Phenotypic Heterogeneity Of Chronic Lymphoproliferative Disorder Of Nk Cells

Background NK cells represent a subset of lymphocytes belonging to the innate immunity branch typically expressing CD16 and CD56 associated to CD3 negativity. Two major subtypes of NK cells can be distinguished through CD16 and CD56 expression: CD56 high /CD16 dim/neg NK cells with low cytotoxic function and CD56 dim /CD16 high NK cells with high cytotoxic function. Recently a subtype of NK cells with memory properties characterized by CD56 dim /CD16 high /CD57+/CD62L- phenotype has been discovered. Chronic Lymphoproliferative Disorders of Granular Lymphocytes are characterized by the clonal expansion of Large Granular Lymphocyte (LGL) that can be CD3 positive (T-LGLL) or CD3 negative (Chronic Lymphoproliferative Disorder of NK Cell, CLPD-NK). The disease generally has indolent course but some patients develop cytopenia, particularly neutropenia, exposing to potentially lethal bacterial infections. Furthermore, NK-CLPD is usually referred as a more indolent disorder with respect to T-LGLL, with lower incidence of cytopenia and treatment need. CLPD-NK therapy does not differ from that of T-LGLL and is usually represented by an immunosuppressive therapy with low dose cyclophosphamide or methotrexate, with cyclosporine A usually being reserved to refractory patients. Somatic STAT3 mutations represent a new diagnostic marker of these disorders, initially reported in T-LGLL in about 40% oh patients, but also present in CLPD-NK in about 30% of cases. Using flow analysis, the aim of the present study was to identify a subset of CLPD NK patients characterized by a more severe disease requiring a shorter follow-up as compared to patients with a more indolent disease. Methods In a cohort of 16 patients affected by CLPD-NK, NK cells were analysed by flow for CD3, CD16, CD56, CD57 and CD62L antigen expression. These patients were studied for the presence of cytopenia and treatment requirement. STAT3 mutation analysis of exon 21 was performed with Sanger sequencing. Finally, p-STAT3 tyr 705 level and total STAT3 level were examined by western blotting. Results In relation to CD16 and CD56 expression, three major NK cells populations can be recognized in CLPD-NK patients: CD56 high /CD16 neg NK cells, CD56 dim /CD16 neg NK cells and CD56 neg/dim /CD16 high . As a consequence, patients can be separated into three groups characterized by the preferentially expansion of one of these populations: 2/16 (13%) with CD56 high /CD16 neg NK population, 4/16 (25%) with CD56 dim /CD16 neg NK population and 10/16 (62%) with CD56 neg/dim /CD16 high NK population. Furthermore, patients with predominance of this last NK cells subset were studied for CD57 and CD62L expression to identify NK cytotoxic subset (CD57-/CD62L low/neg ) and NK memory subset (CD57+/CD62L low/neg ); a NK cytotoxic/memory ratio (C/M ratio) was then calculated. 4 of 10 CD56 neg/dim /CD16 high patients (40%) were characterized by prevalence of NK cytotoxic cells expansion and high C/M ratio (≥3) while the remaining 6/10 patients were characterized by NK memory cells expansion with low C/M ratio (≤1.6). We then evaluated the presence of cytopenia, in particular neutropenia, in our patients9 cohort. Neutropenia was shown in 7/16 (44%) patients with 4/16 (25%) experiencing severe neutropenia. Anemia and thrombocytopenia were less frequent (19% and 6% respectively). Interestingly, 6 out of 7 (86%) neutropenic patients were in the CD56 neg/dim /CD16 high subset and all patients with severe neutropenia belonged to the high C/M ratio subset. Interestingly, 3 out of 4 patients (75%) of this subset required therapy during the natural history of the disease. Concerning STAT3 mutation analysis, no one mutated patient was found in this setting. By western blot analysis, patients with high C/M ratio presented higher p-STAT3 levels than other patients and normal NK cells. Summary Although CLPD-NK represents an extreme heterogeneous disorder, discrete subtypes of disease characterized by different NK cells population expansion can be identified by flow analysis. Interestingly, this splitting allows to identify a subset of patients with prevalence of CD56 neg/dim /CD16 high NK cells with high C/M ratio that are characterized by high level of p-STAT3, high frequency of severe neutropenia and treatment requirement. Disclosures No relevant conflicts of interest to declare.