Immunoglobulin gene segment usage, location and immunogenicity in mutated and unmutated chronic lymphocytic leukaemia

research paper Immunoglobulin gene segment usage, location and immunogenicity in mutated and unmutated chronic lymphocytic leukaemia Katja Mauerer, 1 David Zahrieh, 2 Gullu Gorgun, 1 Aihong Li, 1 Jianbiao Zhou, 1 Sascha Ansén, 1 Laura Z. Rassenti 3 and John G. Gribben 1 1 Department of Medical Oncology, 2 Department of Biostatistics and Computational Biology, Dana- Farber Cancer Institute, Harvard Medical School, Boston, MA, and 3 CLL Research Consortium, University of California San Diego, CA, USA Received 23 January 2005; accepted for publication 3 March 2005 Correspondence: John G. Gribben, DSc, MD, Cancer Research UK Medical Oncology Unit, Barts and The London School of Medicine, Charterhouse Square, London EC1M 6BQ, UK. E-mail: john.gribben@cancer.org.uk Summary The mutational status of the variable region of the immunoglobulin heavy chain gene (IgV H ) is an important prognostic marker in B-cell chronic lymphocytic leukaemia (B-CLL), with mutated patients having improved outcome. To examine the impact of mutational status on V H,D H, and J H gene segment location and immunogenicity, we analysed 375 IgH sequences from 356 patients with B-CLL. Although V H and D H gene usage was different in mutated compared to unmutated patients, there was no impact of gene location on frequency of use or clinical outcome. Surprisingly, somatic mutations did not increase the immunogenicity of the Ig, as assessed by predicted binding affinity of Ig-derived peptides to major histocompatibility Class I and Class II molecules. Even excluding patients using V H 1-69, cases using the V H 1 gene family had a poor outcome. Both mutated and unmutated CLL patients demonstrated evidence of antigen selection. The worst outcome was seen in the subset of 14 unmutated patients with similar HCDR3 amino acid sequence using V H 1-69, D H 3-3 and J H 6, suggesting an antigen-driven process modulating the clinical course. Keywords: chronic lymphocytic leukaemia, V H D H J H gene usage, HCDR3, antigen selection, MHC binding affinity. B-cell chronic lymphocytic leukaemia (B-CLL) is the most prevalent adult leukaemia in Western countries, accounting for approximately 30% of all leukaemias (Rai & Patel, 1995). This clinically heterogeneous disease is characterized by the clonal accumulation of CD5 + B-cells that express intact antigen receptors (Caligaris-Cappio et al, 1982). The immunobiology of this disease has been the subject of extensive research, but remains incompletely understood. During early B cell differentiation, antibody diversity is generated by recombination of the different germline heavy chain variable (V H ), diversity (D H ) and joining (J H ) gene segments in the immunoglobulin (Ig) heavy chain locus and light chain variable (V L ) and joining (J L ) gene segments in the two light chain loci (Tonegawa, 1983; Alt et al, 1987). Additional diversity of the antibody repertoire is provided by nucleotide deletions and insertions at the VD, DJ, or VJ joints and by the usage of three different open reading frames for the D-gene resulting in the creation of the heavy chain complementarity determining region 3 (HCDR3), considered as the core portion, responsible for antigen recognition (Tonegawa, 1983). Final affinity maturation is initiated in the germinal centre, in response to antigen stimulation by somatic hypermutation (SHM). The mutational status of the variable region of the Ig heavy chain gene (IgV H ) has been established as a crucial prognostic factor in B-CLL, with mutated cases showing a significantly longer overall survival compared with patients with tumour cells lacking SHM (Damle et al, 1999; Hamblin et al, 1999). Although the mechanisms responsible for specific V H gene usage are poorly understood, a preferential usage