Increased Dkk-1 plasma levels may discriminate disease subtypes in myeloproliferative neoplasms

Abstract

Alterations in the bone marrow niche induced by abnormal production of cytokines and other soluble factors have been associated with disease progression in classical BCR-ABL1 negative myeloproliferative neoplasms (MPN). Variations in circulating proteins might reflect local disease processes and plasma proteome profiling could serve to identify possible diagnostic and prognostic biomarkers. We employed a human cytokine array to screen for 105 distinct analytes in pooled plasma samples obtained from untreated young MPN patients (<35 years) with different clinical phenotypes and driver mutations, as well as from healthy individuals. Among molecules that exhibited significantly increased levels in MPN patients versus controls, the top of the list was represented by Dickkopf-related protein 1 (Dkk-1), which also showed the highest potential for discrimination between MPN subtypes. In the next step, a quantitative ELISA was used to measure plasma Dkk-1 levels in 30 young-onset MPN-10 essential thrombocythemia (ET), 10 polycythemia vera (PV), 10 pre-fibrotic primary myelofibrosis (pre-PMF)-and 10 controls. The results suggested that plasma Dkk-1 levels could differentiate ET from pre-PMF, in JAK2 V617F-positive as well as in CALR-positive patients, and also ET from PV in JAK2 V617F-positive patients.

Keywords: Dickkopf-related protein 1; circulating biomarker; cytokine array; myeloproliferative neoplasms.

Figure 1

Plasma protein profiling by cytokine array in MPN subtypes and controls. A, Representative images of array membranes corresponding to different MPN subtypes and control. The spots marked in boxes present differences in signal intensity among array membranes. Reference spots were used for signal normalization. B, Expression level of proteins that exhibited at least 1.5 fold change in MPN patients (compared to controls). In cytokine array, protein expression was assessed by densitometric analysis. The mean value of pixel densities in controls was set at 1.0, and the fold change was calculated for each protein. Data are presented as mean fold change ± standard error of the mean (SEM). C, Fold change in protein expression in MPN subtypes (compared to controls). The mean value of pixel densities in controls was set at 1.0, and the fold change was calculated for each protein, in each MPN subtype, in three independent experiments. Data are presented as mean fold change ± standard error of the mean (SEM). *P < .05, **P < .01, ***P < .001, Student's unpaired t test

Figure 2

Relative plasma Dkk‐1 levels (normalized to platelet count) in MPN subtypes and controls. Differences in plasma Dkk‐1 levels across all MPN subtypes and controls (A), between ET and pre‐PMF in JAK2 V617F‐positive patients (B), between ET and pre‐PMF in CALR‐positive patients (C), and between ET and PV in JAK2 V617F‐positive patients (D). Box plots (median, 25%‐75% interquartile range, minimum and maximum values) of normalized plasma Dkk‐1 levels are shown. *P < .05, **P < .01, Mann–Whitney U test