The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is biased and characterized by the existence of subsets of cases with closely homologous ("stereotyped") complementarity-determining region 3 (CDR3) sequences. In the present series, 201 (21.9%) of 916 patients with CLL expressed IGHV genes that belonged to 1 of 48 different subsets of sequences with stereotyped heavy chain (H) CDR3. Twenty-six subsets comprised 3 or more sequences and were considered "confirmed." The remaining subsets comprised pairs of sequences and were considered "potential"; public database CLL sequences were found to be members of 9 of 22 "potential" subsets, thereby allowing us to consider them also "confirmed.

Somatic hypermutation (SHM) features in a series of 1967 immunoglobulin heavy chain gene (IGH) rearrangements obtained from patients with chronic lymphocytic leukemia (CLL) were examined and compared with IGH sequences from non-CLL B cells available in public databases. SHM analysis was performed for all 1290 CLL sequences in this cohort with less than 100% identity to germ line. At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged from the analysis of certain subgroups of CLL sequences defined by: (1) IGHV gene usage, (2) presence of stereotyped heavy chain complementarity-determining region 3 (HCDR3) sequences, and (3) mutational load. Recurrent, "stereotyped" amino acid changes occurred across the entire IGHV region in CLL subsets carrying stereotyped HCDR3 sequences, especially those expressing the IGHV3-21 and IGHV4-34 genes. These mutations are underrepresented among non-CLL sequences and thus can be considered as CLL-biased. Furthermore, it was shown that even a low level of mutations may be functionally relevant, given that stereotyped amino acid changes can be found in subsets of minimally mutated cases. IntroductionDeveloping B cells generate a vast repertoire of antibody specificities through somatic recombination of distinct variable (V), diversity (D) (heavy chain only), and joining (J) genes to form the variable domain exons of immunoglobulins (IG). 1 Unlike heavy chain complementarity determining regions (HCDR) 1 and 2, which are entirely encoded by the IGHV gene, HCDR3 is created de novo by the VDJ recombination process. 1 The skewing of diversity to the HCDR3 implies that HCDR3 sequences are the principal determinants of specificity, at least in the primary repertoire. 2,3 However, HCDR3 diversity is not enough to realize the full potential of antibody diversity. 4 Furthermore, unconventional antigens, such as B-cell superantigens, may be recognized not via the CDRs but rather via the framework regions (FRs). 5 Somatic hypermutation (SHM) of IG variable genes forms a second round of diversification after somatic recombination which increases antibody diversity. 6 SHM has long been thought to occur mainly in the germinal centers (GCs) after antigen stimulation and in a manner dependent on T-cell help. 7 Recent reports, however, suggest that SHM can be T-cell independent and may also occur outside classic GCs. [8][9][10][11][12][13] In recent years, the mutational status of IGHV genes has been established as one of the most important molecular genetic markers in defining prognostic subgroups of chronic lymphocytic leukemia (CLL). CLL patients who carry IGHV genes with 98% identity or more to the closest germ line gene ("unmutated") follow a more aggressive clinical course and have strikingly shorter survival than patients carrying IGHV genes with less than 98% identity to germ line ("mutated"). 14,15 The 98% cutoff was chosen as a shortcut to exclude potential polymorphic variants [16][17][18][19] and has been used by the majority of st...

Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotypedBCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of "CLL-biased" features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset. (Blood. 2012;119(19):4467-4475) IntroductionThe analysis of the Ig genes in chronic lymphocytic leukemia (CLL) has contributed significantly toward deciphering the molecular pathogenesis of the disease. Studies from the 1990s provided the first indications for a possible role of Ag(s) in selecting the CLL progenitor cells, through the discovery of a biased Ig heavy variable (IGHV) gene repertoire, different from that of normal B cells, as well as distinctive Ag-binding sites among unrelated cases. [1][2][3][4][5] By the late 1990s, it emerged that the mutational status of the rearranged IGHV genes directly correlated with patient survival. In particular, patients with unmutated IGHV genes were found to follow a more aggressive clinical course and have significantly shorter survival than patients carrying mutated IGHV genes. 6,7 Yet, there were exceptions to this rule: cases using the IGHV3-21 gene, although mostly expressing mutated Ig, had a survival similar to that of unmutated cases. 8 Intriguingly, approximately half of the IGHV3-21 cases were found to display restricted and, in some instances, essentially identical variable heavy complementarity determining region 3 (VH CDR3) sequences and identical light chains, strongly suggesting recognition of a common antigenic determinant. 9 Soon thereafter, the study of Ig sequences in CLL by groups in both Europe and the United States led to the identification of several other subsets of cases carrying highly similar BCR Igs among both mutated and unmutated cases (stereotyped BCR). [10][11][12][13][14] The identification of stereotypy among unrelated and geographically distant cases was widely accepted as evidence for the Submitted November 26, 2011; accepte...

Through the European Research Initiative on chronic lymphocytic leukemia (CLL) (ERIC), we screened 3490 patients with CLL for mutations within the NOTCH1 (n=3334), SF3B1 (n=2322), TP53 (n=2309), MYD88 (n=1080) and BIRC3 (n=919) genes, mainly at diagnosis (75%) and before treatment (>90%). BIRC3 mutations (2.5%) were associated with unmutated IGHV genes (U-CLL), del(11q) and trisomy 12, whereas MYD88 mutations (2.2%) were exclusively found among M-CLL. NOTCH1, SF3B1 and TP53 exhibited variable frequencies and were mostly enriched within clinically aggressive cases. Interestingly, as the timespan between diagnosis and mutational screening increased, so too did the incidence of SF3B1 mutations; no such increase was observed for NOTCH1 mutations. Regarding the clinical impact, NOTCH1 mutations, SF3B1 mutations and TP53 aberrations (deletion/mutation, TP53ab) correlated with shorter time-to-first-treatment (P<0.0001) in 889 treatment-naive Binet stage A cases. In multivariate analysis (n=774), SF3B1 mutations and TP53ab along with del(11q) and U-CLL, but not NOTCH1 mutations, retained independent significance. Importantly, TP53ab and SF3B1 mutations had an adverse impact even in U-CLL. In conclusion, we support the clinical relevance of novel recurrent mutations in CLL, highlighting the adverse impact of SF3B1 and TP53 mutations, even independent of IGHV mutational status, thus underscoring the need for urgent standardization/harmonization of the detection methods.