of specific IgH rearrangements has been observed in several lymphomas and leukaemias (Rettig et al, 1996; Camacho et al, 2003), perhaps relating to the stage of differentiation of the oncogenic event. In B-CLL cells compared with normal B cells, the most prevalent V H genes are V H 1-69, V H 3-7, V H 3-21 and V H 4-34 (Johnson et al, 1997; Fais et al, 1998; Hamblin et al, 1999). Previous studies have demonstrated a biased utilization of V H 1-69 rearrangements in unmutated cases combined with skewed D H 3-3 and J H 6 gene usage, encoding longer than ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 499 510 doi:10.1111/j.1365-2141.2005.05480.x

K. Mauerer et al average HCDR3s with conserved amino acid motifs (Widhopf & Kipps, 2001; Potter et al, 2003). These findings of the biased repertoire of V H,D H, and J H gene segments, as well as similar amino acid motifs in HCDR3, led to the hypothesis of an antigen component involved in the pathogenesis of B-CLL. In B-lineage acute lymphoblastic leukaemia (ALL) the position of the V H gene segment used on chromosome 14q32 has been implicated in V H D H J H usage and related to stage of B cell differentiation (Mortuza et al, 2001; Li et al, 2004). No previous studies have examined V H gene location in correlation to clinical outcome in B-CLL. In the light of these data, the present study analysed the V H, D H, and J H gene rearrangements of a large cohort of 356 patients with B-CLL to further determine features of V H D H J H - recombination associated with changes in disease progression and outcome and to investigate further evidence of antigen selection in B-CLL. In addition, we sought to examine whether the presence of SHM was associated with increased immunogenicity of Ig-derived peptides presented with major histocompatibility complex (MHC) class I or II, a potential explanation of the better clinical outcome in patients with mutated type CLL. Materials and methods Patients and material A total of 356 patients with typical B-CLL based on clinical criteria and laboratory features according to National Cancer Institute guidelines (Cheson et al, 1996) were included in the study. Sequencing was performed in 189 during the watch and wait phase from patients who continued to attend outpatient haematology clinic at the Dana-Farber Cancer Institute (DFCI) and in a further 167 cases at enrolment onto two sequential chemotherapy first line treatment protocols in patients who had not previously attended DFCI. The median age at diagnosis of these patients was 52 years (range: 22 82 years). Fifty-seven per cent were male, 43% were female. Peripheral blood mononuclear cells (PBMC) were isolated from blood samples by density gradient centrifugation using Ficoll-Paque, lysed, and genomic DNA and/or RNA extracted and purified according to the manufacturer s instructions using the nucleospin kit (BD Biosciences, Palo Alto, CA, USA). The studies were approved by our institutional review board and informed consent obtained from all patients for analysis of samples. PCR amplification of IgH gene rearrangements and direct sequencing of PCR product To identify patient leukaemia-related IgH gene rearrangements, diagnostic PB samples were amplified by polymerase chain reaction (PCR) using a series of six V H leader or seven V H family FR1 consensus primers and a J H consensus primer (Invitrogen, Carlsbad, CA, USA) in a modification of a method previously described (Deane et al, 1991; Provan et al, 1996). Clonal PCR products were excised and purified using QIAquick gel extraction kits (QIAGEN, Valencia, CA, USA). Purified PCR fragments were sequenced in the Dana- Farber/Harvard Cancer Center Core Sequencing Facility (Boston, MA, USA). Sequence reactions were analysed on an Applied Biosystems 3700 capillary sequencer using Big Dye Terminator Chemistry version 2 (Applied Biosystems, Foster City, CA, USA). The