New Insight into Epstein-Barr Virus-Associated Biological Behavior of Aggressive Natural Killer Cell Leukemia
Authors: Zhou Hongsheng , Yin Chanxin , Deng Yongjian , Xue Xiaolei , Li Xiangzhao , Tian Ying , Li Hongsheng , Liao Libin , Fang Dan , Dai Min , Wang Qiang , Xu Na , Huang Fen , Fan Zhiping , Zhang Yu , Wei Yongqiang , Xu Dan , Jiang Qianli , Liu Xiaoli , Sun Jing , Liu Qifa , Xu Bing , Feng Ru , Carter Bing Z , Meng Fanyi 
Published: November 2019

Abstract
Aggressive natural killer cell leukemia (ANKL) is a rare and highly aggressive subtype of mature NK-cell neoplasms. Similar with extranodal NK/T-cell lymphoma, nasal type (ENKL), another subtype of NK-cell neoplasm, ANKL is also an Asian-prevalent and Epstein-Barr virus (EBV)-related neoplasm. In contrast, our knowledge of ANKL, especially about EBV biological behavior in this rare leukemia, lags far behind that of ENKL and other EBV-related hematopoietic malignancies, such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and post-transplant lymphoproliferative disorder (PTLD). Dissection of the virus-host crosstalk in ANKL could contribute to better understanding the mechanism and finding out effective therapy for this neoplasm.
In the present study, we investigated EBV-associated biological behavior in serial ANKL patients, including the clinical presentation, EBV genomic DNA, EBV antigens expression, cytogenetic-molecular aberrations, and leukemia-associated microenvironment. A total of 28 ANKL patients were collected upon review of the clinical database in Nanfang hospital. Different items of EBV infection evidence consisted of EBV viremia (n=9), EBV genomic DNA (n=20), and EBER/EBNA/LMP1/LMP2A expression (n=23). EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) was the predominant clinical feature. Bone marrow smear was infiltrated with large granular lymphocyte (LGL) with LMP1/LMP2a-positive bulb, indicating the presence of EBV viral inclusions bodies. Positron emission tomographic (PET)-computed tomographic (CT) scan revealed bone marrow, liver and spleen as the most frequently involved organs, compared with nose and nasopharynx in ENKL. Cytogenetic analysis demonstrated 7q10-32 (n=4) was the “hotspot” of chromosome aberrations in ANKL. PCR analysis with EBNA-2/LMP1 specific primers on reserved DNA samples (n=20) revealed ANKL cells were infected with type-1 EBV strain with wide-type LMP1 (n=20), compared with 30bp-deleted LMP1 gene in ENKL. Integrated mutation analysis (n=20) identified recurrent mutations in Src homology 2 (SH2) domains of STAT5a (n=7) and p16inka (exon 3/4, n=20), but no mutation in SH2 domains of ID2, STAT1, and STAT3. Immunochemical (IHC) analysis on formalin-fixed paraffin-embedded tissues (n=23) revealed latency type-3 EBV expression in ANKL cells, with latency antigens of EBER, EBNA, LMP-1, and LMP-2. Furthermore, LMP-1/LMP-2-positive leukemia/lymphoma-associated macrophages (LAMs, n=23) were enriched in ANKL microenvironment. Notably, EBV-positive LAMs were significantly associated with poor prognosis and disease progression. Univariate analysis revealed significant difference (p<0.05) in overall survival (OS) between High-Ratio of CD68+LAMs/CD56+ANKLs (HMA, n=13) and Low-Ratio LAM/ANKLs cohorts (LMA, n=10). Furthermore, IHC analysis on paired presentation and progression samples (n=3) showed that EBV-positive LAMs increased in pace with disease progression.
Conclusions: Our data demonstrate that type-1 EBV strain as being latency type-3 expression infected both leukemia cells and microenvironment and thus linked the pathogen-microenvironment-host crosstalk in ANKL. EBV-infected leukemia-associated microenvironment, particularly LAMs, might not only play a critical role in prognosis classification but also contribute to the leukemogenesis of NK transformation in ANKL, which it’s still poorly understood and deserves more research efforts.

Disclosures
Zhou: Guangzhou Pearl River of Science and Technology New Star Project: Research Funding.