Co-occurrence of JAK2-V617 F mutation and BCR::ABL1 translocation in chronic myeloproliferative neoplasms: a potentially confounding genetic combination

Myeloproliferative neoplasms (MPNs) are classified into Philadelphia (Ph) chromosome–positive chronic myeloid leukemia (CML) and Ph-negative MPNs. BCR::ABL1 translocation is the key genetic event of CML, whereas JAK2/MPL/CALR mutations are molecular aberrations of Ph-negative MPNs. Despite initially considered mutually exclusive genetic aberrations, the co-occurrence of BCR::ABL1 and JAK2 has been reported in a limited number of cases. The two genetic alterations may be identified either at the same time or JAK2 aberration may be detected in patients with a previous CML treated with tyrosine kinase inhibitors or, finally, BCR::ABL1 translocation occurs in patients with a history of JAK2-positive MPN. This combination of genomic alterations is potentially confounding with clinical manifestations often misinterpreted either as disease progression or drug resistance, therefore leading to inappropriate patient’s treatment. Our systematic review aims to improve hematologist and pathologist knowledge on this rare subset of patients. Starting from the presentation of two additional cases from our routine daily practice, we focus mainly on clinical, laboratory, and bone marrow histological findings, which may represent useful clues of BCR::ABL1 and JAK2 co-occurrence. The interaction between JAK2 and BCR::ABL1 clones during the disease course as well as therapy and outcome are presented.


Introduction
Myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells with proliferation of one or more of the hematopoietic lineages, classified into distinct categories depending on different features, including the underlying genetic abnormalities (1)(2)(3).MPNs are classified into Philadelphia (Ph)-positive t(9;22) (q34.1;q11.2) chronic myeloid leukemia (CML) and Ph-negative MPNs.BCR::ABL1 gene translocation is the key molecular event defining CML.The reciprocal rearrangement and fusion of the BCR gene on chromosome 22 and ABL gene on chromosome 9 leads to the production of an oncoprotein that can be p190, p210, or p230 depending on the breakpoint of BCR::ABL.The presence of BCR:: ABL1 translocation is strictly associated with CML, being an essential requisite for CML development and diagnosis.On the other hand, mutations in the Janus kinase 2 (JAK2) gene on exon 14, mostly at codon 617 (JAK2 V617F) or other activating JAK2 mutations in exon 12 or, more rarely, mutations in the myeloproliferative leukemia (MPL) gene or in exon 9 of Calreticulin (CALR) gene are observed in Ph-negative MPNs, which may have different clinical and morphological features presenting as either polycythemia vera (PV), primary myelofibrosis (PMF), or essential thrombocythemia (ET).The gain-of-function JAK2 mutation leads to the activation of the JAK/STAT signaling pathway, resulting in cellular proliferation and resistance to apoptosis.The JAK2 V617F mutation is detected in PV (95% of cases), PMF (60%), and ET (50%) (1)(2)(3).Although BCR::ABL1 translocation and JAK2 mutation were initially considered to be mutually exclusive, more recently the simultaneous presence of these genetic alterations has been reported .The co-occurrence of JAK2 V617F mutation and BCR::ABL1 translocation in the same patient is a rare event with a frequency ranging from 0.2% to 2.5% according to different studies (21,38,54).The two genetic alterations may be identified either simultaneously or JAK2 mutation may be found in the setting of a previously diagnosed CML treated with a tyrosine kinase inhibitor (TKI) or finally BCR::ABL1 translocation may develop in patients with a long history of JAK2-positive MPN.Current scientific literature reports 85 cases of coexistence of BCR::ABL1 and JAK2 .Most reports are isolated cases or small case series and, given the paucity of data, clinical and pathological characteristics and prognosis of patients harboring concurrent BCR::ABL1 and JAK2 abnormalities have not been systematically evaluated.In the present report, starting from the presentation of two cases with a long history of JAK2-positive PMF subsequently developing CML with BCR::ABL1 translocation acquisition, we performed a systematic review with the purpose to improve our understanding of this particular setting of MPNs with co-occurring BCR::ABL1 translocation and JAK2 mutation, focusing on clinical and laboratory signs and on bone marrow (BM) morphological features, which may represent clues of coexistence of Ph-negative MPNs and CML.Interaction between JAK2 and BCR::ABL1 clones during the disease course is also discussed as well as treatment and outcome.

