Upregulation of Platelet Transcripts As Biomarkers of Fibrotic Progression in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are a group of clonal hemopoietic disorders that have a proliferative phase (including essential thrombocythemia (ET) and polycythemia vera (PV)), and which can progress to advanced disease such as myelofibrosis (MF) or acute leukemia. This is thought to be due in part to progressively dysfunctional megakaryocytes driving reticulin deposition in the bone marrow. In previous work, we demonstrated that the platelet transcriptome in MPN differs from normal, and the difference is most marked with increased reticulin (Guo et al., 2020). A putative 3-gene signature ( CCND1, H2AFX and CEP55) could discriminate between patients with and without increased reticulin fibrosis. To progress this, we have profiled the transcriptome of platelets from a further 173 MPN patients were analyzed using next generation sequencing. CCND1, H2AFX and CEP55 were upregulated in platelets from patients with MF compared to ET (4.6-fold, padj<0.001, 9.0-fold, padj<0.001 and 10.5-fold, padj<0.001, respectively) and to PV (3.9-fold, padj<0.001, 9.2-fold, padj<0.001, 12.0-fold, padj<0.001). Application of the established 3-gene signature had a positive predictive value of 78%, a negative prediction value of 90% and accuracy of 88% for MF.

To assess the temporal changes in these gene transcripts in platelets and the potential utility to predict fibrotic progression, we then performed longitudinal analyses in two MPN mouse models. Jak2V617F knock-in mice ( n=25) with stable serially transplantable disease, which resembles PV (Mullally et al., 2010), were sacrificed at 4, 8, 12, 16 and 20 weeks post-transplant, and controls ( n=5) at 20 weeks. Blood counts were performed, and spleen and sternum harvested and stained with hematoxylin and eosin, and for reticulin. By 20-weeks, the platelet and leucocyte counts and hemoglobin had increased as expected. There was splenomegaly with extramedullary hematopoiesis and a mild increase in marrow reticulin. There was no significant up-regulation of Ccnd1, H2afx or Cep55 in platelet transcripts at any timepoint compared to the control mice. This data showed stability of the mouse PV phenotype and platelet transcripts, in keeping with the chronic phase of MPN.

Mice with MPLW515L ( n=8) (Pikman et al., 2006) were sacrificed at 3 and 6 weeks post-transplant, and control mice ( n=4) at 6 weeks. At 3-weeks post-transplant Cep55 (131.6-fold, padj<0.001) and H2afx (1.7-fold, padj<0.01) had increased in the MPLW515L mice and there was no increase in reticulin in the bone marrow. At 6-weeks platelet and leucocyte counts had increased, there was splenomegaly with extramedullary hematopoiesis including marked megakaryocytosis and marrow reticulin. On platelet transcript analysis Cep55 (154.6-fold, padj<0.001) and H2afx (2.4-fold, padj<0.001) were significantly increased; there was no significant change for Ccnd1.

Upregulation of Cep55 and H2afx in the MPLW515L mouse occurred prior to the accumulation of bone marrow reticulin and development of MF phenotype. This suggests that up-regulation of Cep55 and H2afx in platelets may be a predictor of disease progression from ET and PV to MF. For MPN patients, a deviation from steady-state platelet transcript levels, specifically for H2AFX and CEP55, may infer a biological transition towards the fibrotic phenotype. This novel approach utilizing platelet transcript analysis may therefore have potential for serially monitoring MPN patients and the early identification of genomic changes that may herald disease transformation to MF.