Molecular Analysis in Patients with Idiopathic Erythrocytosis

Background:

Erythrocytosis corresponds to an increase in the red blood count, hemoglobin (Hb) concentration and hematocrit (Htc) above the reference range adjusted to age, sex and living altitude. Clinical and molecular data on patients suffering from primary erythrocytosis are sparse. Due to its rare incidence, primary erythrocytosis frequently represents a challenge for the diagnosis. The management of such patients is demanding as no clear standards of treatment have been defined.

Objective: We aimed to analyze the clinical and molecular features of patients with idiopathic erythrocytosis at our clinic.

Methods:

We evaluate in our internal registry patients encoded as erythrocytosis (primary or secondary) which were currently in follow-up. All data was obtained from the clinical records of the patients. For patient selection, we focused on those in which a JAK2 determination was available. We identified 102 patients from them 78 patients were JAK2 positive and were excluded in this analysis. 24 patients were initially selected as possible idiopathic erythrocytosis, of which 8 patients were excluded due to possible secondary causes.

Patients in whom the diagnosis of primary erythrocytosis was suspected were studied with a 13 genes NGS panel. The analyzed genes included EPOR (exons 7,8), VHL (orf including exon 1'), EGLN1, EPAS1, EPO (including several regulatory regions), JAK2 (exons 12-16), BPGM, HBB, HBA1, HBA2, HIF3A, OS9, SH2B3 (somatic). Only gene variants of the categories pathogenic, likely pathogenic and variant of unknown significance (VUS) according to ACMG guidelines 2015 are mentioned.

Results:

We identified 16 patients (15%) that did not fulfill the criteria for polycythemia vera nor had a secondary underlying cause of polycythemia and were diagnosed as idiopathic erythrocytosis. The median age was 32 years (r 20-76) and 81% (13/16) were males. The median Hb-value was 173 g/l (r 142-192), Htc 50% (r 43-65%). The median erythrocyte concentration was 5.87 G/l (r 5.18-6.57 G/l). No thromboembolic complications occurred in any of these patients either before or after diagnosis. Eleven patients (69%) were initially treated with venesection, which was interrupted during the follow-up except in two cases. The median follow-up in our clinic was 43 months (r 1 - 132). The median follow up since the interruption of venesection was 23 months (r 12-106). There was no evolution to leukemia, a myeloproliferative disorder or any kind of tumor.

In the results of the molecular analysis we found that the EPO: c.-1306C>A, rs1617640 reference allele was never homozygous. Indeed, all 16 patients showed an alternative allele: nine patients were heterozygous and 8 homozygous. EPO c.*772G>T, rs551238 was similarly distributed: no patients homozygous for the reference allele, 8 heterozygous and 8 patients homozygous for the alternative allele. In 5 patients other gene variants were also detected: in two patients EGLN1: c.1088T>G, p.(Leu363Arg) (the nucleotide is highly conserved, VUS, likely pathogenic), the other three were VHL: c.340+648T>C (reported as quite frequent, so more likely VUS, but it is within the alternate exon 1 region described by Lenglet et al. thus further studies are recommended), EPO: c.-1206C>A (not rare, VUS), EPO: c.*656G>A (downstream variant, very rare, unknown significance) respectively. No other mutations were identified.

Conclusion:

In our clinic idiopathic erythrocytosis showed a population of patients predominantly constituted of young men, had an indolent evolution, neither presented with thrombotic complications before or after the diagnosis, nor with a hematological transformation. The high prevalence of EPO gene variant underlines the role of this association. The five patients with combined mutations did not show a different clinical pattern. The use of NGS to evaluate patients with unclear erythrocytosis might allow an improvement in clinical management avoiding unnecessary interventions.