INTRODUCTION

Myeloproliferative neoplasm (MPN) is a clonal proliferation of one or more of myeloid lineage in the bone marrow. According to the World Health Organisation (WHO), MPN is classified into BCR-ABL positive chronic myeloid leukemia (CML) and BCR-ABL negative MPN (Ph-MPN) which are polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) (Swerdlow et al. 2017). Chronic myeloid leukemia is caused by reciprocal translocation of chromosomes 9 and 22 resulting in the characteristic Philadelphia chromosome (Ph+) forming BCR-ABL fusion oncoproteins which leads to autonomous cellular proliferation via activation of tyrosine kinase.

The Ph- MPN usually involves Janus Kinase 2 (JAK2) V617F mutation with approximately more than 90% occurrence in PV and to a lesser extent, 50% of all cases of MF and ET (Swerdlow et al. 2017). Janus kinase proteins are responsible for cellular signaling. However, due to somatic mutation of JH2 domain in JAK2 there is V617F substitution rendering constitutional activation of the JAK/STAT pathway causing uninhibited cell proliferation and survival (Hubbard 2018). Previous studies suggested that JAK2 V617F and BCR-ABL mutations were mutually exclusive (Pastore et al. 2013). Recent reports described some paradoxical coincidence of these two mutations (Bader & Dreiling 2019). This case report highlighted a rare case of MPN with coexistence of BCR-ABL and JAK2 V617F mutation.

CASE report

The patient was a 70-year-old man with known hypertension, dyslipidaemia and ischemic heart disease, presented with intermittent fever, weight loss and lethargy for a year. Clinical examination showed splenomegaly with no hepatomegaly or lymphadenopathy. His abdominal computed tomography (CT) scan demonstrated marked splenomegaly (22.7 cm).

Full blood count (FBC) showed high red blood cell count (7.9 x 1012/L), raised HCT (52.2%), normal hemoglobin level (15.2 g/dL) with microcytic indices (MCV 66.1 fl, MCH

19.2 pg), leukocytosis (15.3 x 109/L) and mild thrombocytosis (432 x 109/L). The peripheral blood smear revealed leukoerythroblastic picture with anisopoikilocytosis and hypochromic microcytic erythrocytes, pencil cells, tear drop cells and occasional target cells (Figure 1). White cell features included leukocytosis with neutrophilia, basophilia and occasional myelocytes with no blast cells was seen. Platelets increased with presence of large and giant forms.

Bone marrow aspiration and trephine biopsy (Figure 2) demonstrated hypercellular marrow with trilineage hyperplasia and grade 2 to 3 fibrosis (Swerdlow et al. 2017). The granulocytic series was increased with prominent eosinophilia. There was increased thickness of the immature myeloid cells of 5 to 6 cell thickness in the paratrabecular region in contrast to the normal 2 to 3 cell layers (Swerdlow et al. 2017). Megakaryocytes were frequent and arranged in clusters whilst some showed dwarf megakaryocytes and there was no excess of blasts seen. A diagnosis of MPN with fibrosis was made at this point of time.

His molecular cytogenetic study showed presence of BCR-ABL rearrangement in 29% of the cells after analysed by fluorescence in situ hybridisation (FISH) (Figure 3) on 200 nuclei which was sufficient to diagnose CML. This FISH signal pattern was unlike the common pattern, with one green, one red and two yellow signals found in CML. On the contrary, this pattern may suggest a variant type of deletion of the BCR and ABL fusion. Furthermore, conventional karyotyping showed normal male karyotype (46, XY) with only one metaphase spread available.

Interestingly, the peripheral blood for reverse transcriptase polymerase chain reaction (RT-PCR) BCR-ABL fusion gene  was negative whilst his peripheral blood for JAK2 V617F mutation was positive (Figure 4). Venesection was performed followed by regular oral Imatinib 400 mg daily and Hydroxyurea. After three  months of Imatinib and Hydroxyurea initiation, the complete hematological remission was not attained despite compliance to therapy. Fortunately, the cytogenetic remission was finally achieved after two years of treatment which was monitored via FISH using peripheral blood. Polymerase chain reaction method for monitoring purpose was not utilised in view of no primers available for the variant BCR-ABL transcript.

