to a limitation of the sample (such as insufficient volume), were counted as not analysed. Results were classified as “positive” or “negative” for the translocation. Abnormal karyotypes that nevertheless did not reveal the classical t(15;17) translocation or one of its variants were considered “negative” in this context.
Results
Over the timeframe under consideration, 55 patients underwent a combination of two or more of the techniques at diagnosis or suspected relapse: 87.3% had FISH analysis (14.6% in a full unsorted mixed-cellularity BM or PB sample, and 85.4% in fluorescence-activated cell sorting (FACS)-separated APL blast cells); 65.4% had MolB testing; and 67.3% underwent conventional karyotyping. Overall, 22.9% of patients had all three tests, 37.5% had FISH + MolB and 39.6% had FISH + CK.
Only two samples were negative for all three tests.
All MolB and CK testing results were fully concordant. Likewise, all negative MolB results were also negative by FISH; however, 11.1% of positive MolB results were negative by FISH, k = (-1.0), p=1.00; half of these samples were submitted for conventional karyotyping and were positive for the translocation.
All samples with a normal karyotype had a negative FISH test; all abnormal karyotypes without a t(15;17) translocation were negative by FISH; however, 12.5% of abnormal karyotypes with a detectable t(15;17) translocation were also negative by FISH (and positive on MolB), k = (-0.3), p=0.88. In all the above discordant cases, both morphology and cellular immunophenotyping by flow cytometry were not diagnostic for APL, and FISH probe hybridization had been performed on FACS-sorted cells; all cases were suspected relapses.
Conclusion
In our cohort, molecular biology and conventional karyotyping results were fully concordant, with a lower rate of detection for FISH analysis, where approximately 10% positive samples by MolB and CK were undetected, always in the context of a low disease burden, as evidenced by a normal BM smear and immunophenotype, and in a suspected relapse.
FACS-separation of blast cells, especially when the immunophenotype failed to identify these cells as pathologic APL blasts, does not appear to be of benefit and may, in fact, have hindered the detection of the fusion gene by FISH.
Session topic: 4. Acute myeloid leukemia - Clinical
Keyword(s): Cytogenetics, FISH, Molecular cytogenetics, PML-RAR
Abstract: PB1740
Type: Publication Only
Background
“Acute promyelocytic leukemia (APL) with PML-RARA” (WHO, 2016) is characterized by the presence of the aforementioned fusion gene, classically secondary to the t(15;17)(q24.1;q21.2) translocation, although cases of variant or cryptic translocations have been described.
The presence of the translocation and its fusion product can be identified by conventional karyotyping (CK), interphase FISH analysis using adequate probes, and molecular biology (MolB) through polymerase chain reaction (PCR), three techniques with different sensitivities, advantages, disadvantages and limitations, that have been well described in the literature.
Aims
We aim to compare the outcome of the simultaneous application of the three techniques in a real-world setting.
Methods
We reviewed all peripheral blood (PB) or bone marrow aspirate (BM) samples that were analysed in our lab over a five-year period (from January 1st 2013 to February 14th 2018) for a suspected diagnosis of APL at presentation or relapse, and that were submitted for near simultaneous testing using at least two of the three techniques. Samples that were submitted for analysis but that were not processed due to a limitation of the sample (such as insufficient volume), were counted as not analysed. Results were classified as “positive” or “negative” for the translocation. Abnormal karyotypes that nevertheless did not reveal the classical t(15;17) translocation or one of its variants were considered “negative” in this context.
Results
Over the timeframe under consideration, 55 patients underwent a combination of two or more of the techniques at diagnosis or suspected relapse: 87.3% had FISH analysis (14.6% in a full unsorted mixed-cellularity BM or PB sample, and 85.4% in fluorescence-activated cell sorting (FACS)-separated APL blast cells); 65.4% had MolB testing; and 67.3% underwent conventional karyotyping. Overall, 22.9% of patients had all three tests, 37.5% had FISH + MolB and 39.6% had FISH + CK.
Only two samples were negative for all three tests.
All MolB and CK testing results were fully concordant. Likewise, all negative MolB results were also negative by FISH; however, 11.1% of positive MolB results were negative by FISH, k = (-1.0), p=1.00; half of these samples were submitted for conventional karyotyping and were positive for the translocation.
All samples with a normal karyotype had a negative FISH test; all abnormal karyotypes without a t(15;17) translocation were negative by FISH; however, 12.5% of abnormal karyotypes with a detectable t(15;17) translocation were also negative by FISH (and positive on MolB), k = (-0.3), p=0.88. In all the above discordant cases, both morphology and cellular immunophenotyping by flow cytometry were not diagnostic for APL, and FISH probe hybridization had been performed on FACS-sorted cells; all cases were suspected relapses.
Conclusion
In our cohort, molecular biology and conventional karyotyping results were fully concordant, with a lower rate of detection for FISH analysis, where approximately 10% positive samples by MolB and CK were undetected, always in the context of a low disease burden, as evidenced by a normal BM smear and immunophenotype, and in a suspected relapse.
FACS-separation of blast cells, especially when the immunophenotype failed to identify these cells as pathologic APL blasts, does not appear to be of benefit and may, in fact, have hindered the detection of the fusion gene by FISH.
Session topic: 4. Acute myeloid leukemia - Clinical
Keyword(s): Cytogenetics, FISH, Molecular cytogenetics, PML-RAR