An overweight 62-year-old male with hypertension and hyperlipidemia presented with a hemoglobin level of 20.4 g/dL, hematocrit of 0.59, normal white cell and platelet counts, and a mild eosinophilia (Table 1). The patient had no clinical signs of PV, normal spleen size, normal oxygen saturation, no endogenous erythrocyte colony formation, no evidence of abnormal hemoglobin, and no family history of a hematological abnormality. The patient was managed with intermittent venesection alone for nine years with no evidence of the JAK2 p.V617F mutations by allele-specific PCR or the JAK2 exon 12 mutations by high-resolution melt curve analysis, excluding a diagnosis of PV. At 149 months after diagnosis, thrombocytopenia (75 x 109/L) and increasing eosinophilia (1.9 x 109/L) were noted with a low serum erythropoietin (EPO) level of 0.6 IU/L (normal range 2.6-18.5 IU/L). At 154 months after diagnosis, the patient had temporoparietal stroke. Worsening pancytopenia at 157 months prompted bone marrow biopsy which demonstrated 4% myeloblasts (Table 1). Subsequent bone marrow investigations showed progressively increasing myeloblast cell numbers by both morphological and immunophenotypic evaluation with reduced dysplastic megakaryocytes, dyserythropoiesis with basophilic stippling, and binuclear red cell forms, all consistent with MDS progressing to AML (Figure 1). The karyotype at 159 months was normal. The patient was not fit for intensive treatment and was given best supportive care with EPO, intermittent red cell transfusions, and low-dose steroids but died of infection at 161 months.
A targeted NGS approach panel was retrospectively employed to detect mutations possibly contributing to the erythrocytosis and subsequent development of MDS/AML in archival peripheral blood or bone marrow DNA samples from 41, 149, and 161 months. Amplicon libraries covering thirty commonly mutated genes implicated in myeloid malignancies, either covering the entire coding region (CALR, CEBPA, ETV6, EZH2, RUNX1, SH2B3, TET2, TP53, and ZRSR2) or mutational hotspots (ABL1, ASXL1, BRAF, CBL, CSF3R, DNMT3A, FLT3, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PTPN11, SETBP1, SF3B1, SRSF2, and U2AF1), were generated. Sequencing was performed with Ion AmpliSeq  methodology (Thermo Fisher Scientific, Paisley, UK). Calling of somatic mutations was achieved using an algorithm that excluded synonymous mutations, variants located within intronic or untranslated regions, and those present at a variant allele frequency (VAF) of <5%. A minimum target depth of coverage for variant calls was set at 500x as previously described. The nonsynonymous SH2B3 exon 2, single-nucleotide polymorphism (SNP) W262R (c.784T>C, NP_005466.1, rs3184504) was present at all three time points analyzed at VAFs of approximately 50% (Table 1). No mutations were detected in MPL exon 10 or CALR exon 9. A single CBL L380P mutation (c.1139T>C, NP_005179.2) was detected in the bone marrow sample at 161 months. Reinterrogation of sequencing data revealed this CBL mutation to be present a year previously at a VAF of 3.6% (Table 1).