Two cases of myeloproliferative neoplasm with a concurrent JAK2 (V617F) mutation and BCR/ABL translocation without chronic myelogenous leukemia phenotype acquisition during hydroxyurea treatment.

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Myeloproliferative neoplasms (MPNs) are categorized on the basis of BCR/ABL translocation occurrence as either Philadelphia-positive CML or Philadelphia-negative MPNs (Ph-MPNs). MPNs include polycythaemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF) [1]. The JAK2V617F mutation, which is specific for Ph-MPN, occurs in more than 95% PV cases and in approximately 50% ET and PMF cases [1]. The 2008 revision of the WHO classifies MPNs into 2 categories, BCR/ABL positive CML and Ph-MPNs, on the basis of the presence or absence of the Philadelphia chromosome [2].

Although the 2008 WHO classification does not include MPNs with more than 1 genetic aberration as a distinct disease entity, some cases with coexistence of JAK2V617F mutation and BCR/ABL translocation have been recently reported and up to 28 cases have been previously reported [3-20]. Among these reports, the majority of the patients either had pre-existing BCR/ABL-positive CML and developed JAK2V617F mutation while undergoing tyrosine kinase inhibitor treatment [4-7] or developed BCR/ABL-positive CML with a pre-existing JAK2V617F mutation-positive MPN [8-12]. In contrast, a small number of patients showed simultaneous occurrence of both JAK2V617F mutation and BCR/ABL translocation, with a CML phenotype in the bone marrow (BM) with development of symptoms or morphology associated with JAK2V617F mutation, and with MPN only after imatinib treatment [3, 6, 13-15]. We report 2 MPN cases that were simultaneously positive for both JAK2V617F mutation and BCR/ABL translocation and that did not acquire the CML phenotype during hydroxyurea (Korea United Pharm, Seoul, Korea) treatment.

Case 1 was a 36-yr-old man who was admitted in November 2009 with severe thrombocytosis. The patient's hemogram results at admission were as follows: white blood cells, 9.4×109/L, Hb, 13.8 g/dL, and platelets, 830×109/L. The peripheral blood smear (PBS) and BM biopsy revealed a marked increase in the platelet and the clustered megakaryocyte numbers (9.2/high power field) without evidence of dysplasia. Allele-specific PCR for JAK2V617F mutation detection and reverse-transcriptase PCR (RT-PCR) for BCR/ABL fusion transcript detection using custom-designed primers was performed. The PCR and the RT-PCR analyses showed that the patient was positive for JAK2V617F heterozygous mutation and the BCR/ABL fusion transcript (b3a2 type), respectively.

Subsequently, the JAK2V617F mutation allele burden was quantified by real-time quantitative PCR using JAK2 MutaQuant™ (Ipsogen, Marseille, France). The JAK2V617F mutation allele burden was calculated as the percentage of the V617F copy number to the sum of V617F and wild-type copy number. The JAK2V617F mutation allele burden was quantified to be 27.91% (7,200/25,800) at diagnosis. The major BCR/ABL fusion transcripts quantitation was also performed by real-time PCR using the LightCycler t(9;22) Quantification kit (Roche Diagnostics, Mannheim, Germany) and the normalized copy number (NCN) was 0.02 at diagnosis. The patient's karyotype was determined to be 46,XY [20]; however, his interphase FISH analysis result was nuc ish(ABL1x3,BCRx2)(ABL1 con BCRx1)[61/200], representing cryptic BCR/ABL fusion on der(22)t(9;22) in 30.5% of the total cells.

Thus, on the basis of these findings, the patient was diagnosed to have ET with a major BCR/ABL fusion transcript. The patient was treated with hydroxyurea and the initial response during the first year of treatment was promising (BCR/ABL fusion transcript maintained in the range of 0.005 NCN to 0.01 NCN). However, despite continuing treatment, the number of BCR/ABL fusion transcripts increased to 5.0 NCN in the second year of follow-up, which indicated treatment failure. Interestingly, the patient did not show morphological evidence of CML during the follow-up period.

