The chronic lymphocytic leukemia (CLL) immunoglobulin (IG) heavy chain repertoire is known to display biased immunoglobulin variable heavy-chain (IGHV) gene usage, remarkable complementarity determining region 3 (HCDR3) stereotypy as well as distinctive somatic hypermutation (SHM) patterns, at least for subsets of cases. Our aim in the present study was to similarly investigate the IG light chain (LC) genes in terms of mutation frequency and targeting and CDR3 stereotypy to elucidate if the LC may play a significant complementary role in antigen recognition in CLL. We thus examined SHM patterns and secondary rearrangements of the IG LC gene loci in a total of 612 IGKV-J and 279 IGLV-J rearrangements from 725 patients with CLL. Firstly, we observed a highly restricted light chain gene usage in the vast majority of CLL cases with stereotyped HCDR3s. In particular, stereotyped IGHV3-21 CLL cases were characterized by a strikingly biased expression of lambda light chains utilizing the IGLV3-21 gene (36/37 cases of subset#2), whereas all 15 subset #4 cases with stereotyped IGHV4-34 IGs carried an IGKV2-30 rearrangement. In addition, subset-biased light chain CDR3 motifs were identified in groups of sequences utilizing the same IGKV or IGLV gene. For example, all 30 IGKV1-39/1D-39 light chains of subset#1 (using stereotyped IGHV1/5/7 genes) carried notably long KCDR3s (10–11 amino acids) generated by significant N region addition and characterized by the frequent introduction of a junctional proline (26/30 cases). Important differences regarding mutational load were observed in groups of sequences utilizing the same IGKV or IGLV gene and/or belonging to subsets with stereotyped B cell receptors (BCRs). In fact, significant differences were observed with regard to mutational status among groups of sequences utilizing different alleles of certain IGK/LV genes (specifically the IGKV1-5, IGLV1-51 and IGLV3-21 genes). At cohort level, the SHM patterns were typical of a canonical SHM process. A clustering of R mutations in KCDR1 was evident for all IGKV subgroups with the notable exception of the IGKV2 subgroup, which exhibited preferential targeting to the KCDR2, especially in IGKV2-30 rearrangements of cases with stereotyped IGHV4-34/IGKV2-30 BCRs (subset#4). Recurrent amino acid changes at certain positions across the entire IGKV/IGLV sequence were observed at a high frequency (27–67% of cases) in a number of stereotyped subsets, especially those expressing the IGHV3-21/IGLV3-21 BCR (subset #2) and the IGHV4-34/IGKV2-30 BCR (subset #4). Comparison with CLL LC sequences carrying heterogeneous K/LCDR3s or non-CLL LC sequences revealed that these distinct amino acid changes are greatly under-represented in such groups and appear therefore to be “subset-biased”. Finally, a significant proportion of CLL cases (63 cases; 26 kappa- and 37 lambda-expressing) with monotypic LC expression were found to carry multiple potentially functional LC rearrangements. Of note, nineteen of these 63 cases (30%) belonged to subsets with stereotyped BCRs. This finding alludes to the possibility of secondary rearrangements most likely occurring in the context of (auto)antigen-driven receptor editing, particularly in the case of stereotyped subsets. In conclusion, SHM targeting in CLL LCs appears to be just as precise and, most likely, functionally driven as in heavy chains. Secondary LC gene rearrangements and subset-biased mutations in CLL LC genes are strong indications that LCs are crucial in shaping the specificity of leukemic BCRs, in association with defined heavy chains. Therefore, CLL is characterized not only by stereotyped HCDR3 and heavy chains but, rather, by stereotyped BCRs involving both chains, which create distinctive antigen binding grooves.
4370 The cell of origin of chronic lymphocytic leukemia (CLL) has long been sought and immunoglobulin gene analysis provides new clues. The immunoglobulin heavy variable gene (IGHV) status has clinical relevance in CLL, where two subsets, delineated by the absence or presence of somatic mutation, have markedly different prognoses. The unmutated subset (U-CLL), of inferior prognosis, appears to derive from a pre-germinal center B cell. In U-CLL, there is strikingly increased usage of the 51p1-related alleles of the IGHV1-69 gene, often combined with selected IGHD genes and with IGHJ6. Shared sequence “stereotypic” characteristics of the HCDR3 result, and suggest antigen selection of the leukemic clones. In this study, we have analyzed 147 51p1/IGHJ6 rearrangements from 3 healthy individuals (>51yr) and sought sequence patterns parallel to those of U-CLL. A pre-established dataset of 313 51p1/IGHJ6 rearrangements from patients with U-CLL was used as a reference. A high proportion (49/147, 33.3%) of normal sequences revealed stereotypic patterns, several (22/147, 15%) being similar to those described in U-CLL. Additional CLL-associated stereotypes, not yet reported, were detected in 7/147 sequences (4.8%). Stereotypes (13.6%) not detected in CLL were also found in 20/147 (13.6%) 51p1/IGHJ6 combinations. The HCDR2-IGHJ6 sequences were almost exclusively unmutated (143/147, 97,3% sequences had ≥98% homology to germline). Junctional amino acids in normal B cells were heterogeneous, as in the cases of CLL with stereotyped 51p1/IGHJ6 B-cell receptors. Normal B cells expressing 51p1-derived IgM (4.8% of all B-cells) had a phenotype of naïve B-cells, similar to 51p1-negative (CD27-) B cells, i.e. IgM+ IgD+ CD23+ CD38+, with a small percentage of CD5+ B cells, not found in the memory B-cell subset. This snapshot of the naïve B-cell repertoire reveals subsets of B cells closely related to those characteristic of CLL. Conserved patterns in the 51p1-encoded IgM of normal B cells suggest a restricted sequence repertoire shaped by evolution to recognize common pathogens. Proliferative pressure on these cells is the likely route to U-CLL. Disclosures: No relevant conflicts of interest to declare.
