Human Cancer Biology IgG-Switched CLL Has a Distinct Immunogenetic Signature from the Common MD Variant: Ontogenetic Implications Anna Vardi 1 , Andreas Agathangelidis 3 , Lesley-Ann Sutton 4 , Maria Chatzouli 5 , Lydia Scarf  o 3 , Larry Mansouri 4 , Vassiliki Douka 1 , Achilles Anagnostopoulos 1 , Nikos Darzentas 2,6 , Richard Rosenquist 4 , Paolo Ghia 3 , Chrysoula Belessi 5 , and Kostas Stamatopoulos 1,2,4 Abstract Purpose: Immunoglobulin G–switched chronic lymphocytic leukemia (G-CLL) is a rare variant of CLL, whose origin and ontogenetic relationship to the common IgM/IgD (MD-CLL) variant remains undefined. Here, we sought for clues about the ontogeny of G-CLL versus MD-CLL by profiling the relevant IG gene repertoires. Experimental Design: Using purpose-built bioinformatics methods, we performed detailed immuno- genetic profiling of a multinational CLL cohort comprising 1,256 cases, of which 1,087 and 169 expressed IG mu/delta and gamma heavy chains, respectively. Results: G-CLL has a highly skewed IG gene repertoire that is distinct from MD-CLL, especially in terms of (i) overuse of the IGHV4-34 and IGHV4-39 genes and (ii) differential somatic hypermutation (SHM) load. Repertoire differences were also found when comparing subgroups with similar SHM status and were mainly attributed to the exclusive representation in G-CLL of two major subsets with quasi-identical (stereotyped) B-cell receptors. These subsets, namely #4 (IGHV4-34/IGKV2-30) and #8 (IGHV4-39/IGKV1(D)-39), were found to display sharply contrasting SHM and clinical behavior. Conclusions: G-CLL exhibits an overall distinct immunogenetic signature from MD-CLL, prompting speculations about distinct ontogenetic derivation and/or immune triggering. The reasons underlying the differential regulation of SHM among G-CLL cases remain to be elucidated. Clin Cancer Res; 20(2); 323–30. Ó2013 AACR. Introduction The great majority of chronic lymphocytic leukemia (CLL) cases express surface immunoglobulins (IG) of the mu and delta isotypes (IgM þ IgD þ , MD-CLL; ref. 1). However, sub- populations of isotype-switched cells often exist within IgM þ IgD þ CLL clones (2, 3). Indeed, class switch recombination (CSR) seems to be dissociated from somatic hypermutation (SHM) in CLL, at least in certain cases, and, perhaps para- doxically, in vivo CSR is predominantly identified in CLL clones with unmutated IG receptors (U-CLL; refs. 4, 5). Activation-induced cytidine deaminase (AID), which is normally required for both SHM and CSR, is expressed in a small proliferative compartment within the CLL clone, irrespective of the immunoglobulin heavy variable (IGHV) gene mutational status, and can be functionally induced in vitro under conditions that mimic T-cell help (6). Further- more, ongoing CSR and high AID expression define a subgroup of U-CLL displaying even worse prognosis, under- scoring the importance of intense (micro) environmental stimulation for shaping clonal behavior and eventual clin- ical outcome (7). That notwithstanding, the precise signals that induce in vivo CSR in CLL are presently not fully elucidated. CLL cases in which the major clone is isotype-switched are relatively rare (6%–10%; ref. 8). The majority of such cases are immunoglobulin G–switched chronic lympho- cytic leukemia (G-CLL); however, little published data exist about this subgroup. That said, a phenotypic, molec- ular, and functional characterization of 14 unselected G- CLL cases suggested a relationship with CLL bearing mutated IG receptors (M-CLL) and derivation from post- germinal B cells (9). However, the existence of U-CLL with immunoglobulin G (IgG)–switched receptors chal- lenges this scenario and warrants the search for alternative possibilities. Here, we sought evidence about the origin of G-CLL by comprehensively profiling the IG gene repertoires in the largest series studied to date for this purpose. Our focus on Authors' Affiliations: 1 Hematology Department and HCT Unit, G. Papa- nicolaou Hospital; 2 Institute of Applied Biosciences, Centre for Research & Technology Hellas, Thessaloniki, Greece; 3 Division of Molecular Oncology, San Raffaele Scientific Institute, Milan, Italy; 4 Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; 5 Hema- tology Department, Nikea General Hospital, Piraeus, Greece; and 6 Central European Institute of Technology, Masaryk University, Brno, Czech Republic Corresponding Author: Kostas Stamatopoulos, Hematology Department and HCT Unit, G. Papanicolaou Hospital, 57010 Thessaloniki, Greece. Phone: 30-2313-307992; Fax: 30-2313-307579; E-mail: kostas.stamatopoulos@gmail.com doi: 10.1158/1078-0432.CCR-13-1993 Ó2013 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 323 on May 4, 2021. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst November 15, 2013; DOI: 10.1158/1078-0432.CCR-13-1993