Molecular Profiling in Confirming the Diagnosis of Early Myelodysplastic Syndrome

Introduction: Diagnosis of myelodysplastic syndrome (MDS) based on bone marrow morphology can be very difficult, when blasts are not increased. The demonstration of cytogenetic abnormalities in these cases can confirm the diagnosis, providing cytopenia is documented. Cytopenia is usually the major reason for initiating work-up for myelodysplasia and , in general, cases with unicytopenia are the most difficult to make the diagnosis. In principle the recent characterization of the molecular abnormalities underlying the biology of MDS should provide objective biomarkers that can be used to confirm the diagnosis of MDS in the absence of cytogenetic abnormalities.

Toward this goal, we developed a 14-gene panel to detect molecular abnormalities in patients referred to rule out MDS with blast count <5% without cytogenetic abnormalities, but with documented cytopenia.

Method: Cytopenia is defined as having platelets <100,000 /µl, neutrophils <1,800/µl, or hemoglobin <10g/dL. Total nucleic acid was extracted from bone marrow or peripheral blood samples and tested for mutations in any of the following genes: ASXL1, ETV6, EZH2, IDH1, IDH2, NRAS, CBL, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2. Direct bidirectional Sanger sequencing, as well as next generation sequencing were used for testing. Samples from 137 patients fulfilling the criteria described above were analyzed. As cytogenetic abnormalities is a marker of MDS, a control group of 14 patients with cytogenetic abnormalities but no increase in blasts were evaluated using the same molecular panel.

Results: Fifty three of the 137 patients (39%) had a mutation in one or more genes. Of the 137 patients, three had tricytopenia, 14 had bicytopenia and 120 had unicytopenia. Two of the three with tricytopenia (66%) had mutations and nine of 14 with bicytopenia (64%) had mutations. In contrast 42 of the 120 patients with unicytopenia (35%) had mutation in one or more genes. Thirty of the 53 patients with mutation (57%) had one gene mutated and only 4 (13%) of these patients had bi- or tricytopenia. Of the remaining 23 patients with mutations in two or more genes a higher percentage (30%) of patients had bi- or tricytopenia. Compared to patients without mutations in the tested genes, those with mutation had significantly lower number of neutrophils (P=0.006), but higher percentage of monocytes (P=0.0002) and slightly higher percentage of lymphocytes (P=0.06).

Twelve of 14 (86%) patients with cytogenetic abnormalities showed mutation in one or more genes and only three patients of the 14 (21%) had bi- or tricytopenia.

Conclusion: Diagnosis of MDS at early stage of disease (blasts <5%) can be significantly enhanced by adding molecular profiling to cytogenetics studies. Molecular profiling using limited number of genes (14) in patients with cytopenia and suspected of having MDS, but no cytogenetic abnormalities, can confirm the diagnosis of MDS in 39% of cases. Compared to MDS with unicytopenia, MDS with bi- or tricytopenia without increase in blasts, is more likely to be confirmed by molecular testing.