Epstein-Barr virus-negative aggressive natural killer-cell leukaemia with high P-glycoprotein activity and phosphorylated extracellular signal-regulated protein kinases 1 and 2

Aggressive natural killer-cell leukaemia (ANKL) is a rare type of disease with fulminant course and poor outcome. The disease is more prevalent among Asians than in other ethnic groups and shows strong association with Epstein-Barr virus (EBV) and P-glycoprotein (P-gp) expression associated with multidrug resistance. Here we present a case of a 47 year old Caucasian female with a prior medical history of azathioprine treated ulcerative colitis who developed EBV-negative form of ANKL. The patient presented with hepatosplenomegaly, fever and nausea with peripheral blood and bone marrow infiltration with up to 70% of atypical lymphoid cells positive for cCD3, CD2, CD7, CD56, CD38, CD45, TIA1 and granzyme B, and negative for sCD3, CD4, CD5, CD8, CD34 and CD123 indicative of ANKL. Neoplastic CD56+ NK-cells showed high level of P-glycoprotein expression and activity, but also strong expression of phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) MAP kinase. The patient was treated with an intensive polychemotherapy regimen designed for treatment of acute lymphoblastic leukaemia, but one month after admission developed sepsis, coma and died of cardiorespiratory arrest. We present additional evidence that, except for the immunophenotype, leukaemic NK-cells resemble normal NK-cells in terms of P-gp functional capacity and expression of phosphorylated ERK1/2 signalling molecule. In that sense drugs that block P-glycoprotein activity and activated signalling pathways might represent new means for targeted therapy.


Introduction
Mature natural killer (NK) malignancies, according to the World Health Organization classification, comprise extranodal NK/T-cell lymphoma, nasal type (ENKL) and aggressive NK-cell leukaemia (ANKL). 1,2 These are rare types of neoplasms, particularly in Western countries, but with somewhat higher incidence rate in Asian and Central and South American population. Both entities have a highly aggressive clinical course with very poor survival rates. 3 Fernandez et al. 4 and Koizumi et al. 5 were the first to report and give evidence of a new clinical entity named aggressive NK-cell leukaemia. Bone marrow and peripheral blood are common sites of involvement, with equal incidence in males and females. Median age of patients diagnosed with ANKL is between 30 and 40 years. Common symptoms comprise fever, anaemia, hepatosplenomegaly, disseminated intravascular coagulation and lymphadenopathy. 6 Survival rates are extremely low, measuring in weeks and not exceeding 2 years (median 2 months).

Case Report
Further analysis concentrated on P-gp and phosphorylated signalling molecules in CD56+ neoplastic cells ( Figure 2). P-gp activity was assessed using Rhodamine 123 and verapamil as an inhibitor. This functional test demonstrated high P-gp transport activity (ratio of fluorescence intensity, RFI=16.54) which was in concordance with high membrane P-gp expression (D=0.63, Kolmogorov-Smirnov test) by Mrk16 antibody staining (Kamiya Biomedical Company). Monoclonal antibody staining of cytoplasmic phosphorylated molecules Akt, ERK1/2 and p38 revealed strong phosphorylated ERK1/2 expression only (D=0.62, Kolmogorov-Smirnov test).
Enzyme-linked immuno-sorbent assay testing showed negative serum EBV VCA IgM and EBV EA IgG, and positive EBV VCA IgG and EBNA IgG, which was indicative of past EBV infection. In situ hybridisation for EBV-encoded early small RNA1 (EBER-1) in bone marrow neoplastic cells was negative, as well as EBER-1 staining in neoplastic cells found in patient's gastric mucosa.
Upon diagnosis of aggressive NK-cell leukaemia, the patient was treated with a chemotherapy protocol designed for treatment of acute lymphoblastic leukaemia in younger patients consisting of steroids, vincristine, cyclophosphamide, danorubicine and repeated administration of asparaginase. Initially, the patient responded favourably, leukocyte counts dropped, hepatosplenomegaly disappeared and coagulation parameters normalized. During pancytopaenia, signs of meningeal infiltration with leukaemia developed. The patient was treated with repeated intrathecal administration of methotrexate, cytarabine and steroids without improvement. Coma and sepsis ensued and the patient died one month after being admitted to our hospital.

