Comparing the Clinical and Genetic Features By Next-Generation Sequencing and RNA Sequencing of Polycythemia Vera and Post-Polycythemia Vera Myelofibrosis Patients
Authors: Tu Lingrong , Yang Xiudi , Zhu Xiaoqiong , Lu Ying , Qian Honglan , Huang Jian 
Published: December 2024

Among 887 PV and post-PV MF patients in Zhejiang, China, 701 (79.9%) harbored JAK2V617F mutation, 11 (1.2%) JAK2 Exon 12 mutation alone; and 165 (18.8%) were JAK2 negative with reduced Epo levels. Univariate analysis revealed that physical symptoms (P=0.003; HR 2.48, 95% CI 1.36-4.54), splenomegaly (P&lt;0.001; HR 5.15, 95% CI 2.50-10.61), WBC&gt;=11*10^9/L (P=0.033; HR 1.85, 95% CI 1.05-3.27) and LDH(ROC,&gt;287U/L) (P=0.006; HR 5.08, 95% CI 1.58-16.33) were risk factors for progression post-PV MF. Multivariable analysis revealed that physical symptoms (P&lt;0.001; HR 3.50, 95% CI 1.67-7.35), splenomegaly (P=0.030; HR 3.79, 95% CI 1.14-12.61) and LDH(ROC,&gt;287U/L) (P=0.030; HR 3.79, 95% CI 1.14-12.61) were independent risk factors for progression post-PV MF. Among 548 PV and post-PV MF patients who underwent cytogenetics testing, 3.6% (20/548) patients harbored abnormal cytogenetics. Among them, 13 patients were PV, and 7 patients were post-PV MF patients. Different phases harbored different abnormalities. +8 and 1q+ were the most commonly cytogenetic abnormalities in PV patients, and the most commonly single abnormality was +8(3/13,23.1%), and +1q (2/13,15.4%) was the most common component in double abnormalities. -5/5q-, -13/13q-, -17 and 1q+ (all 28.6%, 2/7) were the most commonly cytogenetic abnormalities in post-PV MF patients, and complex karyotype (28.6%, 2/7) was the most commonly cytogenetic abnormality.

To assess the prognostic factors in genetic features, we performed next-generation sequencing (NGS) in 14 post-PV MF and 66 PV patients, including 46 JAK2V617F and 20 JAK2 negative. 36 genes mutations were detected. Expect JAK2 mutation, the most frequently mutated gene was TET2(5/14, 35.7%), followed by AXSL1(4/14, 28.5%) in post-PV MF patients. Expect JAK2 mutation, the most frequently mutated gene was TET2(10/46, 21.7%), followed by KMT2C (6/46, 13.0%), KMT2D (5/46, 10.9%) and TP53(5/46, 10.9%) in JAK2+ PV patients. The most frequently mutated gene was KMT2C (3/20, 15.0%), followed by TET2(2/20, 10.5%) in JAK2- PV patients. Patients with Post-PV MF had a higher median number of mutations associated with cell cycle regulation than JAK2+ PV patients(P=0.01). In PV patients, we also found that TET2 mutation was associated with splenomegaly(P=0.04).

We further performed RNA-Seq analysis in 23 PV samples and 3 post-PV MF samples. Compared to PV, post-PV MF showed 3 up-regulated and 22 down-regulated differentially expressed genes (DEGs), of which AL121758, MTCO1P12 and B4GALT4 were significantly up-regulated. Gene ontology (GO) functional enrichment analysis showed no significantly difference (all q&gt;0.05). KEGG biofunctional analysis showed that DEGs were significantly enriched in Amino acid metabolism(q=0.01). In the GSEA analysis, the enrichment was shown in antigen binding, immunoglobulin complex, phagocytosis recognition, T cell receptor complex etc. in GO pathway. In KEGG pathway, the enrichment was shown in primary immunodeficiency, Fanconi anemia pathway, aminoacyl tRAN biosynthesis, ribosome etc. We also performed RNA-Seq analysis in 15 JAK2+ PV samples and 3 post-PV MF samples. Compared to PV, post-PV MF showed 3 up-regulated and 22 down-regulated differentially expressed genes (DEGs), of which AL121758, AL645608, and MTCO1P12 were significantly up-regulated. Gene ontology (GO) functional enrichment analysis showed that DEGs were enriched in membrane protein proteolysis, sequestering of actin monomers, actin filament depolymerization, actin filament depolymerization etc. KEGG biofunctional analysis showed no significantly difference (all q&gt;0.05). In the GSEA analysis, the enrichment was shown in T cell receptor complex, adaptive immune response, complement activation, classical pathway, immunoglobulin complex etc. in GO pathway. In KEGG pathway, the enrichment was shown in graft versus host disease, Th1 and Th2 cell differentiation, intestinal immune network for iga production, primary immunodeficiency etc.

This research was funded by the Key R&D Program of Zhejiang (No. 2022C03137) and the Zhejiang Medical Association Clinical Medical Research special fund project (No. 2022ZYC-D09). *Correspondence to: Prof Jian Huang, E-mail: househuang@zju.edu.cn;