relevant consensus forward and reverse primers were used as sequence primers to obtain the sequence of both strands. Nucleotide sequences were aligned using the DNAstar software (DNASTAR, Madison, WI, USA). Analyses of V H,D H,andJ H sequences and HCDR3 structure V H, D H, and J H segments were identified with a closest matching known human germline gene using the ImMuno- Gene Tics (IMGT) Database (http://imgt.cines.fr, IMGT, European Bioinformatics Institute, Montepellier, France), the IGBLast search (http://www.ncbi.nlm.nih.gov/igblast/, National Cancer for Biotechnology Information, Bethesda, MD, USA), or V BASE directory using dnaplot (http:// www.mrc-cpe.cam.ac.uk, Centre for Protein Engineering, Cambridge, UK). For D H gene determination, a requirement of a minimum of six nucleotide matches in a row or seven matches interrupted by one mismatch was used. Mutations were measured by comparing the CLL and germline IgH gene at the DNA and protein levels, however the assignment of a patient to the germline or mutated group was based on DNA changes. V H sequences deviating more than 2% from the corresponding germline gene were defined as mutated (Damle et al, 1999; Hamblin et al, 1999). Previously published data on the use of V H,D H, and J H genes in healthy peripheral B cells (PBL) (Brezinschek et al, 1995, 1997) were used as the controls in this study. Assessment of the distribution of replacement and silent mutations within the CDRs and framework (FR) regions and possible impact of antigen selection on the hypermutation process (for cases with >2% deviation from the closest germline V H gene) was performed according to the multinomial model proposed by Lossos et al (2000), taking advantage of their computer program at the http://wwwstat.stanford.edu/immunoglobulin website. The criteria used to define subsets with similar (marked homology) HCDR3s were as follows: (1) use of the same V H,D H, and J H germline genes; (2) the IgH rearrangements showed an amino acid similarity within the HCDR3s of >60% identity; and (3) use of the same D H segment reading frame. HCDR3 length was calculated between codon 95 at the end of FR3 [usually two amino acids (aa) downstream of the conserved cystine] and codon 102 at the beginning of FR4 (a conserved tryptophan in all J H segments) as previously described (Kabat et al, 1991). Acid and basic aa and HCDR3 charge, as defined by an estimated pi, were determined using the ProtParam software (http://us.expasy.org/tools/protparam.html). 500 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 499 510

VDJ usage and immunogenicity in CLL Epitope prediction analysis The IgV H -deduced protein sequences of a subset of 36 mutated and 36 unmutated patients with known human leucocyte antigen (HLA) type were reviewed for nonameric and decameric peptides that could potentially bind to MHC class I and II molecules. Two independent computer prediction analysis tools were used to examine the binding of peptides to HLA-A*0201 molecules, the bimas algorithm (http://thr.cit.nih.gov/molbio/ hla_bind) and the syfpeithi database (http://www.syfpeithi.de). For class I, the analysis was confined to HLA-A*0201 (HLA-A2) because this MHC class I allele is the most common, expressed in approximately 50% of our tumour patients. To increase the sensitivity of our predictions, the threshold for binding was set at a predicted half-life of 1 min using the bimas algorithm and at a predicted score of 15 using the syfpeithi database. In addition, the immunoglobulin sequences were scanned for potential MHC class II binding peptides, using the syfpeithi database and Propred algorithm (http://www.imtech.res.in/ raghava/propred/) (Singh & Raghava, 2001). Statistical analysis The Wilcoxon rank-sum test (Wilcoxon, 1945) was used for testing differences between continuous variables and associations between categorical variables