Background: Epstein-Barr virus (EBV)-related malignancies harbour distinct serological responses to EBV antigens. We hypothesized that EBV serological patterns can be useful to identify different stages of chronic lymphocytic leukemia. Methods: Information on 150 cases with chronic lymphocytic leukemia and 157 frequency-matched (by age, sex and region) population-based controls from a Spanish multicentre case-control study was obtained. EBV immunoglobulin G serostatus was evaluated through a peptide-based ELISA and further by immunoblot analysis to EBV early antigens (EA), nuclear antigen (EBNA1), VCA-p18, VCA-p40 and Zebra. Two independent individuals categorized the serological patterns of the western blot analysis. Patients with very high response and diversity in EBV-specific polypeptides, in particular with clear responses to EA-associated proteins, were categorized as having an abnormal reactive pattern (ab_EBV). Adjusted odds ratios (OR) and 95% confidence interval (CI) were estimated using logistic regression models. Results: Almost all subjects were EBV-IgG positive (>95% of cases and controls) whereas ab_EBV patterns were detected in 23% of cases (N = 34) and 11% of controls (N = 17; OR: 2.44, 95% CI, 1.29 to 4.62; P = 0.006), particularly in intermediate/high risk patients. Although based on small numbers, the association was modified by smoking with a gradual reduction of ab_EBV-related OR for all Rai stages from never smokers to current smokers. Conclusions: Highly distinct EBV antibody diversity patterns revealed by immunoblot analysis were detected in cases compared to controls, detectable at very early stages of the disease and particularly among non smokers. This study provides further evidence of an abnormal immunological response against EBV in patients with chronic lymphocytic leukemia.

This work was supported by the “Acción Transversal del Cancer”, approved on the Spanish Ministry Council on the 11th October 2007, by the Instituto de Salud Carlos III (Spanish Government) (grants PI08/1770, PI08/0533, PI08/1359, PS09/00773-Cantabria, PI11/01810, PI11/02213, RCESP C03/09, RTICESP C03/10, RTIC RD06/0020/0095, RTIC RD12/0036/0056, Rio Hortega CM13/00232, SV-09-CLINIC-1 and CIBERESP), by the Fundación Marques de Valdecilla (API 10/09), by Obra Social CAJASTUR (SV-CAJASTUR-1), by the Recercaixa (2010ACUP 00310), by the Spanish Association Against Cancer (AECC) Scientific Foundation and by the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR)–Generalitat de Catalunya (Catalonian Government) (grants AGAUR 2009SGR1026, 2014SGR756 and 2009SGR1465). The ICGC CLL-Genome Project is funded by Spanish Ministerio de Economía y Competitividad (MINECO) through the Instituto de Salud Carlos III (ISCIII) and Red Temática de Investigación del Cáncer (RTIC RD12/0036/0036) del ISCIII. Sample collection and storage was partially supported by the Instituto de Salud Carlos III FEDER (RD09/0076/00036), Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d’Oncologia de Catalunya (XBTC).