Co-Occurrence of Separate BCR-ABL1-Positve and JAK2V617F-Positive Clones in 23 Patients Reveals Biologic and Clinical Importance

Background:

The simultaneous detection of a BCR-ABL1 rearrangement and a JAK2V617F mutation in the same patient is a very rare event and has previously been described in case reports or very small series of cases only.

Aim:

1) To establish the incidence of cases with concurrent BCR-ABL1 rearrangement and JAK2V617F mutation. 2) Evaluate whether one clone harbours both mutations or whether there are two independent clones. 3) Establish whether these patients have additional concurrent gene mutations and how they influence the evolution of both diseases.

Patients and Methods:

A total of 27,907 patients with suspected myeloproliferative neoplasms (MPN) where studied in parallel for BCR-ABL1 and JAK2V617F mutation from May 2005 to June 2014 at our institution. BCR-ABL1 analysis was performed by multiplex RT-PCR and JAK2V617F mutation analysis by melting curve based LightCycler assay. A total of 23 patients (0.08%) were positive for both mutations. Eleven patients were male and 12 were female with a median age at diagnosis of 72 years (range 46-80 years). Of fifteen patients 2 or more sample time points were available for follow-up analyses (median follow-up: 4 years, range: 5 months - 9 years). Both BCR-ABL1 and JAK2V617F mutation loads were assessed by quantitative real time PCR. In addition, 22/23 cases were analyzed upon detection of co-occurrence of both clones with a pan-myeloid gene panel consisting of 25 genes: TET2, RUNX1, PHF6, ASXL1, CBL, DNMT3A, SF3B1, TP53, BCOR, BRAF, ETV6, EZH2, FLT3 (TKD), GATA1, GATA2, IDH1, IDH2, KIT, KRAS, MPL, NPM1, NRAS, SRSF2, U2AF1, and WT1. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Lexington, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). RUNX1 was sequenced on the 454 Life Sequence NGS platform (Roche 454, Branford, CT). The median coverage per amplicon was 2,215 reads (range 100-24,716). The lower limit of detection was set at a cut-off of 1.5%.

Results:

At the time point of detection of both mutations morphological assessment was available in 12 patients. The remaining 5 showed features typical for CML. Bone marrow blast count was <5% in all cases. Cytogenetics was available in 18/23 cases (78.3%). The classical t(9;22)(q34;q11) was identifiable in 16/18 patients. Two patients had a normal karyotype as they were in complete cytogenetic remission of their CML (due to TKI treatment) at diagnosis of the JAK2 V617F positive clone. In the majority of patients (n=16) the JAK2V617F mutation predated the BCR-ABL1 clone, in 4 patients CML was known before the detection of the JAK2V617 positive clone, in 1 patient both were diagnosed simultaneously and in another 2 patients information in this regard was lacking. BCR-ABL1 transcript types distributed as follows: b2a2 and/or b3a2 (n=18), and e1a2 (n=5).

The continuous quantitative assessment of BCR-ABL1 and JAK2V617F mutational loads in 15 patients showed asynchronous patterns of courses in all cases giving proof of these aberrations representing two different clones in these cases. When treatment with TKI was initiated, the BCR-ABL1 clone decreased while the JAK2V617F clone either remained stable or increased in all 15 cases. Next generation sequencing revealed further mutations in 13/22 analyzed patients (56.5%). One mutation was detected in 8 patients, 4 patients revealed 2, and one patient even 3 different additional mutations. In detail, mutations in the following genes were detected: TET2 (n=8), ASXL1 (n=4), RUNX1 (n=2), CBL (n=1), DNMT3A (n=1), PHF6 (n=1) SF3B1 (n=1) and TP53 (n=1). These mutations were traced and quantified retrospectively. Data suggests that they were most probably present in the JAK2V617F positive clone. This again supports the theory of both clones being independent of each other.

Conclusions:

1) Co-occurrence of BCR-ABL1 and JAK2V617F is a very rare event (0.08%). 2) BCR-ABL1 and JAK2V617F represent two different clones. 3) Additional gene mutations are detected in 56% of these cases and all seem to be within the JAK2V617F positive clone. 4) Clinically, the BCR-ABL1 clone is easily controlled with TKI, however, the combined management of the JAK2V617F clone is more challenging especially when a fibrotic phase of the disease takes over. The long-term effect of JAK2-inhibitors in the management of these patients is yet to be established.