Granulocyte whole exome sequencing and endothelial JAK2V617F in patients with JAK2V617F positive Budd‐Chiari Syndrome without myeloproliferative neoplasm

Budd-Chiari Syndrome (BCS) is characterized by hepatic venous outflow obstruction. JAK2-positive myeloproliferative neoplasms (JAK2 MPN) are one of the most frequent thrombotic conditions underlying a diagnosis of BCS (Smalberg et al, 2012). A subset of BCS patients harbour the JAK2 mutation in peripheral blood (PB) granulocytes without evidence of overt MPN (JAK2 MPN-neg). While the occurrence of other myeloid-associated mutations in genes are common in patients with JAK2 MPN, it is unknown if such mutations are present in patients with JAK2 MPN-neg. In addition, it remains to be determined if there are differences in JAK2 allele burden between JAK2 MPN and JAK2 MPN-neg. Recently, it has been demonstrated that a subset of patients with JAK2 MPN have endothelial cells (ECs) that are positive for JAK2(Teofili et al, 2011) and that these cells may possibly contribute to the prothrombotic state. However, the presence of JAK2-positive endothelial colony-forming cells (E-CFC) in JAK2 MPN-neg patients with BCS has not been evaluated. The objective of this study was to assess the presence of JAK2 in BM E-CFC from JAK2 MPN-neg patients with BCS and to evaluate the JAK2 allele burden and the presence of additional mutations. We selected patients diagnosed with BCS and the presence of JAK2 mutation in the absence of other thrombophilic conditions at Hospital Israelita Albert Einstein, Sao Paulo, Brazil, between March 2013 and June 2015. Patients were investigated for the presence of an associated MPN and diagnosed according to the 2001 World Health Organization criteria. This study was approved by the Institutional Review Board and conducted according to the Principles of the Declarations of Helsinki. Paired DNA (sorted CD66b-granulocytes/skin biopsy) was subjected to whole exome sequencing (WES) on the HiSeq 2000 platform (Illumina Inc., San Diego, CA) using the SureSelect library preparation kit (Agilent, Santa Clara, CA). Somatic variant calls were generated by combining the output of SomaticSniper (http://gmt.genome.wustl.edu/packages/somatic-sniper/), MuTect (https://www.broadinstitute.org/cancer/cga/mutec) and Pindel (www.sanger.ac.uk/ science/tools/pindel) plus additional in-house criteria (minimum coverage at both tumour/germline ≥8 reads; ratio of allele fraction tumour:germline >2). Bone marrow (BM) mononuclear cells were obtained by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Approximately 20 ml of BM was washed twice with phosphate-buffered saline and 2% fetal bovine serum and then suspended in Endocult liquid medium (STEMCELL Technologies Canada Inc., Vancouver, BC, Canada), as previously described (Wu et al, 2012), but extending the culture time by 7 days. ECs grown in culture were then evaluated by flow cytometry (ECs were considered CD146+, CD144+, CD34 , CD117 ) (Ozdogu et al, 2007). ECs were isolated by fluorescence-activated cell sorting (FACS) in a FACSAria (BD Biosciences, San Jose, CA). DNA was extracted from sorted ECs and tested for the presence of the JAK2 mutation by an allele-specific polymerase chain reaction (PCR). Data on JAK2 allele burden was extracted from WES results obtained from a cohort of 77 patients with JAK2-positive MPN (including 4 patients with JAK2 MPN-neg) (Santos et al, 2014) and from 110 patients previously reported by Nangalia et al (2013). We identified 32 cases of BCS, 10 (31 2%) of who were positive for the JAK2 mutation. Three of these patients passed away before inclusion in the study and 7 patients were included; their baseline features are summarized in Table I. Two patients were diagnosed with primary myelofibrosis (PMF) (Patients 1 and 7) during evaluation and the remaining 5 cases were JAK2 MPN-neg. Samples from Patients 1–6) were analysed by WES. In the 5 JAK2MPN-neg patients (Patients 2–6), the only known pathogenic somatic mutation found was JAK2. We found no additional myeloid-associated mutations in these patients. The median JAK2 variant allele frequency Table I. Demographic features of BCS patients and JAK2 alleleburden.