Case descriptions 2.1 Case 1
A 56-year-old woman presented to our center because of increasing white blood cell count (WBC 48.1 × 10 9 /L).The patient had a 13-year history of a Ph-negative, JAK2-positive MPN consistent with pre-fibrotic PMF.At the time of PMF diagnosis the patient had splenomegaly (spleen: 14 cm in diameter), with platelet (PTL) count up to 800 × 10 9 /L, whereas WBC and hemoglobin (Hb) level were within normal limits.Peripheral blood (PB) smear showed dacryocytes, poikilocytes, erythroblasts, and neutrophils with hyper-segmented nuclei.BM aspirate revealed giant megakaryocytes (MKs) and small MKs with hyperchromatic nuclei.BM biopsy showed an hypercellular marrow (80% cellularity) with reduced erythropoiesis and hyperplastic, normal-maturing granulopoiesis; variably sized MKs, including large elements with bulbous nuclei admixed with small-sized forms with hyperchromatic nuclei, were increased and showed loose and dense clusters (Figures 1, 2).Reticulin fibrosis grade 0 was observed.Chromosomal analysis demonstrated a normal female karyotype (46XX) and molecular testing revealed V617F mutation in JAK2 gene (allelic burden: 83.98%), whereas CALR and MPL mutations were absent as well as BCR::ABL1 translocation.Due to repeated cerebral thrombo-embolic episodes, the patient received antiaggregant therapy with acetylsalicylic acid (ASA) and hydroxyurea (HU), followed 8 years later by interferon (IFN) alpha with good control of the disease.
At the time of referral to our center, blood tests showed progressively increasing leukocytosis up to 48 × 10 9 /L, with normal Hb level (13.3 g/dL) and normal PTL count (416 × 10 9 / L).Chromosomal analysis demonstrated Ph chromosome; fluorescent in-situ hybridization (FISH) analysis detected a BCR:: ABL1 fusion in 99% of interphase nuclei indicative of t(9:22)(q34; q11) translocation; quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) for BCR::ABL1 fusion transcripts demonstrated 177% BCR::ABL1 fusion transcript of p210 variant.At this time, JAK2 V617F remained positive with an allelic burden of 21.48%; MPL and CALR remained negative.BM biopsy revealed hypercellularity with high myeloid: erythroid ratio and increased forms of intermediate maturation (metamyelocytes and band neutrophils); MKs were increased in number and predominantly small sized with scarce tendency to form loose clusters (Figure 3); no increase in CD34-positive hematopoietic precursors was noted.Reticulin fibrosis grade 0 was present.BM histopathology combined with clinical data and molecular results were suggestive of CML chronic phase (CP) with concomitant JAK2 mutation and BCR:: ABL1 rearrangement.The patient was started on TKI Imatinib at 400 mg/die with a complete cytogenetic response (CCyR) and BCR:: ABL1 transcript lower than 10%, 3 months later.Despite a good response of the Ph-positive MPN, JAK2 V617 allelic burden increased up to 89.83%.The marrow was hypercellular with granulocytic predominance and an increase in variably sized MKs with clustering.Grade 0 fibrosis was present.The pathological High power view of BM highlighting a dense cluster of variably-sized MKs (Hematoxylin and eosin, 400× magnification).features were consistent with persistency of the JAK2-positive MPN.IFN-alpha (3 MU/every 10 days) was therefore reintroduced in association with Imatinib (400 mg/die) and ASA, obtaining a good control of the disease with deep molecular response (DMR) of CML, but JAK2 still positive at a constant allelic burden (20%) at 7 years from CML diagnosis.

