ANKL Is Strongly EBV-Related, Frequent In Chromosome Aberrations and Gene Mutations, LMP2A/LMP1-CDK6-MDM2-CD44-Positive and Might Be Sensitive To L-Asparaginase-Containing Regimen

Aggressive natural killer leukemia (ANKL), previously being classified as large granular lymphocyte (LGL) leukemia or malignant histiocytosis, is a rare subset neoplasm with high aggressive clinical course. Until now, apart from clinical feature, the frequent chromosome aberrations, somatic genes mutations, signal pathway and the optimal treatment regimen are still poor understood.

A retrospective analysis was performed upon review of clinical database, including additional immunohistochemistry (IHC) staining and integrated mutation analysis on reserved samples. Twelve samples of extranodal NK/T-cell lymphoma-nasal type (ENKL-NT) were used in IHC analysis for control.

A total of 26 cases were collected during 2001 to 2013, including 17 males and 9 females, median age of 28 years old (range 4-76 years old). Most patients presented with acute-onset high swinging fever (n=26), deteriorating jaundice (n=19) and pancytopenia (n=22) at diagnosis. The organ most frequently involved organs were bone marrow (n=25), liver (n=23), spleen (n=22) and gastrointestinal (n=6). Disseminated intravascular coagulopathy (DIC) was present in 15 out of 26 patients, significantly associated with liver involvement and jaundice (p<0.01). Epstein-Barr virus (EBV) viremia was present in 7 of 8 tested cases (5.12x10*,2-5.41x10*,6 copies/mL). The characteristic morphological appearance of ANKL in bone marrow smears were deep purple-red staining granules in cytoplasm and prominent vacuoles located in cytoplasm and/or nucleus. FACS demonstrated ANKL cells were CD2+cCD3+CD7+CD11b+/-CD11c+/-CD29+CD38+/-CD45+CD56+CD86+BCL-2+, CD3-TCR-cCD79a-cMPO-CD5-CD10-CD19-CD25-CD33-CD83-CD123-. Surprisingly, ANKL cells were negative for CD21, an established EBV receptor. Additional IHC on 14 reserved bone marrow samples revealed ANKL cells were strongly positive for LMP2A (n=14), LMP1 (n=11) and EBER (n=10). In ENKL-NT, LMP2A and LMP1 were moderately positive in 5 and 4 out of 12 cases, respectively. EBERs were detected in all ENKT-NT cases. Moreover, IHC analysis showed ANKL cells were strongly stained by CDK6 (n=13), MDM2 (n=14), CD44 (n=13), IFN-γ (n=14), slightly stained by CDK4 (n=2), negative for SH2 domain-containing inositol 5’-phosphatase-1 (SHIP-1), which is responsible for 2B4-mediated inhibition in NK cells. In ENKL-NT samples, CDK6 (n=11), MDM2 (n=9) and CD44 (n=8) were positive; furthermore, SHIP-1 was slightly positive in 4 samples. Chromosome aberrations were present in 7 patients, including dup1(q22q25), inv(3)(p21p25), del(3)(p13), t(3;11)(q21;q23), i(7)(q10), del(7)(q32), del(14)(q24), der(15)t(7;15)(q10;q10), -8, -12,+13,-18,+19,-22. Multiple somatic mutations were detected in 7 cases, including TET2 (D1938E, S69R, V292L, E1207D, G1391C, T1393K, Y1998X), FBXW7 (S427L,Q428K, D431E, R505C, Q508K, G517V, D775Y), CEBPA (E57D, N292K, S85I), FLT3 (M837I, E604X), KRAS (G77R, Q22H, G13D), PAX5 (P93T, A111S), PHF6 (R15S, S155X, V5F), DNMT3A (P602H, H613D), EGFR (E736X, S155X), IDH2 (K166M),SH2B3 (E190X), c-Kit (G812C), JAK1 (D775Y), NOTCH1 (A1701S). Only 1 out of 10 patients obtained partial remission after anthracycline-based or platinum-based regimens (CHOP, CHOP-Bleomycin, CDOP, EPOCH, HyperCVAD and ESHAP). Notably, two cases rapidly achieved durable complete remission after L-asparaginase-containing regimen (CHOP/PEG-L-asp and VDLP). Two patients received allogeneic hematopoietic stem cell transplantation, one maintained durable remission and another died of invasive pulmonary infection before engraftment. The median overall survival of 26 cases was only 7.5 days (range 2-330 days).

ANKL is Trojan story about EBV and NK cells, being presented with strong evidence of EBV infection/transformation, highly aggressive clinical course and prominent chromosome/genomic instability, which deserves more research efforts to dissect the role of EBV, molecular cytogenetics make-up, signaling pathway of LMP1/LMP2A-PI3K/AKT-CDK6-MDM2 and optimal regimen such as L-asp-contained protocol for this rare leukemia subset.

Zhou:Guangzhou Pearl River of Science & Technology New Star (No. 2011J2200069 to HS.Zhou): Research Funding. Liu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding.