C-Mannosyl tryptophan is a novel biomarker for thrombocytosis of myeloproliferative neoplasms

Abstract

C-Mannosyl tryptophan (CMW), a unique glycosylated amino acid, is considered to be produced by degradation of C-mannosylated proteins in living organism. Although protein C-mannosylation is involved in the folding and secretion of substrate proteins, the pathophysiological function in the hematological system is still unclear. This study aimed to assess CMW in the human hematological disorders. The serum CMW levels of 94 healthy Japanese workers were quantified using hydrophilic interaction liquid chromatography. Platelet count was positively correlated with serum CMW levels. The clinical significance of CMW in thrombocytosis of myeloproliferative neoplasms (T-MPN) including essential thrombocythemia (ET) were investigated. The serum CMW levels of the 34 patients with T-MPN who presented with thrombocytosis were significantly higher than those of the 52 patients with control who had other hematological disorders. In patients with T-MPN, serum CMW levels were inversely correlated with anemia, which was related to myelofibrosis (MF). Bone marrow biopsy samples were obtained from 18 patients with ET, and serum CMW levels were simultaneously measured. Twelve patients with bone marrow fibrosis had significantly higher CMW levels than 6 patients without bone marrow fibrosis. Collectively, these results suggested that CMW could be a novel biomarker to predict MF progression in T-MPN.

Keywords: C-Mannosyl tryptophan; C-Mannosylation; Essential thrombocythemia; Post-essential thrombocythemia myelofibrosis; Prefibrotic primary myelofibrosis; Thrombocytosis of myeloproliferative neoplasms.

Figure 1

Measurement of C-mannosyl tryptophan (CMW) in the serum samples from healthy workers. Serum CMW levels were quantified using ultra-performance liquid chromatography (UPLC). The fluorescence intensity (excitation at 285 nm/emission at 350 nm) and the identity of CMW were confirmed and quantified. (A) Serum a (thin line) and b (dotted line) were from healthy workers with high and low platelet counts, respectively. (B) Comparison of serum b alone and serum b supplemented with synthesized CMW. The arrows indicate the peak corresponding to CMW.

Figure 2

The consort flow diagram of study participants. Thrombocytosis of myeloproliferative neoplasms (T-MPN) group includes newly diagnosed essential thrombocythemia (ET), previously diagnosed ET, and those with subsequently confirmed myelofibrosis (MF). Control group represents patients with hematological disorders excluding T-MPN.

Figure 3

(A) Serum CMW levels in patients with control (n = 52) and patients with T-MPN (n = 34). The circles show CMW levels, and red line shows the medians. (B) Excluding patients with grade 3b and higher chronic kidney disease, the serum CMW levels of patients with control (n = 52) and those with T-MPN (n = 25). (C) The receiver operating characteristic (ROC) curve of serum CMW levels to the differential diagnoses for patients with T-MPN (n = 34). The area under curve (AUC) was 0.702; sensitivity, 0.529; and specificity, 0.846. Based on these values, the CMW cutoff value was 451.4 nM. *p < 0.05, **p < 0.01.

Figure 4

(A) Comparison of representative serum samples from T-MPN patient with MF-2 (thin line) versus T-MPN patient with MF-0 (dotted line). The serum CMW levels were quantified using UPLC. The arrow indicates a peak corresponding to CMW. (B) Serum CMW levels of patients with MF-0/1 (n = 11) and with MF-2/3 (n = 7). The circles show CMW levels, and red line shows the medians. (C) Serum CMW levels of patients with MF-0 (n = 6) and with MF-1/2/3 (n = 12). (D) The ROC curve of serum CMW levels for diagnosing T-MPN with MF-1/2/3. The AUC was 0.875; sensitivity, 0.750; and specificity, 1.000. Based on these values, the CMW cutoff value was 517.9 nM. ns; not significant. **p < 0.01.