We report a case of a 49-year-old man with no significant medical history. The patient was referred to our center in October 2011. The blood count was abnormal with anemia (Hb 11.2 g/dL) and a platelet count of 78000/mm3. The white blood cell count was 11500/mm3 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. 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.
FISH analysis (Figure 2) using dual fusion probes for BCR (22q11.2) and ABL1 (9q34) regions (LSI BCR/ABL ES Dual Color Translocation Probe, Abbott Molecular; Vysis, Des Plaines, IL, USA) excluded the BCR-ABL1 fusion and showed an extra signal of the BCR probe on chromosome 9p. Moreover, a FISH assay was performed using JAK2 probes obtained from bacterial artificial chromosome (BAC) (RP11-39K24 AL161450, Sanger Institute, Cambridge, UK) 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 (Life Technologies, Carlsbad, CA, USA) identified an in-frame 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 Graft-versus-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 35-month follow-up, the patient remains alive with undetectable BCR-JAK2 transcript levels in the blood and no transplant-related complications (Figure 4).