We examined SHM features in 1967 IGH rearrangements from 1939 patients with CLL. The sequences were divided into four “identity groups”; “truly unmutated” (100% identity to germline; 677 sequences), “minimally mutated” (99–99.9% identity; 133 sequences), “borderline mutated” (98–98.9% identity; 93 sequences) and “mutated” (<98% identity; 1064 sequences). At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged on analysis of subgroups of sequences. In particular, the IGHV repertoire of the four “identity groups” differed considerably, with the IGHV1-69 and IGHV1-2 genes predominating among “truly unmutated” and “minimally mutated” sequences, respectively. In contrast, other genes were mostly used in “mutated” rearrangements (eg, IGHV4-34/3-23/3-7). Of note, IGHV3-21 and IGHV3-48 had the highest proportion of “borderline mutated” rearrangements. In selected groups of sequences, a remarkable preservation of the germline configuration was observed in superantigenic binding motifs, prompting speculation that subsets of CLL cells could also receive stimulation signals by superantigenic-like interactions. In detail, the vast majority of IGHV4-34 sequences retained germline conformation at the four FR1 positions of the IGHV4-34-specific I/i binding motif. Similarly, IGHV3-21 sequences displayed remarkably few alterations of the IGHV3-specific staphylococcal protein A binding motif. Following established criteria, we identified 530/1967 sequences (27%) as belonging to 113 different subsets with stereotyped HCDR3. The distribution of sequences among subsets differed significantly according to mutational status: 43% of “truly unmutated” sequences belonged to a subset, compared to only 16% of the “mutated” group (p<0.001). Of note, among certain IGHV genes (ie, IGHV1-2/3-21/4-34/4-4), shared “stereotyped” amino acid (AA) changes (i.e. the same AA replacement at the same position) occurred across the entire IGHV sequence significantly more frequently in cases with stereotyped vs. heterogeneous HCDR3s and therefore, could be considered as “subset-biased”. Stereotyped AA changes were also observed in subsets of minimally mutated cases, indicating that even a low level of mutations may be functionally relevant. Comparison to public-database non-CLL sequences revealed that certain stereotyped AA changes were over-represented in CLL and thus could also be considered as “CLL-biased”. The very precise targeting and distinctive features of SHM in subgroups of CLL patients provide further evidence for the important role of selection by specific antigen(s) in CLL leukemogenesis.
2337 Poster Board II-314 Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen (Ag) recognition through the clonotypic B cell receptors (BCRs). However, it is still unclear whether Ag involvement is restricted to the malignant transformation phase or whether the putative Ag(s) may continuously trigger the CLL clone. Valuable insight into these issues may be gleaned from the study of intraclonal diversification (ID) within the immunoglobulin (IG) genes through ongoing somatic hypermutation (SHM). Definitive data regarding ID within IG genes in CLL remains limited and conflicting. In the present study we systematically explored the presence of ID within IG genes of CLL, not only at cohort level but also in subgroups defined by BCR stereotypy and IG gene mutational status. We thus conducted a large-scale subcloning study of both IG heavy and light variable genes, in a total of 1496 and 1008 subcloned sequences from 71 and 56 CLL cases, respectively. The analysis was intentionally biased to cases expressing IGHV4-34/IGKV2-30 IGs (subset #4) and IGHV3-21/IGLV3-21 IGs (subset #2) that exhibit distinctive, subset-biased SHM patterns. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and at least 14 colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample (ii) confirmed mutation (CM) - a mutation observed more than once among subcloned sequences from the same sample. Analysis of heavy chain sequences revealed that 40% (28/71) of cases carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, whilst 32% (23/71) of cases carried only UCMs. The remaining 28% (20/71) of cases carried sets of identical IGHV-D-J subcloned sequences. Although most cases showed no or low levels of ID, an intense and, likely, functionally driven ID was evident in selected cases, especially those belonging to subset #4. The distinct ID in subset #4 was statistically significant when compared to all other groups defined by IGHV gene usage and mutation status, BCR stereotypy or heavy chain isotype. Subsequent analysis of the clonotypic light chains revealed that the impact of ID was generally low, with the outstanding exception again relating to subset #4. In fact, of 22 IGKV-J rearrangements exhibiting CMs, 11 (50%) utilized the IGKV2-30 gene and notably 10/11 (91%) of these were expressed by subset #4 cases. In such cases, the expressed IGKV2-30 gene was affected by an active and precisely targeted ID, analogous to their partner IGHV4-34 gene. These findings suggest that the SHM mechanism may continuously operate in certain subsets of CLL patients, particularly those cases expressing stereotyped IGHV4-34/IGKV2-30 BCRs typical of subset #4. In such cases, the observed ID patterns attest to the very precise targeting of the SHM process and may be considered as evidence for a “stereotyped response” to an active, ongoing interaction with Ag(s). Disclosures: No relevant conflicts of interest to declare.
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