EBV infection status in 54 therapy-naïve patients with chronic lymphocytic leukemia

Purpose: detection of human Epstein-Barr virus (EBV) genomic sequences, viral load and the EBV transcripts (BZLF1, LMP1, LMP2A, and EBNA2) in peripheral blood (PB) mononuclear cells (PBMCs), the plasma levels of IgG antibodies against EBNA1, the levels and avidity of IgG antibodies against the EBV capsid antigen (anti-EBV-CA-IgG), and the expression of CCR1, CCR2, and CD38 on PB CD19+CD5+ lymphocytes in 54 therapy-naïve patients with chronic lymphocytic leukemia (CLL).
Data include information about demographic and clinical examination parameters of 54 therapy-naïve CLL patients at the time of enrollment: age, gender, CLL disease clinical stage (according to the Rai classification, updated version of the European Research Initiative on CLL, ERIC), Complete Blood Cell Count (leukocytes, lymphocytes, monocytes, neutrophils, platelets, RBC), hemoglobin level in PB, clinical flow cytometry data (CD19+, CD5+, and CD23+ cells in PB lymphocytes), Time to the first treatment (TTFT) in months, immunoglobulin gene heavy chain variable region (IGHV) mutation status, ZAP70 mRNA expression.
Blood was collected the same time as assessment of the primary diagnosis in the clinical flow cytometry (FC) laboratory at Riga East University Hospital. Plasma and PBMCs, after density gradient centrifugation, were aliquoted and stored at -80°C prior to use.
Quantification of the EBV DNA copy number in PBMC DNA samples was determined by real time PCR using the commercial kit EBV Real-TM Quant (Sacace Biotechnologies S.r.l., Como, Italy). The plasma levels of the IgG antibodies against the EBV capsid antigen (anti-EBV-CA) and the EBV nuclear antigen 1 (anti-EBNA1) were determined using the commercial enzyme‐linked immunosorbent assay (ELISA) Euroimmun kits (EI 2791-9601 G and EI 2793-9601 G; Euroimmun Medizinische Labordiagnostika, Lubeck, Germany). The avidity of the anti-EBV-CA IgG antibodies were determined, using the Euroimmun ELISA kit: Avidity Determination Anti-EBV-CA ELISA (IgG) (EI 2791-9601-1 G). Absorbance was measured at 450 nm, using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific Inc.).
The IGHV mutation status was defined according to the International CLL IPI working group (iwCLL) and the European Research Initiative on CLL (ERIC) recommendations, applying the ERIC published protocol [Agathangelidis et al., 2018].
The mRNA expression of the EBV genes was assessed using the first-strand cDNA and the nested PCR applying primers that had been published previously.
The presence of CD191 (CCR1), CD192 (CCR2), CD38 on the PBMC populations was assessed by seven-color FC, using a BD FACSAriaIIIu analyzer and the Diva8.2 software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).