Presence of JAK2V617F Mutation in Endothelial Cells from Budd-Chiari Syndrome Patients Is Not Correlated with Ph-Negative Myeloproliferative Neoplasm

Introduction

The role of the endothelial cell in the pathogenesis of Ph-negative MPNs is still not elucidated. Some have reported the presence of the JAK2V617F mutation in endothelial colony forming cells (ECFC) isolated from peripheral blood in patients with Ph-negative MPNs (Teofili L et all, Blood 2011). Others, however, did not find such an association (Piaggio G et al, Blood 2009). In patients with Budd-Chiari Syndrome (BCS), the JAK2V617F mutation has been found in endothelial cell (ECs) isolated by micro dissection from liver biopsies in patients both with and without MPN (Sozer S et al, Blood 2009). Besides the JAK2V617F mutation, other mutations (e.g. ASXL1, TET2, DNMT3A, SRSF2) have been described in patients with Ph-negative MPNs, but their presence has not been evaluated in patients with BCS who carried the JAK2V617F mutation.

Objectives

1. To evaluate the presence of the JAK2V617F mutation in CECs from patients with BCS both with and without concomitant Ph-negative MPNs; 2. To determine the mutational landscape of granulocytes in patients with BCS who harbored the JAK2V617F mutation but did not have the clinical diagnosis of a Ph-negative MPN.

Methods

We identified 10 patients from our institution who had a diagnosis of BCS and harbored the JAK2^V617F mutation in granulocytes. Three patients died from hepatic failure before they could be evaluated by bone marrow biopsy, so 7 patients remain for the analysis. All patients were investigated for the presence of Ph-negative MPNs with bone marrow trephine biopsy. ECs assays were performed according to the method of Hill. Briefly, Ficoll-Paque density gradient–isolated mononuclear cells were plated on fibronectin coated 6-well dishes with EndoCult medium (Stem Cell Technologies) for 48 hours, when non adherent cells were recovered and re-plated in a new dish at 10⁶/mL concentration. After an additional 5 days, adherent cells were plucked and analyzed by flow cytometry. The ECs population was sorted using a FACS Aria BD Biosciences sorter according to the following phenotype: CD45-PerCP-negative, CD31-FITC-positve, VEGFR2-PE-positive, CD34-PECy7-positive, CD133-APC-negative. The presence of the JAK2^V617F mutation was investigated by allele-specific PCR. Paired DNA (sorted CD66b-granulocytes/skin biopsy) from 3 patients with JAK2V617F-positive BCS without a clinical diagnosis of Ph-negative MPN was subjected to whole exome sequencing on a Illumina HiSeq 2000 platform using Agilent SureSelect kit. Tumor coverage was 150x and germline coverage was 60x. Somatic variants calls were generated by combining the output of Somatic Sniper (Washington University), Mutect (Broad Institute) and Pindel (Washington University), followed by in-house filters to reduce false positive calls.

Results

We were able to obtain CECs from all 7 patients. The purity of the CECs populations obtained was over 96% in all cases. Among the 7 patients with BCS, five did not have any clinical feature of a Ph-negative MPN, with a normal bone marrow biopsy. Results are summarized in the table. The JAK2V617F mutation was positive in the CECs from 5 cases, including 3 patients who only had BCS. In one patient with BCS solely the reaction did not work, and in another the JAK2 was wild-type in the ECs. The mutation was positive in CECs from both patients with myelofibrosis and BCS. Three patients with BCS solely were evaluated by whole exome sequencing. The only known pathogenic abnormality found was the JAK2V617F mutation, albeit at a low allele fraction (5%, 6% and 12.6%).

Conclusion

The presence of the JAK2V617F mutation in CECs from patients with BCS who did and did not have a diagnosis of Ph-negative MPN suggest that the mutation plays an important role in the development of vascular complications in these patients. Further studies with a larger number of patients are needed to precisely define the importance of CECs in the pathogenesis of MPNs. The sole presence of the JAK2V617F mutation in circulating granulocytes at a very low allele fraction in patients with BCS without Ph-negative MPNs suggest that these patients have a pre-malignant clone that would probably remain undiagnosed had it been not for the development of hepatic venous thrombosis.