Case 2
An 82-year-old woman was referred to our center because of increasing leukocytosis (WBC 53.78 × 10 9 /L) with mild anemia (Hb 10.7 g/dL) and normal PTL count (399 × 10 9 /L).The patient had been diagnosed JAK2 V617F-positive (allelic burden: 27%) PMF 7 year earlier at another institution and had been treated with HU for few weeks only, due to drug intolerance (cutaneous edema).The patient had voluntarily interrupted hematologic follow-up for approximately 6 years before presenting to our center.At the time of initial PMF diagnosis, BCR::ABL1 was negative.At the time the patient was referred to us, BM biopsy showed a markedly hypercellular marrow (95% cellularity) with significantly increased myeloid to erythroid ratio, normally maturing myeloid lineage and no increase in CD34-positive hematopoietic precursors.The erythroid lineage was markedly reduced as well as MKs, which were predominantly of small size.Grade 3 reticulin fibrosis was present.Quantitative RT-PCR identified BCR::ABL1 p210 fusion gene (95%).Karyotyping showed 46XX and cytogenetic analysis revealed t(9;22)(q34;q11).FISH analysis identified BCR::ABL1 fusion in 90% of interphase nuclei.JAK2 V617F mutation was confirmed (allelic burden: 30%).Splenomegaly (spleen: 14.5 cm in diameter) was identified.Therefore, the final diagnosis was CML in CP, arising in the background of a JAK2-positive MPN with grade 3 reticulin fibrosis consistent with fibrotic PMF.The patient was started on Imatinib at 100 mg/die, subsequently increased to 200 mg/die.Four months later, BCR::ABL1 fusion gene declined to 18%.Despite a discrete CML control, due to increasing thrombocytosis (PTL count > 1.000 × 10 9 /L), a new BM biopsy was performed 5 months later.The marrow was hypercellular (80% cellularity) with a significant prevalence of the myeloid lineage and an increase in variably sized MKs even with hyperchromatic nuclei and a moderate tendency of dense clustering.Grade 3 reticulin fibrosis was present.JAK2 V617F mutation was confirmed with 15% allelic burden.The pathological features were in keeping with persistency of the JAK2-positive MPN with fibrosis.Molecular analysis showed persistence of BCR::ABL1 fusion gene (15%).In addition to Imatinib (200 mg/die), the patient was put on recombinant IFN alpha-2b (750.000U/die).Twelve months later, PTL count decreased to 500 × 10 9 /L with 5% JAK2 allelic burden and a major molecular response (MR3) was achieved (BCR-ABL1 fusion gene: 0.07%).The patient is still on treatment with both drugs with a good control of the disease at 24 months from CML diagnosis.negative JAK2-positive MPN; (3) retrospective, observational casecontrol studies, case reports and/or series, literature review.The exclusion criteria were as follows: (1) studies not published in English; (2) lack of concurrent JAK2 mutation and BCR::ABL1 translocation (3) clinically non-relevant, incidentally detected BCR-ABL1 translocation, in the context of a Ph-negative JAK2-positive MPN; (4) clinically non-relevant, incidentally detected JAK2 mutation, in the context of a Ph-positive MPN.Two independent reviewers (M.Zanelli, AB) identified papers on the basis of title, abstract, and key words; then, they evaluated whether the selected papers met the inclusion criteria by reading the article full texts.Moreover, reference lists from each retrieved article were checked to find additional reports.From selected papers, the following information was collected: author's surname, year of publication, patient's age and sex, disease course, clinical and laboratory findings suggestive of a second coexistent MPN, BM histological features suggestive of a second coexistent MPN, interaction between JAK2 and BCR::ABL1 clones, treatment, and outcome.A third independent reviewer (AS) revised all collected results and solved discrepancies.
4 Results of literature data including our cases

Study selection and characteristics
The final literature search, performed in August 2023, identified 678 potential items of interest (Figure 4).After removing irrelevant publications (615), 65 records were further analyzed.Fifteen of these were excluded according to exclusion criteria, while 50 fulltext articles were assessed for eligibility and included into qualitative synthesis.The included articles were case reports/case series (4-53).

