Clinical and Molecular Characteristics of Megakaryocytes in Myelodysplastic Syndrome

Abstract

Objective Myelodysplastic syndrome (MDS) is a malignant clonal disorder of hematopoietic stem cells which is characterized by morphologic dysplasia. However, the pathological characteristics of megakaryocytes (MKs) in MDS patients with gene mutation are not well established. Methods Bone marrow MK specimens from 104 patients with primary MDS were evaluated, and all patients were distributed into two groups according to gene mutation associated with functional MKs. The morphologic and cellular characteristics of MKs and platelets were recorded and compared. Results The more frequently mutated genes in MDS patients were TUBB1 (11.54%), VWF (8.65%), NBEAL2 (5.77%), and the most common point mutation was TUBB1 p.(R307H) and p.(Q43P). Patients with MK mutation showed a decrease in adenosine diphosphate-induced platelet aggregation, high proportion of CD34 ⁺ CD61 ⁺ MKs (10.00 vs. 4.00%, p = 0.012), and short overall survival (33.15 vs. 40.50 months, p = 0.013). Further, patients with a higher percent of CD34 ⁺ CD61 ⁺ MKs (≧20.00%) had lower platelet counts (36.00 × 10 ⁹ /L vs. 88.50 × 10 ⁹ /L, p = 0.015) and more profound emperipolesis ( p = 0.001). By analyzing RNA-sequencing of MKs, differentially expressed mRNA was involved in physiological processes including platelet function and platelet activation, especially for MDS patients with high percent of CD34 ⁺ CD61 ⁺ MKs. The high levels of expression of CD62P, CXCL10, and S100A9 mRNA, shown by RNA sequencing, were validated by PCR assay. Conclusion High proportion of CD34 ⁺ CD61 ⁺ MKs was a poor prognostic factor in MDS patients with MK mutation. CD62P, CXCL10, and S100A9 may be the potential targets to evaluate the molecular link between gene defects and platelet function.

Keywords: CD34; CD62P; MDS; MK; TUBB1; emperipolesis.

Fig. 1

Emperipolesis in CD34 ⁺ CD61 ⁺ megakaryocytes (MKs). ( A ) The “rose cluster” phenomenon formed by MK and neutrophils ( red arrow ). ( B ) Neutrophils passing through the cytoplasm of MK ( red arrow ). ( C and D ) POX particles inside the cytoplasm of MK ( red arrows ).

Fig. 2

Platelet aggregation and activation . ( A ) Platelet aggregation function in myelodysplastic syndromes patients with or without megakaryocyte mutation. ( B ) The expression of CD62P and Annexin V on unstimulated platelet surface. ( C ) The expression of CD62P and Annexin V on stimulated platelet surface.

Fig. 3

Characterization of protein-coding RNA in megakaryocytes (MKs). ( A ) A heatmap showing distinctly defined expression profiles of mRNA in MKs. ( B ) Kyoto Encyclopedia of Genes and Genomes pathway analysis in MKs.

Fig. 4

Enrichment plots from gene set enrichment analysis of MKs. ( A – D ) Gene set enrichment analysis (CSEA) results showing upregulated pathways. ( A ) Platelet aggregation, normalized enrichment score (NES) = 2.50, p  < 0.0001. ( B ) Homotypic cell–cell adhesion, NES = 2.50, p  < 0.0001. ( C ) Regulation of platelet activation. NES = 2.47, p  < 0.0001. ( D ) Protein activation cascades NES = 2.09, p  < 0.0001. ( E – H ) GSEA results showing downregulated pathways. ( E ) TNFA signaling, NES = −2.1, p  < 0.0001. ( F ) Interferon alpha response, NES = −2.09, p  < 0.0001. ( G ) Interferon-gamma signaling, NES = −2.03, p  < 0.0001. ( H ) IL6-JAK-STAT3 signaling, NES = −1.79, p  < 0.0001.

Fig. 5

Reverse transcription polymerase chain reaction analysis of the potential gene targets in MDS with MK mutation. ( A ) The level of CXCL10 gene expression. ( B ) The level of CD62P gene expression. ( C ) The level of S100A9 gene expression.