Transformation of an Unclassified Myeloproliferative Neoplasm with a Rare BCR-JAK2 Fusion Transcript Resulting from the Translocation (9;22)(p24;q11)

BCR-ABL1 negative myeloproliferative neoplasms (MPNs) are known to contain alterations of the tyrosine kinase JAK2 (located on 9p24) that result in constitutive activation of the encoded protein. JAK2 fusions are reported in acute and chronic leukemias of myeloid and lymphoid phenotypes. Here, we report an unclassified case of MPN (MPN-U) showing a t(9;22)(p24;q11), which generates a BCR-JAK2 fusion gene by fusing the BCR at intron 13 to JAK2 at intron 17 on the derivative chromosome 22. Most reported JAK2 fusions cases reveal an aggressive clinical course and long-term remissions have only been achieved after allogeneic stem cell transplantation (ASCT). To the best of our knowledge, this is the thirteenth case reported worldwide to describe a BCR-JAK2 fusion transcript in MPN-U. The present report revealed a sustained complete clinical, hematologic, and cytogenetic remission 35 months after diagnosis and ~24 months after ASCT. Regarding BCR-ABL1   negative MPN patients this case report provides strong support for a role of JAK2 activation in the oncogenesis and suggests a possible diagnostic and therapeutic target that should be investigated.


Introduction
Some myeloproliferative neoplasms (MPNs) are Philadelphia-(Ph-) negative, lacking the reciprocal t(9;22)(q34;q11) and its resultant BCR-ABL1 fusion gene. Currently, the most frequent genomic abnormality observed in Ph-negative MPN is a dominant gain-of-function V617F mutation in the JH2 kinase-like domain of JAK2 [1]. However, there are rare additional mechanisms described in Ph-negative MPNs that activate JAK2, such as chromosomal translocations that cause constitutive dimerization through the replacement of amino terminal sequences with a fusion partner [2,3]. Indeed six different fusion partners have been associated with JAK2 (RPN1, SSBP2, PAX5, PCM1, BCR, and ETV6).
Here, we report a rare case of unclassified MPN (MPN-U) with a t(9;22)(p24;q11) leading to a 5 BCR/3 JAK2 fusion gene producing a fusion transcript that juxtaposed BCR exon 13 and JAK2 exon 17 and subsequently rapidly transformed into a myeloid granulocytic sarcoma. We also describe, 35 months after diagnosis and ∼24 months after ASCT, a prolonged and sustained complete clinical, hematologic, and cytogenetic remission after undergoing allogeneic stem cell transplantation (ASCT). October 2011. The blood count was abnormal with anemia (Hb 11.2 g/dL) and a platelet count of 78000/mm 3 . The white blood cell count was 11500/mm 3 with 30% lymphocytes, 2% monocytes, 2% eosinophils, 0% basophils, 29% neutrophils, and 37% promyelocytes, myelocytes, and metamyelocytes. Clinical examination was unremarkable. The bone marrow aspiration and biopsy associated with initial molecular blood and medullary analyses led to diagnose an MPN-U. It did not reveal any BCR-ABL1 rearrangement neither V617F JAK2 mutation. In February 2012, the patient presented to the emergency room with a sudden onset of pyramidal tract deficiency syndrome and with an increase of leukocytosis and blood myeloid precursors. The MRI scan revealed a thoracic spinal epidural compression extending from T4 to T10. Emergent laminectomy was done. Histological analysis was performed on the laminectomy specimen and demonstrated the presence of a granulocytic (myeloid) sarcoma. Radiation therapy was then performed. Cytogenetic examination of the bone marrow aspiration of the patient was performed on two unstimulated short-term cultures (24 hrs and 48 hrs). The karyotype was obtained by conventional R-banding analysis [4]. Chromosome analysis ( Figure 1) showed t(9;22)(p24;q11) as the sole abnormality in 60% of the analyzed metaphases (12/20). In 10% of the analyzed metaphases (2/20), it showed the latter translocation in addition to der(22) t(9;22)(p24;q11). The last 30% of the analyzed metaphases (6/20) were normal. Mutations of exons 12, 13, and 14 and, in particular, the V617F JAK2 gene mutation were not found. Considering the t(9;22)(p24;q11), that the exons 12, 13, and 14 and the V617F JAK2 mutations were absent, and that JAK2 had previously been shown to fuse with BCR in MPN-like patients, the best fusion gene candidates were JAK2 in 9p24 and BCR in 22q11. (a) Nucleus with two red signals (red arrow, ABL probe) on chr9 and two green signals (green arrow, BCR probe) on chr22, indicating that BCR-ABL1 FISH for the Ph-chromosome did not reveal any fusion signals, revealing a normal hybridization pattern negative for t(9;22)(q34;q11.2) BCR-ABL1 fusion. (b) Two red signals (red arrows, ABL probe) were present on the long arm of both the normal and the derivative chr9. Three green signals were present, indicating an extra signal of the BCR probe, suggestive of an extra chromosome 22 or additional chromosome material containing the 22q11.2 region: two intense green signals were on the normal chr22 (green arrows, BCR probe) and one reduced intensity green signal was localized on the derivative chr22 and on the short arm of derivative chr9 (dotted green arrow, telomeric part of the BCR probe). Notice that one of the derivative chromosome signals was too feint to be seen in some nuclei. labelled with spectrum red (Vysis, Downers Grove, IL, USA) and showed a split of the probe between 9p24 and 22q11. According to the cytogenetic examination and FISH results which showed a t(9;22)(p24;q11), a BCR-JAK2 fusion was suggested.
Blood from the patient was collected in EDTA, and RNA was isolated from 107 cells using the TRIzol kit (Life Technologies, Carlsbad, CA, USA). cDNA synthesis was performed using Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen, Life Technologies, Carlsbad, CA, USA). The breakpoint Sanger sequencing using ABI 3130 Case Reports in Hematology First nucleotide of exon 17 of Last nucleotide of exon 13 of 22q11 9p24 9 22 (Life Technologies, Carlsbad, CA, USA) identified an inframe fusion of the last nucleotide of BCR exon 13 with the first nucleotide of JAK2 exon 17 (Figure 3). There was no loss or insertion of a base at these breakpoints.
To follow up the minimal residual disease, a specific primer-probe assay was designed. Real time quantitative PCR (QPCR) using TaqMan chemistry was performed on an Applied 7500 (Life Technologies, Carlsbad, CA, USA) with the following sequences: forward primer 5 -GCT GAC CAA CTC GTG TGT GAA-3 , reverse primer 5 -TCA GGT GGT ACC CAT GGT ATT CT-3 and the probe FAM 5 -CAG CAT TCC GCT GAC CAT CAA TAA GGA-3 . QPCR expression levels of BCR-JAK2 were carried out relative to the expression of the housekeeping gene ABL1. Molecular monitoring was able to detect low levels of disease. Hence, the assay was >4 logs more sensitive than conventional cytogenetic, detecting one copy of BCR-JAK2 to 10000 copies of ABL1 (0.0001%) and allowing us to follow up the effectiveness of treatment. The patient underwent acute myeloid leukemia-like chemotherapy induction and consolidation achieving a chronic phase in May 2012. An ASCT from a matched human leukocyte antigen-(HLA-) unrelated donor (MUD) was then undertaken in August 2012 with a TBI-Endoxan regimen conditioning and without any Graftversus-host disease complications. The QPCR follow-up of BCR-JAK2 expression in both the bone marrow and peripheral blood mononuclear cells showed complete hematological and molecular (<0.0001%) remission 3 months later. With 35month follow-up, the patient remains alive with undetectable BCR-JAK2 transcript levels in the blood and no transplantrelated complications (Figure 4).   JH2 of JAK2. The constitutive activation of this chimeric protein is mediated by oligomerization through the coiled-coil domain of BCR and by disruption of the autoinhibitory role of the inhibitory regions (IR) of the pseudo-kinase domain JH2 of JAK2. In fact, there are three inhibitory regions (IR1, -2, and -3) within JH2. IR3, at the C terminus of JH2, directly inhibits JH1. IR2, in the C-terminal lobe of JH2, and IR1, extending from the N-terminal to the C-terminal lobe, enhance the IR3-mediated inhibition of JH1. Hence, the disruption of IR by mutation, deletion, or translocation increases basal JAK2 activity. Consequently, the BCR-JAK2 chimeric protein is entirely or partially deprived of IR1, which may result in the upregulation of JAK2 activity [5]. Preclinical studies implied a possible role of c-ABL1 in Jak2 activation in various Phnegative myeloid malignancies [6] and demonstrated that the BCR-JAK2 fusion gene induces STAT5 activation and inhibits BCRxL gene expression, thereby promoting tumorigenic properties and increasing cell survival [7].