Epidemiology
To date, 50 studies have analyzed MPNs with concomitant JAK2 and BCR::ABL1, with a total of 85 cases .Including our two cases, a total of 87 cases have been described so far.Patient characteristics, course of the disease, clinical and laboratory data, as well as BM histological features suggestive of concomitant JAK2 and BCR::ABL1, interaction between JAK2 and BCR::ABL1 clones, treatment and outcome, are summarized in Supplementary Table S1.
Of note, our case n°1 with CML (CP) histology was re-biopsied after having achieved a CCyR of CML with TKI; at this time, JAK2 allele burden increased and histology switched to a PMF phenotype.Our case n°2 was re-biopsied at the time of CML partial MR and the histology switched from CML+fibrosis to a phenotype suggestive of JAK2+MPN with fibrosis, in particular the MKs from small-sized, non-clustering forms changed to variably sized, clustering MKs.

Group 1: BCR::ABL1 and JAK2 clone interaction
In Group 1, the following interaction between BCR::ABL1 and JAK2 clones were observed at the time of CML occurrence and during the course of the disease: the majority of cases (18/43; 41.86%) showed an opposite growth of the two genomic alterations with BCR::ABL1 decrease under TKI and JAK2 Graphic showing the frequency of cases with opposite growth of BCR::ABL1 and JAK2 clones under TKI treatment in the three different groups of patients.

Group 1: treatment and outcome
In this paragraph, as well as in the following paragraphs on treatment for Groups 2 and 3, the type of Ph-negative MPN preceding CML is indicated in brackets for each single case.

Group 2 (CML preceding Ph-negative MPN): clinical symptoms and laboratory signs at second disease (Ph-negative MPN) occurrence
In Group 2 (20 cases), the Ph-negative MPNs occurring as second disease after CML, were distributed as follows: seven cases were JAK2+MPN, NOS; six cases PMF; five cases PV and two cases ET.
Laboratory signs detected in the two cases of ET occurring after CML were as follows: PTL increase in 2/2 cases despite CCyR of CML (25); WBC increase in 1/2 despite CCyR of CML (25).
4.8 Group 2: histological features of BM biopsy performed at second disease (Phnegative MPN) occurrence BM biopsy performed at the time of Ph-negative MPN occurrence showed different features according to the type of Phnegative MPN.
BM biopsy performed in six cases of PMF occurring after CML, showed the typical PMF histology (4,9,10,15,46,48).Of note, in 2/ 6 cases, PMF histological features became clearly evident after CMR of CML with TKI; at the time PMF histology was detected, BCR:: ABL1 was negative and JAK2 positive, at a constant allele burden in one case (4) and at an increased allele burden in the other case (9).Moreover, in 1/2 cases with PMF histology obvious after TKI, the presence of fibrosis at initial CML diagnosis could have been a possible clue for PMF, although fibrosis alone is not sufficient for PMF diagnosis as fibrosis may be found even in CML; in this case, BCR::ABL1 and JAK2 were both present from initial CML diagnosis, however PMF features became obvious after CMR of CML (4).
BM biopsy in five cases of JAK2-positive MPN, NOS occurring after CML with available histology, showed histological features consistent with Ph-negative MPN in all cases, in particular increase of large, clustering MKs was detected (15,22,38,39).Of note, in 2/5 cases Ph-negative phenotype became evident after TKI, with BCR:: ABL1 decrease and JAK2 increase (15,22); in addition, in one of these two cases, BM performed at the time of initial CML diagnosis retrospectively revised showed not only MKs with hypo-lobate nuclei, typical of CML, but even large and occasionally clustering MKs with bulbous nuclei (22).

Group 3: histological features of BM biopsy
In Group 3, BM histology showed the following features subdivided according to the type of Ph-negative MPN associated with CML.
BM biopsy data were incomplete in two cases of coexistent CML +PPV-MF and only grades 1 and 2 fibrosis was reported (37).

