Hypothesis: Molecular disease monitoring in AML using peripheral blood and plasma is feasible.
Aim: To develop a molecular monitoring test for ongoing disease assessment of AML. The specific aims:
1: To determine the optimal monitoring sample source (blood, plasma, bone marrow)
2: To compare the concordance of mutation monitoring on blood and plasma to bone marrow aspirate
Method:
Patients and samples:
Patients were recruited from Royal Perth Hospital. Serial matched bone marrow aspirate (BMA) and peripheral blood samples of 10 AML patients were studied. DNA and cell-free DNA (cfDNA) were extracted using Qiagen extraction kits.
Mutation Discovery – Next Generation Sequencing (NGS)
The mutation landscape for each patient was analysed using diagnostic BMA DNA with NGS – 120-gene customised panel on genes associated with AML.
Mutation Monitoring – Targeted NGS
Based on diagnostic mutation profile, mutations were monitored in patient-specific. Monitoring was performed on follow up BMA DNA, blood DNA and plasma cfDNA samples. Mutation burden was tracked sequentially. This is performed using customised gene panel for each patient.
All libraries were prepared using Ion AmpliSeq Library Kit and quantified using a 2100 Bioanalyser. Equimolar amounts of libraries were pooled and sequenced on an Ion Proton Sequencer (ThermoFisher Scientific). Torrent Suite 5.10 and Ion Reporter 5.10 Software (ThermoFisher Scientific) were used for coverage analysis, variant calling and annotation.
Result:
AML-related mutations can be detected on plasma cfDNA and peripheral blood DNA.
Plasma cfDNA generates near identical results (number, types and levels of mutations) compared to BMA DNA at follow up.
Mutation levels on blood are lower than plasma and BMA.
Mutation kinetic (changes of levels over time) on plasma correlates with clinical status
Conclusion:
Plasma is an optimal sample source for molecular disease monitoring in AML
Molecular disease monitoring in AML using plasma cfDNA is feasible.