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Background About 30% of cases of chronic lymphocytic leukaemia (CLL) carry quasi-identical B-cell receptorimmunoglobulins and can be assigned to distinct stereotyped subsets. Although preliminary evidence suggeststhat B-cell receptor immunoglobulin stereotypy is relevant from a clinical viewpoint, this aspect has never beenexplored in a systematic manner or in a cohort of adequate size that would enable clinical conclusions to be drawn.Methods For this retrospective, multicentre study, we analysed 8593 patients with CLL for whom immunogeneticdata were available. These patients were followed up in 15 academic institutions throughout Europe (in CzechRepublic, Denmark, France, Greece, Italy, Netherlands, Sweden, and the UK) and the USA, and data were collectedbetween June 1, 2012, and June 7, 2013. We retrospectively assessed the clinical implications of CLL B-cell receptorimmunoglobulin stereotypy, with a particular focus on 14 major stereotyped subsets comprising cases expressingunmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain variable genes. The primary outcome ofour analysis was time to fi rst treatment, defi ned as the time between diagnosis and date of fi rst treatment.Findings 2878 patients were assigned to a stereotyped subset, of which 1122 patients belonged to one of 14 majorsubsets. Stereotyped subsets showed signifi cant diff erences in terms of age, sex, disease burden at diagnosis,CD38 expression, and cytogenetic aberrations of prognostic signifi cance. Patients within a specifi c subset generallyfollowed the same clinical course, whereas patients in diff erent stereotyped subsets—despite having the sameimmunoglobulin heavy variable gene and displaying similar immunoglobulin mutational status—showedsubstantially diff erent times to fi rst treatment. By integrating B-cell receptor immunoglobulin stereotypy (forsubsets 1, 2, and 4) into the well established Döhner cytogenetic prognostic model, we showed these, whichcollectively account for around 7% of all cases of CLL and represent both U-CLL and M-CLL, constituted separateclinical entities, ranging from very indolent (subset 4) to aggressive disease (subsets 1 and 2).Interpretation The molecular classifi cation of chronic lymphocytic leukaemia based on B-cell receptorimmunoglobulin stereotypy improves the Döhner hierarchical model and refi nes prognostication beyondimmunoglobulin mutational status, with potential implications for clinical decision making, especially withinprospective clinical trials.Funding European Union; General Secretariat for Research and Technology of Greece; AIRC; Italian Ministry ofHealth; AIRC Regional Project with Fondazione CARIPARO and CARIVERONA; Regione Veneto on ChronicLymphocytic Leukemia; Nordic Cancer Union; Swedish Cancer Society; Swedish Research Council; and NationalCancer Institute (NIH).

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset #2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset #2.Within subset #2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset #2/IGHV3-21 was enriched for IGHV-unmutated cases (P = .002). Subset #2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset #2/IGHV3-21 (22 vs 60 months, P = .001). No such difference was observed between non-subset #2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset #2 emerges as uniformly aggressive, contrasting non-subset #2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL. © 2015 by The American Society of Hematology 10.1182/blood-2014-09-600874.

We recently reported that chronic lymphocytic leukemia (CLL) subgroups with distinct clonotypic BCRs present discrete patterns of TLR expression, function, and/or tolerance. In this study, to explore whether specific types of BCR/TLR collaboration exist in CLL, we studied the effect of single versus concomitant BCR and/or TLR stimulation on CLL cells from mutated (M-CLL) and unmutated CLL (U-CLL) cases. We stimulated negatively isolated CLL cells by using anti-IgM, imiquimod, and CpG oligodeoxynucleotide for BCR, TLR7, and TLR9, respectively, alone or in combination for different time points. After in vitro culture in the absence of stimulation, differences in p-ERK were identified at any time point, with higher p-ERK levels in U-CLL versus M-CLL. Pronounced p-ERK induction was seen by single stimulation in U-CLL, whereas BCR/TLR synergism was required in M-CLL, in which the effect was overall limited in scale. An opposite pattern was observed regarding induction of apoptosis, as studied by Western blotting for the cleaved fragment of poly(ADP-ribose) polymerase, and the active isoform of caspase-8, with M-CLL responding even to single stimulation, contrasting with U-CLL that showed minimal response. Our findings suggest that concomitant engagement of BCR and TLR leads to differential responses in CLL depending on the mutational status of the BCR. Differential intensity and duration of responses in M-CLL versus U-CLL indicates that the differences in signal transduction between the two subgroups may be primarily quantitative rather than qualitative. Copyright © 2014 by The American Association of Immunologists, Inc.

