Evaluation of Bone Marrowmmorphology in Addition to JAK2 Allele Burden Is Essential for Assessing Disease Status in Recombinant Interferon Alpha-Treated Polycythemia Vera Patients
Authors: Margolskee Elizabeth Michelle , Krichevsky Spencer , Orazi Attilio , Silver Richard T. 
Published: October 2019

Abstract
Introduction
Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) associated with the JAK2 V617F mutation. Recombinant interferon alfa-2b (rIFNα-2b) or pegylated rIFN2α (pegrIFNα-2a) treatment in PV is effective in inducing hematologic and molecular responses. Reduction in JAK2V617Fallele burden (%V617F) has been used increasingly as a surrogate marker of disease response to treatment and as a unique criterion for discontinuation of therapy. To date, no studies have evaluated the relationship between changes in %V617F and established indicators of disease activity such as bone marrow cellularity and degree of fibrosis. Having observed persistent marrow hypercellularity or progressive fibrosis in PV patients despite a significant reduction in %V617F, we therefore systematically examined the correlation of %V617F with marrow morphology. We also assessed immunohistochemical expression of pSTAT5 as a marker of JAK2 activation.
Methods
The diagnosis of PV was based on the WHO criteria which include presence of erythrocytosis and/or an increased Cr51 red cell mass, JAK2V617F mutation and typical bone marrow findings such as hypercellularity due to panmyelosis and megakaryocyte morphology consistent with PV. Patients with two marrow examinations and quantitative JAK2 levels measured at a median of 1.2 months from the biopsy date were eligible for inclusion. The JAK2V617Fallele burden in peripheral blood samples was determined by pyrosequencing. Clinical, hematologic and molecular responses were graded according to ELN criteria. Marrow fibrosis was scored according to the WHO three-tiered semi quantitative grading system. Immunohistochemical staining for p-STAT5 was performed on clot sections in a subset of patients.
Results
We identified 15 patients for inclusion. The median %V617F at the time of the initial biopsy was 62.2% (18.2-100%), which decreased to a median of 33% (0-93.7%) on subsequent evaluation (p=0.02). Patients were initially treated with peg rIFNα-2a 45-90 mcgm weekly (n=13) or rIFNα-2b (n=2), 1x106units thrice weekly, and gradually increased based upon response and tolerance. The median duration of treatment between biopsy was 4.2 years (0.8 - 6.6). All patients achieved either a complete (CHR) (n=8) or a partial (PHR) (n=7) hematologic response. Of these 15 patients, 3 achieved a complete molecular response (CMR), 4 a partial molecular response (PMR), and 8 patients no molecular response (NMR). We observed no correlation between clinical and molecular response: of the 8 patients who achieved CHR, 2 achieved a CMR and 6 did not. There was no correlation between clinical response and fibrosis or cellularity; of 8 patients who achieved CHR, 5 showed persistent/increased hypercellularity and 6 had persistent/increased fibrosis.
We also found no correlation between marrow morphologic changes and molecular response. Of the 3 patients with CMR, all had persistent hypercellularity (range: 60-90%) and increased/persistent fibrosis. Among the 4 patients who achieved a PMR, 2 had increased cellularity and 2 decreased cellularity. All had increased or persistent fibrosis. Among the 8 patients with NMR, 2 had an increase in marrow cellularity, 2 no change, and 4 an insignificant decrease. Fibrosis increased in 2, was unchanged in 3, and decreased in 3. Thus, there were no significant correlations between changes in %V617F and cellularity (κ=0.02) and fibrosis (κ=0.04). Immunohistochemical staining for p-STAT5 expression in megakaryocyte nuclei showed no correlation between degree of positivity and %V617F.
Conclusions
Although we observed a statistically significant decrease in %V617F after treatment with rIFNα, there were no parallel changes in marrow morphology. In all patients with CMR, we observed persistent or worsening marrow disease, suggesting to us a need for continued therapy. In patients who achieved either CHR or PHR, we observed persistent hypercellularity and persistent/progressive fibrosis. Our results question the use of JAK2V617F allele burden as the sole criterion for discontinuation of rIFNα therapy. The prognostic significance of failing to attain a morphologic response in patients who have achieved a hematologic and/or molecular response remains unresolved. Resolution of these issues with longer follow-up is important, as premature discontinuation of rIFNα may allow the pathogenic mechanisms of the disease to progress unfettered.

Disclosures
Orazi: Novartis: Honoraria.