Discussion
It was difficult to define the best therapy. JAK2 inhibitors alone or in combination with chemotherapy may not be effective against the BCR-JAK2 fusion gene malignancies [7,8]. Compared with JAK2 mutations, JAK2 fusions are probably associated with more aggressive diseases such as  BCR-JAK2 in this case is therefore likely to be the result of a small insertion of BCR into the JAK2 locus on the der(18). acute leukemias (myeloid or lymphoblastic), atypical CML (aCML), and myelofibrosis [9]. In our review of literature (Table 1) seven patients presented with aCML/MPN-U, one patient presented with AML, and one patient with ALL. As it is usually observed in myeloid neoplasms, a predominance of the male gender is reported. Three of the reported patients were unsuccessfully treated with tyrosine kinase inhibitors (imatinib or ruxolitinib). In addition, it is important to note that the mortality rate was 50% in cases where the follow-up was described. In our opinion, ASCT is likely the only curative strategy in these MPN-Us. In fact, JAK2 translocations have been described in acute and chronic leukemias of myeloid and lymphoid phenotypes. Hence, we can hypothesize that MPN-U associated with rearrangements of JAK2 is a hematopoietic stem cell (HSC) disease that is only curable by HSC transplantation [10].

Conclusion
We described a rare Ph-negative case of MPN with a BCR-JAK2 transcript and a reciprocal t(9;22)(q34;q11) that was detected juxtaposing BCR exon 13 and JAK2 exon 17. This rare entity underlies often an aggressive clinical course with rapid progression to blast phase within the first 2 years after diagnosis (Table 1). To the best of our knowledge, this is the thirteenth case reported worldwide. Furthermore we report here the first described isoform fusion transcript juxtaposing BCR exon 13 and JAK2 exon 17. It revealed one of the longest sustained complete clinical, hematologic and cytogenetic remissions in a BCR-JAK2 fusion MPN-U. These rare BCR-JAK2 fusions suggest common pathways between JAK2 activation and the natural history of lympho/myeloproliferative hematologic malignancies. One should take into consideration JAK2 fusions when investigating Ph-negative MPN patients.