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...

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We recently reported that Swedish V H 3-21-using chronic lymphocytic leukemia (CLL) patients showed restricted immunoglobulin gene features and poor prognosis despite V H mutation status. To investigate this further, we analyzed the V H and V L gene rearrangements in 90 V H 3-21 ؉ patients from Sweden, Germany, Italy, United States, Finland, and Australia and correlated these data with survival and other prognostic markers. Sixty-three percent exhibited mutated V H genes and 37% unmutated V H genes. Fifty (56%) patients displayed a short and homologous heavy-

B-cell chronic lymphocytic leukemia (CLL) consists of 2 prognostic entities where cases with mutated immunoglobulin V H genes have better outcome than unmutated cases. V H -mutated CLLs display longer telomeres compared with unmutated cases and telomere length has been indicated to predict outcome, although the prognostic value of telomere length has not been fully established in CLL. We analyzed telomere length, V H gene mutation status, and clinical parameters in a large series of CLL. Telomere length was assessed by quantitative polymerase chain reaction (PCR), giving a very good correlation to telomere length estimated by Southern blotting (P < .001). The prognostic information given by mutation status (n ‫؍‬ 282) and telomere length (n ‫؍‬ 246) was significant (P < .001, respectively). Telomere length was a prognostic factor for stage A (P ‫؍‬ .021) and stage B/C (P ‫؍‬ .018) patients, whereas mutation status predicted outcome only in stage A patients (P < .001). Furthermore, mutated CLLs were subdivided by telomere length into 2 groups with different prognoses (P ‫؍‬ .003), a subdivision not seen for unmutated cases (P ‫؍‬ .232). Interestingly, the V H -mutated group with short telomeres had an overall survival close to that of the unmutated cases. Thus, by combining V H mutation status and telomere length, an improved subclassification of CLL was achieved identifying previously unrecognized patient groups with different outcomes. IntroductionThe end structures of the chromosomes, the telomeres, are composed of a repetitive DNA sequence, (TTAGGG) n , and specific proteins bound to the DNA. 1,2 The telomeres protect the chromosomes from degradation, prevent end-to-end fusions, and are crucial for control of replicative senescence. In humans, the telomeres have an average length of 5 kilobases (kb) to 15 kb and in normal somatic cells the telomeric DNA shortens with 100 base pair (bp) to 200 bp every cell division due to the end-replication problem. [3][4][5] The erosion of the telomeres can be counteracted by telomerase, an enzyme that adds TTAGGG repeats to the chromosomal ends. 6 Telomere maintenance, usually executed by telomerase, is a prerequisite for an extended or infinite cellular lifespan, and studies of telomere dynamics have been in focus ever since it was shown that telomerase activity is present in almost 90% of all malignant tumors. [7][8][9] The nearly exclusive presence of telomerase in tumor cells has made the enzyme a theoretically attractive target for cancer therapy. In contrast, telomerase is restricted to a few normal cell types in humans, including germ cells, stem cells, activated lymphocytes, and epithelial cells with high reproductive demand. [10][11][12][13][14] Additionally, during the germinal center (GC) reaction in lymph nodes a high level of telomerase activity is expressed in the GC B cells accompanied by a unique telomere lengthening process not observed in other cells in vivo. [15][16][17] Telomere length reduction has been found with increasing age and was recently impli...

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