Discussion
JAK2 and BCR::ABL1 are driver genomic alterations leading to different types of MPNs.Despite historically considered mutually exclusive driver mutant genes, the coexistence of JAK2 V617 (or rarely JAK2 exon 12) and BCR::ABL1 has been reported mainly as isolated case descriptions or small case series .In 2016, Martin-Cabrera et al. reported the incidence of 0.2% of BCR:: ABL1/JAK2 V617F double-positive cases in a large cohort of 10.875 MPN cases (54).In 2018, Soderquist et al. estimated a frequency of 0.4% among 1570 MPNs tested for both genomic alterations (39).The previously reported higher frequency of 2.5% by Pieri et al. might be overestimated and likely related to the different cohorts selected in these studies as Pieri et al. evaluated only BCR::ABL1-positive cases, whereas Martin-Cabrera et al. and Soderquist et al. selected all MPN cases (21,39,54).Rare cases with coexistence of CALR and BCR::ABL1 have been reported, but their discussion goes beyond the scope of the present paper (55, 56).
In our systematic review, we selected cases with clinically relevant dual driver mutations representing co-existent Phpositive CML and Ph-negative MPN with JAK2 mutation (4-53).It is well-known that very low level of BCR::ABL1 transcripts may be found in some healthy individuals without clinical features of CML (57).Some Ph-negative MPNs, particularly ET, have been detected to have very low level of BCR::ABL1 transcripts of no clinical and pathologic significance (38) and likewise, the presence of JAK2 mutation, usually at low allelic burden, in the context of a CML may not change the clinical features of CML (58,59).These situations likely represent examples of clonal hematopoiesis of indeterminate potential (CHIP) (38,59).
In our systematic review, the majority of BCR::ABL1/JAK2 V617F double-positive cases fell into the group of Ph-negative MPN preceding CML (49.42%), followed by the group of CML and Ph-negative MPN occurring simultaneously (27.58%) and by the group of CML preceding Ph-negative MPN (22.98%).When Phnegative MPN preceded CML, PV was the most commonly involved disease (20/43; 46.51%), followed by ET (12/43; 27.90%) and PMF (8/43; 18.60%).On the other hand, when CML preceded Ph-negative MPN, the most frequently detected was MPN, NOS (7/ 20; 35%) followed by PMF (6/20; 30%), PV (5/20; 25%) and ET (2/ 20; 10%).When Ph-negative MPN and CML occurred simultaneously, PMF was the most frequent disease (10/24; 41.66%) followed by ET (6/24; 25%) and by MPN, NOS (3/24; 12.5%) and PV (3/24; 12.5%) (see Table 1).Of note, MPNs preceding CML are more often non-fibrotic forms (PV, ET), whereas when CML is diagnosed before MPN, fibrosis is observed in most MPN cases at the time of diagnosis.It might be speculated that the onset of a MPN in the context of a CML under TKIs may be masked by the myelosuppressive effect of TKIs and, hence, only the fibrotic evolution gives sufficient force for the clinical and histological emergence of the MPN clone.On the other hand, the different distribution of MPNs may represent a real biological difference.More studies on large number of cases are needed for the understanding of this issue.
Hematologists and pathologists may face this confounding combination of genomic alterations, which can lead to clinical manifestations easily misinterpreted either as disease progression or resistance to treatment, hence, causing an inadequate management of the patient.
A modification in symptoms or laboratory data, during the course of Ph-negative MPNs, is commonly suspected to represent a sign of disease progression as Ph-negative MPNs may undergo myelofibrotic progression or transformation to acute leukemia or, more rarely, develop unusual types of progression with the occurrence of either monocytosis with the acquisition of (myelodysplastic/myeloproliferative) MDS/MPN-like features or leukocytosis with the acquisition of chronic neutrophilic leukemia-like features (1)(2)(3)60).Likewise, a change in clinical manifestations in patients affected by CML is often interpreted as drug-resistance or a possible evolution to acute leukemia (1-3).However, when facing a change in the clinical course of either CML or Ph-negative MPN, hematologists and pathologists need to exclude even the possibility of a combination of BCR::ABL1 translocation and JAK2 mutation.