DISCUSSION

This case depicted a rare clinical entity in the MPN spectrum. World Health Organisation classified MPN into 2 major groups; (i) CML which is Ph/BCR-ABL positive MPN, and (ii) Ph/BCR-ABL negative MPN mostly involving JAK2 V617F mutation (Swerdlow et al. 2017). The hallmark of CML is the Philadephia chromosome caused by reciprocal translocation of chromosomes 9 and 22 resulting in the formation of BCR-ABL fusion oncogene and later oncoproteins p190, p210, or p230 depending on the breakpoints. In CML, the most commonly found oncoprotein is p210 transcript also known as the major fragments (Mousinho et al. 2018). This fusion oncoprotein leads to ABL tyrosine kinase activation causing unopposed proliferation of the immature and mature granulocytes leading to triphasic clinical course (chronic, accelerated, blast phase) if left untreated (Swerdlow et al. 2017).

On the contrary, the leukemogenic event of the Ph-negative MPN is the somatic point mutations in JAK2 gene, specifically JH2 domain with V617F mutation comprising approximately 95% of cases of PV and 50% cases of ET and MF. The JAK2 V617 mutation activated the JAK/STAT pathway leading to signal transduction, activating transcription thus promoting the cellular proliferation and restricted apoptosis of the affected cell lineages (Hubbard 2018).

The prevalence of double mutation of BCR-ABL and JAK2 V617F is 0.4% based on a multi-institutional study from the Bone Marrow Pathology Group (Soderquist et al. 2018). There have been reports illustrating the double mutation of BCR-ABL and JAK2 V617F may arise from a single cell clone (Hubbard 2018). However, others reported that there are independent clones harboring each mutation. Nonetheless, there is no definitive clonal analysis done to prove the theory (Bader & Dreiling 2019). 

On a clinical note, patient’s symptoms were non-specific to MPN subtypes. The patient’s initial manifestation was mainly constitutional with striking splenomegaly. Typically, CML patients are detected during chronic phase upon routine medical screening with hyperleukocytosis. To a lesser extent, some will present at accelerated or blast phase with anemic symptoms, leukostasis, infection, or bleeding. Major symptoms of PV include hyperviscosity syndrome including cardiovascular diseases such as stroke. Whilst in ET, usual presentation is asymptomatic with persistent thrombocytosis or symptoms of thrombosis or hemorrhage.

Based on previous studies, double mutation MPN typically affects elderly   population as in our case. According to Hummel et al. (2012) study, two out of three patients with double mutations were detected during routine medical check-up with persistent thrombocytosis and moderate leukocytosis leading to hematological review and MPN investigation. Imaging also revealed significant splenomegaly. Similarly, a case series by Mousinho et al. (2018) on two patients with double mutations showed that they were asymptomatic during their initial presentation and both of them were under the investigation for MPN due to sustained moderate thrombocytosis and leukocytosis. Interestingly, both patients did not have splenomegaly as evidenced by imaging modalities.

Morphologically, it was difficult to distinguish the specific types of MPN which explained why the case was concluded as MPN with fibrosis. Moreover, the cytogenetic and molecular results of the patient were not available upon morphological evaluation. There were no classical atypical bizarre megakaryocytes commonly found in ET. Nonetheless, the panmyelosis and megakaryocytic changes may suggest PV. However, the presence of fibrosis in this case cannot be attributed to either the end spectrum of MPN subtypes for instance Post PV-MF, Post ET-MF or fibrosis following CML because  there was no prior documentation. One of the morphological hints for possible coexistence of BCR-ABL and JAK2 V617F mutations was mixed bone marrow cytological features for example the presence of both dwarf and cloud-like forms of megakaryocytes based on previous study (Soderquist et al. 2018). 