were assessed by a Fisher exact test (Cox, 1970). The Jonckheere Terpstra test (Pirie, 1983) was used to test for ordered differences in response among classes. No statistical adjustment was made for performing multiple tests. All probability values are two-sided. The 189 patients enrolled during watch and wait were assessable for the time to treatment endpoint. Patients time to treatment reflects follow-up information as of July 2004. The method of Kaplan & Meier (1958) was used to estimate time to treatment curves and the log-rank test (Mantel, 1966) was used to compare curves. Results V H gene usage, spatial relationship, and mutation analysis In the present study, 375 IgH rearrangements of a large cohort of 356 B-CLL patients were analysed; 19 (5%) patients showed biallelic V H rearrangements in keeping with previous observations (Rassenti & Kipps, 1997). A total of 202 (54%) of the 375 IgV H genes analysed had less than 2% difference from the most similar germline gene, with 84 (42% of the unmutated subgroup) in 100% germline configuration. A total of 173 (46%) of the 375 V H sequences were classified as mutated, with a median of 5Æ3% (range: 2Æ1 13Æ5%) mutations. Twenty per cent of the V H genes had between 2% and 5% mutations and 26% had more than 5% mutations. The most common V H family subtype used was V H 3 (41%), in keeping with the size of this V H family in the germline (Cook & Tomlinson, 1995; Matsuda et al, 1998), followed by V H 1 (33%), and V H 4 (17%), as expected (Duke et al, 2003; Messmer et al, 2004a). Compared with the pattern of V H family usage in adult peripheral blood derived B-cells reported by Brezinschek et al (1995), B-CLL showed higher than expected usage of V H 1 genes (33% vs. 13%), and lower than expected usage of V H 3 genes (41% vs. 56%). Overall, the most frequently encountered gene was V H 1-69, representing 18% of 375 genes detected and 56% of the V H 1 genes, followed by V H 3-23 (7%) and V H 4-34 (7%), in line with previous observations (Hamblin et al, 1999; Duke et al, 2003). Mutations detected in the leukaemic cells varied according to V H family and specific V H gene use. As shown in Fig 1A, V H 1 gene family (50%) was overused, while V H 3 (32%) and V H 4 (10%) were underused in unmutated CLL compared to mutated B-CLL and normal B cells. In the V H 1 family, the lack of mutations was clearly skewed by the striking over-usage of V H 1-69 in the unmutated patient group (Fig 1B) (Deane & Norton, 1991; Johnson et al, 1997; Hamblin et al, 1999). The most commonly used genes in the mutated group (Fig 1B) were V H 3-7 (7%), V H 3-23 (10%), V H 3-48 (8%) and V H 4-34 (10%), in keeping with previous studies (Duke et al, 2003; Ghia et al, 2004). The frequency of the usage of the specific V H segments by their position on chromosome 14 in a telomeric-to-centromeric direction is shown in Fig 1B. When this region was divided into four clusters, each of approximately 200 kb, we observed a privileged use of the D H -distal V H segments compared with normal B-cells (P ¼ 0Æ05). However, this was accounted for almost entirely by the marked over-usage of V H 1-69, used in 33% of unmutated sequences and 2% of mutated sequences. Excluding the overused V H 1-69 in the group of patients with unmutated IgH genes, we observed a negative relationship between frequency of V H gene usage and distance from the J H segments (P ¼ 0Æ02), with a pattern more similar to that seen in B-ALL (Mortuza et al, 2001; Li et al, 2004). V H gene segment usage in mutated CLL was not different to that observed in healthy B cells (P ¼ 0Æ4). B-cells with receptors that have been selected by antigen often display a higher frequency of replacement (R) compared with silent (S) mutations in the CDRs than in the FRs (Jukes & King, 1979; Shlomchik et al, 1987). The B-CLL mutations analysed here followed a similar pattern, with an