title: New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies.
abstract: BACKGROUND
  CD20 is a B cell lineage-specific marker expressed by normal and leukemic B cells and targeted by several antibody immunotherapies. We have previously shown that the protein from a CD20 mRNA splice variant (D393-CD20) is expressed at various levels in leukemic B cells or lymphoma B cells but not in resting, sorted B cells from the peripheral blood of healthy donors.
  
  
  RESULTS
  Western blot (WB) analysis of B malignancy primary samples showed additional CD20 signals. Deep molecular PCR analysis revealed four new sequences corresponding to in-frame CD20 splice variants (D657-CD20, D618-CD20, D480-CD20, and D177-CD20) matching the length of WB signals. We demonstrated that the cell spliceosome machinery can process ex vivo D480-, D657-, and D618-CD20 transcript variants by involving canonical sites associated with cryptic splice sites. Results of specific and quantitative RT-PCR assays showed that these CD20 splice variants are differentially expressed in B malignancies. Moreover, Epstein-Barr virus (EBV) transformation modified the CD20 splicing profile and mainly increased the D393-CD20 variant transcripts. Finally, investigation of three cohorts of chronic lymphocytic leukemia (CLL) patients showed that the total CD20 splice variant expression was higher in a stage B and C sample collection compared to routinely collected CLL samples or relapsed refractory stage A, B, or C CLL.
  
  
  CONCLUSIONS
  The involvement of these newly discovered alternative CD20 transcript variants in EBV transformation makes them interesting molecular indicators, as does their association with oncogenesis rather than non-oncogenic B cell diseases, differential expression in B cell malignancies, and correlation with CLL stage and some predictive CLL markers. This potential should be investigated in further studies.
authors.affiliation: INSERM UMR1098, Établissement Français du Sang Bourgogne Franche Comté, Université de Franche-Comté, SFR FED4234, 25020 Besançon, France.
authors.forename: Clémentine
authors.initials: C
authors.lastname: Gamonet
authors.affiliation: INSERM UMR1098, Établissement Français du Sang Bourgogne Franche Comté, Université de Franche-Comté, SFR FED4234, 25020 Besançon, France.
authors.forename: Elodie
authors.initials: E
authors.lastname: Bole-Richard
authors.affiliation: INSERM UMR1098, Établissement Français du Sang Bourgogne Franche Comté, Université de Franche-Comté, SFR FED4234, 25020 Besançon, France.
authors.forename: Aurélia
authors.initials: A
authors.lastname: Delherme
authors.affiliation: EA3181 et Service de Dermatologie, Université de Franche Comté, CHU de Besançon, Besançon, France.
authors.forename: François
authors.initials: F
authors.lastname: Aubin
authors.affiliation: EA3181 et Service de Dermatologie, Université de Franche Comté, CHU de Besançon, Besançon, France ; CHRU, Department of Rheumatology, Université de Franche-Comté EA 4266, INSERM CIC-1431, 25000 Besançon, France ; EA 4266, Université de Franche-Comté, Besançon, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Toussirot
authors.affiliation: INSERM UMR1098, Établissement Français du Sang Bourgogne Franche Comté, Université de Franche-Comté, SFR FED4234, 25020 Besançon, France ; EA3181 et Service de Dermatologie, Université de Franche Comté, CHU de Besançon, Besançon, France.
authors.forename: Francine
authors.initials: F
authors.lastname: Garnache-Ottou
authors.affiliation: INSERM UMR1098, Établissement Français du Sang Bourgogne Franche Comté, Université de Franche-Comté, SFR FED4234, 25020 Besançon, France ; EA3181 et Service de Dermatologie, Université de Franche Comté, CHU de Besançon, Besançon, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Godet
authors.affiliation: Inserm U1037, Université Toulouse 3-ERL CNRS, CHU Purpan, Toulouse, France.
authors.forename: Loïc
authors.initials: L
authors.lastname: Ysebaert
authors.affiliation: Hématologie Clinique, CHU Estaing, 1 Place Lucie Aubrac, 63003 Clermont-Ferrand Cedex 1, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Tournilhac
authors.affiliation: Hematologie, CHU Bretonneau, Tours, France.
authors.forename: Dartigeas
authors.initials: D
authors.lastname: Caroline
authors.affiliation: INSERM UMR1098, Établissement Français du Sang Bourgogne Franche Comté, Université de Franche-Comté, SFR FED4234, 25020 Besançon, France ; Hematology Department, CHU Jean Minjoz, 25020 Besançon, France.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Larosa
authors.affiliation: INSERM UMR1098, Établissement Français du Sang Bourgogne Franche Comté, Université de Franche-Comté, SFR FED4234, 25020 Besançon, France ; EA3181 et Service de Dermatologie, Université de Franche Comté, CHU de Besançon, Besançon, France ; Hematology Department, CHU Jean Minjoz, 25020 Besançon, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Deconinck
authors.affiliation: INSERM UMR1098, Établissement Français du Sang Bourgogne Franche Comté, Université de Franche-Comté, SFR FED4234, 25020 Besançon, France ; EA3181 et Service de Dermatologie, Université de Franche Comté, CHU de Besançon, Besançon, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Saas
authors.affiliation: INSERM UMR1098, Établissement Français du Sang Bourgogne Franche Comté, Université de Franche-Comté, SFR FED4234, 25020 Besançon, France ; EA3181 et Service de Dermatologie, Université de Franche Comté, CHU de Besançon, Besançon, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Borg
authors.affiliation: INSERM UMR1098, Établissement Français du Sang Bourgogne Franche Comté, Université de Franche-Comté, SFR FED4234, 25020 Besançon, France.
authors.forename: Marina
authors.initials: M
authors.lastname: Deschamps
authors.affiliation: INSERM UMR1098, Établissement Français du Sang Bourgogne Franche Comté, Université de Franche-Comté, SFR FED4234, 25020 Besançon, France ; Laboratoire de Thérapeutique Immuno-Moléculaire et cellulaire des cancers, INSERM UMR1098, Etablissement Français du Sang-Bourgogne/Franche-Comté, 8, rue du Docteur Jean-François-Xavier Girod, 25020 Besançon Cedex, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Ferrand
keywords: Alternative splicing; B malignancies; CD20; CLL; EBV transformation