According to a study by Hummel et al. (2012), the morphology of double mutations cases illustrated an expansion of the megakaryocytic series with atypical forms such as lagre, hyperchromatic and bulbous nuclei and they were arranged in clusters. Most of the cases also showed moderate to marked fibrosis when stained with the reticulin and trichrome stains which had similar finding in our patient. (Hummel et al. 2012).

Cytogenetically, the diagnostic FISH analysis of the bone marrow was done with specific probes for BCR-ABL (Vysis LSI BCR-ABL Dual Color Dual Fusion Translocation Probe) on 200 nuclei which demonstrated presence of 29% of BCR-ABL translocation.

Interestingly, the FISH signals were atypical, revealing only one yellow fusion signal, one red (normal chromosome 22) and one green signal (normal chromosome 9) termed 1F1R1G as opposed to the common one green, one red and two fusion signals pattern of BCR-ABL translocations in CML. The FISH result may depict the presence of BCR-ABL fusion deletion hence the atypical pattern (Švabek et al. 2018). Additionally, this case demonstrated that FISH analysis has the ability to detect subpopulation of cells with rare minor breakpoint of BCR-ABL translocations that may not be amplified by molecular methods. Some chromosomal changes are submicroscopic thus masking the translocation and could be revealed only by molecular analysis or by FISH (Haidary et al. 2019).

The RT-PCR was performed for the BCR-ABL transcripts identification but the result was negative. This incidence may be due to the unique FISH signal pattern mentioned previously which was caused by cryptic translocation (Švabek et al. 2018). According  to a study involving a case with simultaneous BCR-ABL and JAK2 V617F mutation, they detected e1a2 BCR-ABL transcripts instead of the expected major fragments (Bader & Dreiling 2019; Xu et al. 2013). Our laboratory used commercially available primers which were specific for major BCR-ABL fragments (b2a2, b3a2) but cannot detect variant BCR-ABL with rare breakpoints. Similarly, a study by Azma et al. (2012) revealed that the variant translocations of BCR-ABL cannot be detected via RT-PCR technique and they recommended advanced molecular methods such as sequencing to be performed in suspected variant cases.

With regards to the molecular genetic study, JAK2 V617F  mutation was determined by multiplex amplification refractory mutation system (ARMS PCR) (Applied Biosystem Gene AMP 9700) using allele-specific primers. The findings were presented as qualitative results showing mutant type or wild type of JAK2 V617F mutation. In this case, the JAK2 V617F mutation was confirmed by the amplified DNA of 229 and 279 bp which was heterozygous (Figure 4). Previous study suggested to consider CML cases that are associated with other MPN subtypes is a rather poor prognosis due to higher tendency to progress to MF and blast transformation (Soderquist et al. 2018; Lurlo et al. 2014; Xu et al. 2013). Some authors suggested that the WHO should amend the diagnostic criteria for MPN to consider this “double mutation” as a possible novel clinical entity (Bader & Dreiling 2019). The BCR-ABL and JAK2 V617F genetic combination also raise some issues regarding the best treatment strategy. Tyrosine kinase inhibitors such as Imatinib alone may not be sufficient to treat these complex cases unless it is used in concert with JAK2-inhibitor drugs (Yi & Kim 2019). Other adjunctive treatments including cytoreduction using Hydroxyurea may be needed to tailor to patient’s symptoms and cell counts. However, clinicians must be aware of profound drug-induced cytopenia which could be detrimental towards the patients.

CONCLUSION

In conclusion, it is imperative to scrutinise CML cases for concomitant JAK2 V617F mutation especially among patients with atypical clinical or laboratory findings. Therefore, close monitoring with clinical and ancillary technique especially FISH and molecular methods such as DNA sequencing are crucial to help achieve complete hematological, cytogenetic and deep molecular response alongside targeted therapy.