elevated R/S ratio in CDRs (3Æ45) and decreased R/S ratio in FRs (1Æ39, Table I). Overall, FR regions accumulated more mutations (61%) than CDR regions (39%), however the targeting preference of R mutations in the CDRs and away from the FRs in B-CLL sequences were consistent with an antigen driven SHM process. Of note, this was also true in those cases with <2% mutations (R/S ratio 5Æ20 in CDRs versus 1Æ57 in FRs), which are, in the current usage, defined as unmutated (Damle et al, 1999; Hamblin et al, 1999). In fact, the absolute R/S ratio of the CDRs was even higher in unmutated versus mutated cases (5Æ20 vs. 3Æ40), suggesting that even in the unmutated cases, there was evidence of antigen selection. Using the multinomial distribution model (Lossos et al, 2000), evidence for antigen selection was indicated in 65% of the mutated rearrangements. ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 499 510 501

K. Mauerer et al A Per cent (%) 60 50 40 30 20 PBLs 10 0 V H 1 V H 2 V H 3 V H 4 V H 5 V H 6 B 35 30 25 20 15 Per cent (%) Cluster A 200 kb 200 kb 200 kb 200 kb PBLs Cluster B Cluster C Cluster D 10 5 0 3-74 3-72 2-70 1-69 3-66 3-64 4-59 1-58 3-53 5-51 3-49 3-48 1-46 3-43 4-39 4-34 3-33 4-31 3-30 4-28 1-24 3-23 3-21 1-18 3-15 3-13 3-11 3-9 1-8 3-7 2-5 4-4 1-3 1-2 6-1 V H locus Telomere DJ H loci Centromere Fig 1. (A) V H family usage profile in mutated and unmutated CLL patients compared with healthy controls (PBLs). (B) V H gene segment usage profile by physical location on chromosome 14 in 375 rearrangements from 356 patients with B-CLL. Table I. Targeting and base change characteristics of mutations in B-CLL. R/S ratio Number of sequences Number of mutations Number of mutations CDR Number of mutations FR Total CDR FR Overall 375 2758 1070 1688 1Æ92 3Æ45 1Æ39 173 2549 1006 1543 1Æ90 3Æ40 1Æ37 202 209 64 145 2Æ12 5Æ20 1Æ57 D H gene usage and spatial relationship Among the 375 IgH sequences, D H segments could be identified clearly in 353 (94%) sequences. Four sequences had two D H segments for a total of 357 D H gene segments. The D H 3 gene family was used most frequently (46%), followed by D H 2 (19%), and D H 6 (15%). D H 2 and D H 3 were overrepresented whereas D H 4 and D H 5 gene segments were under-represented in B-CLL HCDR3 regions compared with usage in PBLs (Brezinschek et al, 1995). The D H 3 family showed over-usage in the unmutated group (57%) compared with mutated CLL (31%) and healthy controls (38%; Fig 2A). The telomeric-to-centromeric position of these genes is shown in Fig 2B. Four clusters were assigned, each of approximately 15 kb. In contrast to a previous report (Duke 502 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 499 510

VDJ usage and immunogenicity in CLL A Percent (%) B Percent (%) C Percent (%) 60 50 40 30 20 10 35 30 25 20 15 10 5 0 0 D H 1 D H 2 D H 3 D H 4 D H 5 D H 6 D H 7 PBLs 1-1 2-2 3-3 4-4 5-5 6-6 2-8 3-9 3-10 4-11 5-12 6-13 2-15 3-16 4-17 5-18 6-19 1-20 2-21 3-22 4-23 5-24 6-25 1-26 7-27 V H locus 60 50 40 30 20 10 0 Cluster A 15kb Telomere PBLs Cluster B Cluster C Cluster D 15kb 15kb 15kb J H loci Centromere PBLs J H 1 J H 2 J H 3 J H 4 J H 5 J H 6 Fig 2. (A) D H family usage profile in mutated and unmutated CLL patients compared with healthy controls (PBLs). (B) D H gene segment usage profile by physical location on chromosome 14 in 357 rearrangements from 356 patients with B-CLL. (C) J H family usage profile in mutated and unmutated CLL patients compared with healthy controls. et al, 2003), no significant correlation between the frequency of gene usage and distance from J H segments was observed either overall or in the mutated CLL group, suggesting that mechanisms other than gene location are likely to govern the selection of gene segments. Molecular processes via recombination activation genes (RAG1/2) and specific recombination signal sequences (RSS) are probable