Background: About 30% of cases of chronic lymphocytic leukaemia (CLL) carry quasi-identical B-cell receptorimmunoglobulins and can be assigned to distinct stereotyped subsets. Although preliminary evidence suggeststhat B-cell receptor immunoglobulin stereotypy is relevant from a clinical viewpoint, this aspect has never beenexplored in a systematic manner or in a cohort of adequate size that would enable clinical conclusions to be drawn.Methods: For this retrospective, multicentre study, we analysed 8593 patients with CLL for whom immunogeneticdata were available. These patients were followed up in 15 academic institutions throughout Europe (in CzechRepublic, Denmark, France, Greece, Italy, Netherlands, Sweden, and the UK) and the USA, and data were collectedbetween June 1, 2012, and June 7, 2013. We retrospectively assessed the clinical implications of CLL B-cell receptorimmunoglobulin stereotypy, with a particular focus on 14 major stereotyped subsets comprising cases expressingunmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain variable genes. The primary outcome ofour analysis was time to fi rst treatment, defi ned as the time between diagnosis and date of fi rst treatment.Findings: 2878 patients were assigned to a stereotyped subset, of which 1122 patients belonged to one of 14 majorsubsets. Stereotyped subsets showed signifi cant diff erences in terms of age, sex, disease burden at diagnosis,CD38 expression, and cytogenetic aberrations of prognostic signifi cance. Patients within a specifi c subset generallyfollowed the same clinical course, whereas patients in diff erent stereotyped subsets-despite having the sameimmunoglobulin heavy variable gene and displaying similar immunoglobulin mutational status-showedsubstantially diff erent times to fi rst treatment. By integrating B-cell receptor immunoglobulin stereotypy (forsubsets 1, 2, and 4) into the well established Döhner cytogenetic prognostic model, we showed these, whichcollectively account for around 7% of all cases of CLL and represent both U-CLL and M-CLL, constituted separateclinical entities, ranging from very indolent (subset 4) to aggressive disease (subsets 1 and 2).Interpretation: The molecular classifi cation of chronic lymphocytic leukaemia based on B-cell receptorimmunoglobulin stereotypy improves the Döhner hierarchical model and refi nes prognostication beyondimmunoglobulin mutational status, with potential implications for clinical decision making, especially withinprospective clinical trials.

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset # 2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset # 2. Within subset # 2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset # 2/IGHV3-21 was enriched for IGHV-unmutated cases (P =.002). Subset # 2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset # 2/IGHV3-21 (22 vs 60 months, P =.001). No such difference was observed between non-subset # 2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset # 2 emerges as uniformly aggressive, contrasting non-subset # 2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

We recently reported that chronic lymphocytic leukemia (CLL) subgroups with distinct clonotypic BCRs present discrete patterns of TLR expression, function, and/or tolerance. In this study, to explore whether specific types of BCR/TLR collaboration exist in CLL, we studied the effect of single versus concomitant BCR and/or TLR stimulation on CLL cells from mutated (M-CLL) and unmutated CLL (U-CLL) cases. We stimulated negatively isolated CLL cells by using anti-IgM, imiquimod, and CpG oligodeoxynucleotide for BCR, TLR7, and TLR9, respectively, alone or in combination for different time points. After in vitro culture in the absence of stimulation, differences in p-ERK were identified at any time point, with higher p-ERK levels in U-CLL versus M-CLL. Pronounced p-ERK induction was seen by single stimulation in U-CLL, whereas BCR/TLR synergism was required in M-CLL, in which the effect was overall limited in scale. An opposite pattern was observed regarding induction of apoptosis, as studied by Western blotting for the cleaved fragment of poly(ADP-ribose) polymerase, and the active isoform of caspase-8, with M-CLL responding even to single stimulation, contrasting with U-CLL that showed minimal response. Our findings suggest that concomitant engagement of BCR and TLR leads to differential responses in CLL depending on the mutational status of the BCR. Differential intensity and duration of responses in M-CLL versus U-CLL indicates that the differences in signal transduction between the two subgroups may be primarily quantitative rather than qualitative.

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 immunogenetic 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. ©2013 AACR.