Case 2 was a 58-yr-old man diagnosed with leukocytosis and splenomegaly on admission. The patient's hemogram results at admission were as follows: white blood cells, 19.7×109/L, Hb, 13.0 g/dL, and platelets, 285×109/L. The PBS showed an occasional presence of tear-drop cells and immature granulocytes with blasts (Fig. 1A). The BM biopsy showed extensive myelofibrosis (grade 2-3) with a cellularity of 90% and an increased number of dysplastic megakaryocytes (Fig. 1B). The myelofibrosis was demonstrated by the reticulin silver stain (Fig. 1C). At diagnosis, the JAK2V617F mutation analysis showed a heterozygous mutation (Fig. 1D) and the JAK2V617F mutation allele burden was quantified to be 69.66% (2,640/3,790). The RT-PCR analysis revealed the presence of BCR/ABL fusion transcript (b3a2 type) (Fig. 1E). The patient's karyotype was determined to be 46,XY, t(9;22)(q34;q11.2)[4]/46,XY[16]. The quantification result for the major BCR/ABL fusion transcript in BM was found to be 1.0 NCN at diagnosis, which was 50-fold higher than that of case 1. The patient was treated with hydroxyurea for 6 months. However, the BCR/ABL fusion transcript levels remained at the levels at diagnosis (1.0-1.6 NCN). Similar to the findings for case 1, the morphological evidence of CML was not evident during hydroxyurea treatment. The drug treatment was changed to dasatinib and after 7 months of dasatinib treatment the patient did not show conversion, which was indicative of successful treatment.

Fig. 1

The hematological and molecular characteristics of case 2. (A) A peripheral blood smear revealed tear-drop cells, immature granulocytes, and blasts (Wright stain, ×400). (B) The patient's bone marrow biopsy showed extensive myelofibrosis (H&E stain, ×100), demonstrated by reticulin silver stain (C, ×100) with cellularity of 90% and increased dysplastic megakaryocytes. (D) Mutational analysis of JAK2V617F revealed bands that corresponded to both the mutant and wild-type JAK2 (sized 299 bp and 229 bp, respectively). (E) Along with the myeloproliferative neoplasms (MPNs) diagnosis tests, reverse transcriptase (RT)-PCR analysis of BCR/ABL fusion transcripts was performed and a single 305-bp band, representing the major BCR/ABL translocation (b3a2 type), was obtained.

Of the previously reported 28 cases with both JAK2V617F mutation and BCR/ABL translocation, 15 patients had BCR/ABL-positive CML and acquired a JAK2V617F-positive MPN phenotype after tyrosine kinase inhibitor treatment. Although 4 out of the 15 patients were diagnosed with CML and were shown to possess JAK2V617F mutation, 3 of the 4 patients did not show a morphology associated with JAK2V617F mutation-positive MPN until after treatment with imatinib. In contrast, 9 of the 28 patients showed JAK2V617F mutation-positive MPN phenotype at initial diagnosis and developed BCR/ABL-positive CML during hydroxyurea treatment. Finally, at initial diagnosis, 4 patients showed mixed phenotype associated with both BCR/ABL-positive CML and JAK2V617F mutation-positive MPN [3].

In recent studies, 2 hypotheses have been proposed to explain the coexistence of both BCR/ABL translocation and JAK2V617F mutation in some patients. The first hypothesis which has been favored in the several literatures proposes that a single clone possesses one aberration and the patient's phenotype (e.g. CML feature) is dependent on the dominant clone (e.g. BCR/ABL translocation positive) which is determined by the selective pressure exerted by the specific treatment (e.g. hydoxyurea) prescribed for the other clone (e.g. JAK2V617F mutation positive) [4-12]. The second hypothesis proposes that a single clone concurrently possesses both the BCR/ABL translocation and JAK2V617F mutation [3, 13-15]. This hypothesis was supported by a recent study, which reported that the BCR/ABL translocation occurred in a pre-existing JAK2V617F mutation-positive clone [18]. The data obtained in this study supports the second hypothesis as shown by the detection of concurrent JAK2V617F mutation and BCR/ABL translocation at the initial diagnosis of MPN, and the lack of phenotype switch, especially to the CML phenotype, during hydroxyurea treatment. Hence, a comprehensive molecular genetic analysis is needed to elucidate the pathogenesis of these hematological chimeras.

In addition, the 2 patients showed different outcomes according to both the initial level of BCR/ABL fusion transcripts and the introduction of a tyrosine kinase inhibitor during the hydroxyurea treatment. The patient with low initial BCR/ABL fusion transcript levels experienced a relatively good initial response to hydroxyurea treatment, although the treatment failed 2 years later. In contrast, the patient with high BCR/ABL fusion transcript levels did not initially respond well to hydroxyurea treatment, but a dramatic response was achieved after a treatment change to dasatinib. On the basis of these findings, we speculate that treatment with a tyrosine kinase inhibitor can be effective and therefore recommend this approach in JAK2V617F-positive MPN patients with a concurrent BCR/ABL translocation, particularly if the initial BCR/ABL fusion transcript level is high.

In conclusion, we report 2 cases of MPN with concurrent JAK2V617F mutation and BCR/ABL translocation without CML phenotype acquisition during the hydroxyurea treatment. Treatment with tyrosine kinase inhibitors can be effective, particularly if the initial BCR/ABL fusion transcript level is high in these patients. Further molecular genetic analysis is needed to elucidate the pathogenesis of these hematological chimeras.