Gene Expression Profile In Essential Thrombocythemia: Search For New Markers For Triple Negative Cases

Abstract

Essential thrombocythemia (ET) is a BCR-ABL1-negative myeloproliferative neoplasm characterized by JAK2, CALR and MPL mutations which trigger the cytokine receptor/JAK2 pathway. Whole exome sequencing studies have shown that in most ET cases, just one mutation in one of the three MPN driver genes is found. In approximately 15% of chronic thrombocytosis cases classified as ET, the genetic cause remains unknown and, since they do not present mutations in any of the three driver genes, they are known as triple negative. Therefore, epigenetic or other transcription modulation mechanisms may be implicated. New molecular markers are needed to characterize triple negative cases, for that reason we studied the expression levels of genes related to MPN pathogenesis and evolution such as RB1, ASXL1, TET2, and EZH2.

Aims

To analyze the mRNA expression profiles of CALR, ASXL1, RB1 and TET2 genes in a series of ET patients with known mutational status of JAK2V617F, CALR and MPL.

Methods

Our series consisted of 73 ET patients, 53 females and 20 males, with a mean age of 61 years (range 45–77) diagnosed and treated between 1996–2017 at the Hospital Universitario de Gran Canaria Dr. Negrín. mRNA expression levels were determined by real time PCR in a LightCycler 480 Instrument II (Roche) using ABL as a control gene. Results were normalized with a cDNA pool from the peripheral blood of 10 healthy donors, which was introduced as an internal control in each experiment. The R Core Team software (2017) was used for statistical analyses.

Results

There was a positive, marginally significant, association between EZH2 and ASXL1 expression levels (p= 0.057). EZH2 expression levels were significantly lower in triple negative cases compared to those with mutations in any of the three driver genes (mean 0.75 ± 0.3 SD vs. 1.03 ± 0.53 SD, respectively; p = 0.003, t Student test). Levels of platelets were significantly higher in patients with mutations in any of the three driver genes compared to triple negative cases (777.82 ± 302.48 SD and 652.31 ± 231.4 SD, respectively; p = 0.03, Mann–Whitney U test). Levels of hemoglobin were also higher in mutated patients compared to triple negative ET patients (134.91 ± 31.7 SD and 134.03 ± 11.1 respectively, p = 0.04, Mann–Whitney U test).

Conclusion

The positive association between EZH2 and ASXL1 expression could be explained by their collaborative role in H3K27 methylation activity. A low expression level of EZH2 in triple negative cases indicates that EZH2 deficiency may be involved in the pathogenesis of a portion of triple negative cases. Finally, lower levels of platelet/hemoglobin in triple negative cases could reflect a subgroup of ET patients misclassified as triple negative while they may have reactive thrombocytosis.