Deep Sequencing of Peripheral Blood Plasma DNA As a Reliable Test for Confirming the Diagnosis of Myelodysplastic Syndrome

Background: In patients presenting with cytopenia, myelodysplastic syndrome (MDS) should be considered, but confirmation of diagnosis requires bone marrow biopsy and morphologic and cytogenetic evaluation. It is extremely difficult to rely on subjective morphologic features to confirm the diagnosis of MDS, when the karyotype is normal and blasts are not increased. Objective criteria for the diagnosis of MDS are needed in these cases. With recent advances in the characterization of molecular abnormalities in MDS, diagnosis of early MDS is becoming more objective by documenting the presence of MDS-specific molecular abnormalities in cases with appropriate clinical presentation. Since MDS is a disease of excessive apoptosis in bone marrow, DNA resulting from the apoptosis is abundant in circulation. We explored the potential of using cell free DNA in peripheral blood plasma using next generation sequencing (NGS) to confirm the diagnosis of early MDS without the need for marrow biopsy.

Methods: Total nucleic acid was extracted from the plasma of 16 patients presenting with cytopenia and confirmed diagnosis of early MDS (blasts <5%) by the presence of mutations in one or more MDS-specific genes in DNA from cells in bone marrow. Plasma samples from 4 age-matched normals were used as negative controls. We performed targeted sequencing of 14 genes (581 amplicons) using Illumina MiSeq platform. This panel included the following genes: ASXL1, ETV6, EZH2, IDH1, IDH2, NRAS, CBL, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2. NGS and Sanger sequencing was used for testing. Results of cell free DNA in plasma were compared to that from cells or whole blood.

Results: Deep sequencing of cell free DNA in plasma from the 16 patients with early MDS showed at least one or more mutated gene confirming the diagnosis of MDS. Three patients (19%) showed mutation in one gene and the remaining 13 patients (81%) showed mutations in two or more genes. Cell free DNA in plasma from normal controls showed no evidence of mutations. When NGS data of cell free DNA from plasma was compared with Sanger sequencing data of DNA from cells in bone marrow, 10 of the 16 patients (63%) showed mutations in cell free DNA in plasma not detected by Sanger sequencing in DNA from cells in bone marrow. All mutations detected by NGS in cell free DNA in plasma were below the detection level of the Sanger technique and most likely represent subclones. NGS allowed the measurement of relative tumor load in plasma. Tumor load in plasma as detected by NGS was significantly (P=0.008) higher than that detected in cellular DNA, suggesting higher sensitivity of the former in detecting minimal residual disease and a better tool for monitoring therapy. Without exception, all detected mutations showed higher tumor load in plasma as compared with DNA from cells or whole blood, supporting the concept that plasma is enriched in tumor-specific DNA.

Conclusion: NGS of cell free DNA in plasma using limited number of MDS-specific genes, when used in patients with cytopenia, presents an objective test for the confirmation of the diagnosis of MDS. Plasma is enriched in tumor-specific DNA in patients with MDS. Furthermore, mutation analysis of cell free DNA in plasma can detect subclones with mutations and can predict the emergence of new clones. Analysis of cell free DNA in plasma using NGS provides important data on tumor load, which can be used to monitor therapy, and predict progression, and also reduces the need for performing bone marrow biopsies.