Clues to the likelihood of the coexistence of CML and Phnegative MPN may be obtained from laboratory exams, modification in clinical symptoms as well from BM morphology.The appearance of constitutional symptoms, splenomegaly, PTL increase, RBC increase or cytopenia in patients with CML in MR should lead clinicians to rule out a coexisting Ph-negative MPN and mutations of JAK2, MPL, CALR should be evaluated.Likewise, in patients affected by a known Ph-negative MPN, an unexpected WBC or PTL increase or a change in symptoms should alert the clinician to exclude the emergence of CML.BM histology may reveal a shift of phenotype from CML to Ph-negative MPN or vice versa during treatment.It is well known that MK size and clustering may allow the distinction between the different types of MPNs with small-sized, hypo-lobate, non-clustering MKs more commonly found in CML and large, hyper-lobate clustering forms more typical of Ph-negative MPNs.The appearance of large hyperlobate and clustering MKs with or without fibrosis as well as the identification of MKs with hybrid features (both small-sized, hypolobate and large-sized hyper-lobate forms) in the marrow of patients with a known CML should prompt testing for mutated JAK2/CALR/MPL.On the other hand, patients with a known Phnegative MPN showing BM histological features reminiscent of CML should undergo cytogenetic and molecular testing to identify Ph chromosome and BCR::ABL1 transcripts.Routinely performed BM biopsies during the course of Ph-negative and Ph-positive MPNs are essential to identify a possible shift of phenotype.The role of well-trained hematopathologists in the evaluation of BM biopsies is essential for the correct diagnosis; moreover, a strict integration between pathological features and molecular data is critical in particular in this setting of patients.
The clonal composition of MPNs harboring both BRC::ABL1 and JAK2 V617F has been discussed in various reports, with some studies favoring the presence of two independent clones (8) and others supporting the hypothesis that the two genetic events occur in the same clone (15)(16)(17).Despite being rather evident that each of the two theories may be possible, the presence of two independent clones seems to represent the more common event.Interestingly, we observed an opposite growth of BCR::ABL1 and JAK2 under TKI treatment, with BCR::ABL1 decrease and JAK2 increase in the majority of patients of our systematic review (81.8% in concomitant CML and Ph-negative MPN; 65% in CML preceding Ph-negative MPN; 41.86% in MPN preceding CML).The increase in JAK2 mutation burden during Ph-positive clone suppression by TKI treatment favors the hypothesis of two independent clones.Unfortunately, data on outcome in this subset of patients carrying both BCR::ABL1 and JAK2 are often incomplete or unavailable; therefore, it is rather difficult to draw conclusions on the optimal treatment.Whether a TKI associated with JAK2-inhibitor represents an optimal therapeutic strategy requires further evaluation.In those patients with dual anomalies, CML seems to be rather easy to manage, with often a good response of BCR::ABL1 burden to different types of TKIs.The JAK2-positive MPNs have been treated with various therapies including HU, IFN, anagrelide and JAK2-inhibitors such as ruxolitinib, but the paucity of followup data does not allow to understand which is the optimal management strategy.Promising results with allo-SCT have been recently reported (see Table 3, Supplementary Materials), although data on a larger number of cases are needed (46).

Funding
The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.

FIGURE 1
FIGURE 1Medium power view of BM biopsy showing hypercellular marrow with normal-maturing prevalent granulopoiesis and clusters of variably sized MKs including large forms with bulbous nuclei (Hematoxylin and eosin, 200x magnification).

FIGURE 3
FIGURE 3Medium power view of BM biopsy showing hypercellularity with high myeloid: erythroid ratio and mainly small MKs with scarce tendency to form clusters (Hematoxylin and eosin, 200× magnification).

FIGURE 4 PRISMA
FIGURE 4    PRISMA flow chart of literature search.

TABLE 1
Distribution of Ph-negative MPNs in the three groups of patients with concomitant or sequential JAK2-positive MPN and CML (see Supplementary Material).

TABLE 2
Data on different TKIs used in concomitant or sequential JAK2-positive MPNs and CML (see Supplementary Material).