Contributions
Abstract: PB1695
Type: Publication Only
Background
A key prognostic factor in acute leukemia is the monitoring of minimal residual disease (MRD). Methods are based on flow cytometry, as well as molecular biology methods using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR methodology enables us to follow MRD-specific markers or non-specific markers such as WT1.
Aims
Assessment of suitable molecular-biological markers of MRD in patients with AML, and in patients with rare hematologic malignancy Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN).
Methods
Expression of the non-specific marker WT1 was tested according to the European Leukemia Net protocol (ELN) using a WT1 ProfileQuant kit (Qiagen). To monitor the expression of Chondroitin Sulphate Proteoglycan 4 (CSPG4), one of the Leukemia Associated Antigens (LAA), we used a TaqMan Gene Expression Assay (ThermoFisher). The data were normalized to the expression of the ABL control gene TaqMan Gene Expression Assay with results in relative units (ru); upper normal limit is ru=1.00. Similarly, we measured the expression of the XAGE1 gene, another LAA. Additionally, an in-house methodology was established to obtain CSPG4 absolute quantification, showing CSPG4 copy number normalized to 104 copies of the ABL reference gene. Leukemia-Associated Immunophenotype (LAIP) measured by flow cytometer also monitored the MRD.
Results
In 170 patients with AML, CSPG4 expression with a median expression of 0.517 was measured at diagnosis. Significantly increased expression of this marker was found in the AML M5 subtype (n=17, median=20.51, P˂0.0001), in AML possessing a MLL translocation (n=12, median=59.15, P˂0.0001), and in 4 patients with BPDCN (median=74.26, P=0.0001); all related to CSPG4 values measured in peripheral blood of healthy donors (n=22, median=0.262). The median expression of the CSPG4 in BPDCN patients was nearly 2 orders above the normal upper limit. No specific MRD marker is available in patients with BPDCN, and WT1 expression at diagnosis is uniformly low. We validated the measurement of CSPG4 expression in 4 patients by comparison with WT1 and XAGE1 expressions, reaching a high correlation (P=0.05, P=0.0006, P=0.0002, and P<0.0001). The MRD level was monitored in all patients at diagnosis, after induction therapy, after consolidation therapy, and after allogeneic stem cell transplantation (ASCT). The expression of this MRD marker correlated with the clinical status of the patients, as well as with flow cytometric data. Subsequently, a CSPG4 absolute quantification methodology was developed and validated.
Conclusion
MRD monitoring in patients with AML has prognostic significance. By monitoring the expression of CSPG4, we found that it was overexpressed in patients with AML with MLL translocation, with AML M5 subtype, and in a rare BPDCN entity. Common features of the AML patients with MLL translocation and the BPDCN patients are poor prognosis and low WT1 expression at diagnosis. We validated the methodology for monitoring of this alternative marker at the mRNA level instead of WT1. The CSPG4 expression was in agreement with the clinical and flow cytometric data of the patients. Further prospective monitoring of this marker is necessary.Conclusion:CSPG4 appears to be a potentially suitable molecular marker of MRD in AML M5, AML with MLL translocation and BPDCN. Supported by MH CZ-DRO UHKT 00023736
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): AML, Minimal residual disease (MRD)
Abstract: PB1695
Type: Publication Only
Background
A key prognostic factor in acute leukemia is the monitoring of minimal residual disease (MRD). Methods are based on flow cytometry, as well as molecular biology methods using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR methodology enables us to follow MRD-specific markers or non-specific markers such as WT1.
Aims
Assessment of suitable molecular-biological markers of MRD in patients with AML, and in patients with rare hematologic malignancy Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN).
Methods
Expression of the non-specific marker WT1 was tested according to the European Leukemia Net protocol (ELN) using a WT1 ProfileQuant kit (Qiagen). To monitor the expression of Chondroitin Sulphate Proteoglycan 4 (CSPG4), one of the Leukemia Associated Antigens (LAA), we used a TaqMan Gene Expression Assay (ThermoFisher). The data were normalized to the expression of the ABL control gene TaqMan Gene Expression Assay with results in relative units (ru); upper normal limit is ru=1.00. Similarly, we measured the expression of the XAGE1 gene, another LAA. Additionally, an in-house methodology was established to obtain CSPG4 absolute quantification, showing CSPG4 copy number normalized to 104 copies of the ABL reference gene. Leukemia-Associated Immunophenotype (LAIP) measured by flow cytometer also monitored the MRD.
Results
In 170 patients with AML, CSPG4 expression with a median expression of 0.517 was measured at diagnosis. Significantly increased expression of this marker was found in the AML M5 subtype (n=17, median=20.51, P˂0.0001), in AML possessing a MLL translocation (n=12, median=59.15, P˂0.0001), and in 4 patients with BPDCN (median=74.26, P=0.0001); all related to CSPG4 values measured in peripheral blood of healthy donors (n=22, median=0.262). The median expression of the CSPG4 in BPDCN patients was nearly 2 orders above the normal upper limit. No specific MRD marker is available in patients with BPDCN, and WT1 expression at diagnosis is uniformly low. We validated the measurement of CSPG4 expression in 4 patients by comparison with WT1 and XAGE1 expressions, reaching a high correlation (P=0.05, P=0.0006, P=0.0002, and P<0.0001). The MRD level was monitored in all patients at diagnosis, after induction therapy, after consolidation therapy, and after allogeneic stem cell transplantation (ASCT). The expression of this MRD marker correlated with the clinical status of the patients, as well as with flow cytometric data. Subsequently, a CSPG4 absolute quantification methodology was developed and validated.
Conclusion
MRD monitoring in patients with AML has prognostic significance. By monitoring the expression of CSPG4, we found that it was overexpressed in patients with AML with MLL translocation, with AML M5 subtype, and in a rare BPDCN entity. Common features of the AML patients with MLL translocation and the BPDCN patients are poor prognosis and low WT1 expression at diagnosis. We validated the methodology for monitoring of this alternative marker at the mRNA level instead of WT1. The CSPG4 expression was in agreement with the clinical and flow cytometric data of the patients. Further prospective monitoring of this marker is necessary.Conclusion:CSPG4 appears to be a potentially suitable molecular marker of MRD in AML M5, AML with MLL translocation and BPDCN. Supported by MH CZ-DRO UHKT 00023736
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): AML, Minimal residual disease (MRD)