Elevated plasma EDA fibronectin in primary myelofibrosis is determined by high allele burden of JAK2 V617F mutation and strongly predicts splenomegaly progression

Abstract

In primary myelofibrosis, extra-domain A fibronectin (EDA-FN), the result of alternative splicing of FN gene, sustains megakaryocyte proliferation and confers a pro-inflammatory phenotype to bone marrow cell niches. In this work we assessed the levels of circulating EDA-FN in plasma samples of 122 patients with primary myelofibrosis. Patients with a homozygous JAK2V617F genotype displayed the higher level of plasma EDA-FN. Increased EDA-FN levels were associated with anemia, elevated high-sensitivity C-reactive protein, bone marrow fibrosis and splanchnic vein thrombosis at diagnosis. While no correlation was observed with CD34+ hematopoietic stem cell mobilization, elevated blood level of EDA-FN at diagnosis was a predictor of large splenomegaly (over 10 cm from the left costal margin) outcome. Thus, EDA-FN expression in primary myelofibrosis may represent the first marker of disease progression, and a novel target to treat splenomegaly.

Keywords: extra domain A; fibronectin; neoangiogenesis; primary myelofibrosis; splenomegaly.

Figure 1

Plasma EDA-FN levels are determined by high allele burden of JAK2V617F mutation. Plasma EDA-FN levels (mean ± 1.96 standard error) of subjects with PMF stratified according to the driver mutations genotypes. JAK2V617F mutants with >50% allele burden (homozygous genotype) had the highest mean EDA-FN plasma concentration (N=15, mean value = 6.73 μg/mL, range, 2.16 - 19.16). The mean value of EDA-FN in homozygous JAK2V617F genotype was higher than in other genotypes together (N=103, mean value = 4.73 μg/mL, P=0.015) and in heterozygous JAK2V617F genotype (N=59, mean value = 4.85 μg/mL; P=0.036). Triple negative subjects had the lowest plasma EDA-FN concentration (N=9; mean value = 1.29 μg/mL; range, 0.75 to 6.28); their value was not different from those of healthy controls and was significantly lower than those with MPL mutation (N=5, mean value = 5.11 μg/mL, range, 2.57 to 10.82; P=0.016) and CALR mutations (N=30; mean value = 5.65 μg/mL, range, 0.65 to 20.11; P=0.039).

Figure 2

Plasma EDA-FN levels predict splenomegaly progression. Kaplan Meier analysis of the progression to large splenomegaly in patients with EDA-FN lower and greater than the cut-off value of 5.3 μg/mL. The difference was statistically significant (P=0.027).

Figure 3

EDA-FN is increased in spleen of PMF patients. (A) Confocal microscopy analysis of FN containing EDA segment (Ab IST-9) (red) in spleen biopsies of healthy control (HC) and Primary Myelofibrosis (PMF) patients. Nuclei were stained with Hoechst 33258. Objective 10x, Scale Bar = 100μm. (B) EDA-FN immunoreactivity expressed as % of EDA-FN antibody staining area in spleens of HC (N=2) and PMF patients (N=3) normalized on total surface. (C) Confocal microscopy analysis of EDA-FN localization (red) in proximity of CD34+ vessels (green) in spleen sections of HC and PMF patient. Hoechst 33258 was used to stain nuclei. Objective 60x, Scale bar = 50 μm. (D) Quantification of total CD34+, CD34+/EDA-FN- and CD34+/EDA-FN+ vessels in spleen biopsies of HC (N=2) and PMF patients (N=3). At least 10 fields per sample were analyzed. Objective 20x, Scale bar = 100 μm.