Discussion
As mentioned above, aggressive NK-cell leukaemia is uncommon disease, but occurs with higher frequency in Asians and Central/South Americans than in other ethnic groups. The vast majority of such cases are associated with EBV which is found in clonal episomal form implying active aetiologic role in NK-cell-derived malignancy development. 12 Here we emphasize the clinical and scientific importance of reporting much rarely EBV-negative form of ANKL in Caucasian patient. Namely, 13-15% of all ANKL cases are EBV-negative, raising certain suspicions in regards of proposed pathogenic mechanisms. 13,14 Consequently, presumptions of different clinical course and outcomes of patients with EBV-positive and EBV-negative ANKL were made. The published reports on this issue show conflicting results: one group reported no significant prognostic value of EBV-positivity, 13 whereas the other presented evidence of significantly longer survival of EBV-negative ANKL patients in comparison to EBV-positive ones (11.5 vs 1.5 months, respectively). 14 Obviously, larger cohorts of patients will be more conclusive, since both reports include only two EBV-negative ANKL cases. The patient we are reporting had a very aggressive and rapid clinical course despite EBV-negativity.
Taking into account the poor outcome of aggressive NK-cell leukaemia (median survival <2 months), it is important to improve knowledge of pathophysiology of this disease and particularly of the biology of leukaemic NK cells. In this respect, it is well known that NK cells in peripheral blood of healthy individuals have high P-gp expression and activity, the highest of all lymphoid cells. 11,12 Even though it is known that P-gp acts as a protector of haematopoietic stem cells against toxins, its role in mature NK cells still needs to be elucidated. Our case confirms a strong P-gp activity and expression in malignant NK-cells. Since MDR-1 protein is involved in multidrug resistance mechanism, this might at least partially explain why ANKL is so resistant to chemotherapy. 15 Accordingly, ANKL therapy protocols could be created in a way that they combine P-gp activity modulators and standard chemotherapy. Alternatively, they could avoid chemotherapeutics known to be Pgp substrates.
Furthermore, signalling pathways are very important in tumorigenesis. Utilisation and constitutive activation of certain signalling molecules enable malignant leukaemic cells to suppress normal haematopoiesis and continu-Case Report ously proliferate. Peripheral blood NK cells and NK cell lines demonstrate ERK1/2 (p44/p42 mitogen-activated protein kinase) phosphorylation. This pathway plays critical role in NKcell cytotoxicity; it drives lytic activity and mobilisation of granzyme B. [16][17][18] However, it has been proposed that there are two different pathways responsible for ERK1/2 activation. Sky/Zap70 -PI3K -Rac -Pak -MEK -ERK cascade regulates human PB NK cell cytolysis, while Ras -Raf -MEK -ERK cascade regulates growth, survival and gene expression in human NK LDGL. 19,20 Constitutive activation of ERK1/2 molecule was found in our patient's ANKL cells, confirming previous statements, although it remained unclear which upstream mediators led to ERK1/2 activation. Thus, the use of signalling pathway inhibitors might prove to be useful in elucidation of upstream signalling pathway in ANKL.
Patient's previous medical history revealed UC azathioprine therapy during the period of 17 months. It has been reported that patients who receive such treatment are at an increased risk of developing colorectal cancer. 21 However, association of increased risk of lymphoma with immunosuppressive therapy of UC has not been unambiguously demonstrated. 22,23 The meta-analysis performed by Kandiel et al. 24 showed an approximately fourfold increased risk of lymphoma in UC patients treated with azathioprine. Caution is neces-sary with interpretation of these results, since an increased risk could be a consequence of the severity of the disease itself, or combination of both.

Conclusions
In conclusion, aggressive NK-cell leukaemia is chemotherapy resistant disease with extremely poor prognosis. However, leukaemic NK-cells resemble normal NK-cells in terms of P-gp functional capacity and expression of phosphorylated ERK1/2 signalling molecule. In this regard, drugs that block P-glycoprotein activity and activated signalling pathways might represent new means for targeted therapy.