candidates (Oettinger et al, 1990; McBlane et al, 1995). CLL cases, however, overused J H distal D H genes (P <0Æ0001) and were mostly accounted for by overuse of D H 2-2 (15%) and D H 3-3 (27%). J H gene usage Usage of the J H 4 gene family was 37%, followed by J H 6 (33%), and J H 5 (18%), in line with previous studies (Hamblin et al, 1999; Duke et al, 2003). Compared with healthy B cells (Brezinschek et al, 1995), J H 5 was more frequently encountered (18% vs. 7%). There was a marked over-usage of J H 6in the unmutated patient group (42%) compared with the mutated CLL patients (23%; P <0Æ0001). The results are summarized in Fig 2C. HCDR3 length, charge, and structural homology The median HCDR3 length for the 375 sequences was 48 bp. Analysing the HCDR3 length of the mutated and unmutated CLL patients separately, we confirmed a significantly longer HCDR3 length in unmutated CLL (median: 57 bp, range: 21 90 bp) than in mutated CLL patients (median 41 bp, range: 16 81 bp; P ¼ 0Æ0001) (McHeyzer- Williams et al, 1993; Brezinschek et al, 1997). The germline CLL patients showed a median HCDR3 length of 54 bp (range 28 78). Moreover, in unmutated B-CLL cells, the HCDR3 region contained more negatively charged amino acids, resulting in a lower calculated isoelectric point (median pi 3Æ8) compared with mutated cases (median pi 4Æ8; P ¼ 0Æ005). These V H gene mutation-related differences in HCDR3 length and charge may reflect selection for specific structural motifs that facilitate antigen binding. In keeping with the notion that unmutated cases might also be antigen driven, we identified patients with identical HCDR3 regions. The most striking sequence homology was noted for V H 1-69-expressing CLL samples that used the D H 3-3 gene segment along with J H 6. By cluster analysis of the amino acid sequence of these 21 V H 1-69 expressing samples, we identified four distinct CLL samples from unrelated patients with identical heavy chain sequences including identical HCDR3s, 21 aa in length (motif VSGSTIFGVVIQKIYYYYMDV, Fig 3). The remaining 17 sequences using V H 1-69, D H 3-3 and J H 6 fell into two groups based upon their use of the alternative reading frames from D H 3-3. Seven of these used the hydrophobic reading frame from D H 3-3 as those cases described above, and had marked CDR3 homology (Fig 3). The other 10 used the hydrophilic D H 3-3 reading frame and nine of those 10 cases reached the criteria of similar HCDR3 regions with marked homology (Fig 3). Moreover, we identified two further rearrangement patterns shared by three and two distinct CLL samples respectively featuring completely identical V H sequences. Three distinct CLL samples used V H 3-11, D H 3-10 and J H 1 with identical HCDR3, 13 aa in length (motif PPNYYYGSGSYIH) and two cases used V H 5-51, D H 3-3 and J H 4 with identical HCDR3, 19 aa in length (motif HASYYDFWSGYFLPSHFDY). All 25 patients with such identical or similar HCDR3 regions were unmutated. ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 499 510 503

K. Mauerer et al FR3 CDR3 FR4 V H1-69, D H3-3, J H6 N1 D N2 J 100% homology, D segment: hydrophobic RF Germline G Y C A R I T I F V V I I Y Y Y Y G M D V W G CLL 36... S....... Q I.... _..... V S G K CLL 88... V S G S....... Q K I.... _..... CLL 146... V S G S....... Q K I.... _..... CLL 186... V S G S....... Q K I.... _..... FR3 CDR3 FR4 N1 D N2 J Marked homology, D segment: hydrophobic RF Germline C A R I T I F G V V I I Y Y Y Y Y G M D V W G CLL 85... S....... Q I T T T T * R L G.. V S G K CLL 117... I G G T...... N L G P.... _..... CLL 174... A D........ V Q D.......... CLL 179... S G D....... N........... G A CLL 264... C...... N V..... _..... Y CLL 268... V A..... N T Y..... _..... CLL 309... V Q..... T H Y..... _..... FR3 CDR3 FR4 N1 D N2 J Marked homology, D segment: hydrophilic RF Germline C A R Y Y D F W S G Y Y Y Y Y Y Y G M D V W G CLL 84... D F E G G G....... P N.......... CLL 107... G S N G........ P N.......... CLL 184... V S G G....... S T G D......... CLL 319... A G N....... H A A..... _..... CLL 4... D S G M V N...... S P A........... CLL 159... G G G N...... W R A L.......... CLL 241... L P P....... S Q S L Y........... CLL 277... W I Q L D....... I R P S........... CLL 92... E G S........ P N.......... FR3 CDR3 FR4 V H3-11, D H3-10, J H1 N1 D J J 100% homology Germline S F Q H C A R Y Y Y G G S Y Y N E Y W G CLL 67........... _ I... P P N CLL 127... P P N........ _ I... CLL 336... P P N........ _ I... FR3 CDR3 FR4 V H5-51, D H3-3, J H4 N1 D N2 J 100% homology Germline W D C A R Y Y D F S G Y Y Y F Y W G CLL 329........... F H..... H A S L P S CLL 356... H A S........ F L P S H..... Fig 3. Alignment of HCDR3 amino acid sequences obtained from identical (100% homology) and similar (marked homology) Ig heavy chains expressed in three subsets of unrelated and unmutated CLL patients. Amino acid sequences are shown flanked by the 3 end of FR3 and the 5 end of FR4. A dot indicates homology to the germline sequence. Stopcodons are shown as asterisks (*) and deletions from germline sequences are indicated as dashes ( ). V H D H J H gene rearrangement and clinical outcome The impact of V H D H J H gene rearrangement on prognosis was analysed in a subset of 189 B-CLL patients whose sequences were obtained at presentation during the watch and wait phase. Since the remaining patients were sequenced specifically at the time of treatment and therefore represent a skewed data set, they were excluded from this analysis. Treatment was recorded for 98 patients and 91 patients remained untreated as of July 2004. As expected, the median time from diagnosis to treatment for unmutated patients (median: 2Æ7 years) was significantly shorter than that of mutated patients (median: 5Æ8 years; P < 0Æ0001; Fig 4A). Analysing the association of the time from diagnosis to treatment with the usage of specific V H segments by their position on chromosome 14 in a telomeric to centromeric direction, we observed a worse outcome of patients using D H distal V H genes (P ¼ 0Æ001). However, this was accounted for entirely by the poor outcome of patients using V H 1-69 (P ¼ 0Æ45, when these patients were excluded from analysis). Irrespective of specific gene segment usage, patients using V H 1 family members had a significantly shorter time to treatment (median: 3 years), compared with the other V H families (V H 2 to V H 6, median 5Æ5 years; P <0Æ001; Fig 4B), and even when the much-overused V H 1-69 gene was excluded from the analysis, a significantly shorter time from diagnosis to treatment for the V H 1 family members persisted (3Æ9 years vs. 5Æ5 years; P ¼ 0Æ05; Fig 4C). No significant association between D H gene location and time to treatment could be detected (P ¼ 0Æ22). Patients utilizing D H 2 and D H 3 had significantly shorter time to treatment (3Æ8 vs. 6Æ0 years; P ¼ 0Æ003) reflecting the over usage of these gene families in the unmutated patient group. J H usage was not associated with time to treatment (P ¼ 0Æ83). We noted a trend (P ¼ 0Æ08) in favour of a shorter time to treatment in the 14 unmutated patients with identical HCDR3 or marked HCDR3 sequence homologies sharing V H 1-69 in combination with D H 3-3 and J H 6 compared with unmutated patients with heterogeneous HCDR3 rearrangements utilizing V H 1-69 along with other D H J H gene usage (0Æ8 vs. 3Æ0 years; Fig 4D). Prediction of HLA epitopes in immunoglobulin Since SHM could result in increased immunogenicity of the Ig, the heavy chain rearrangements from a subset of 36 mutated and 36 unmutated patients were translated and analysed, using bioinformatics, for potential binding peptides to their MHC class I and II. From these sequences, we identified more than 700 nonameric and decameric peptides using a low binding threshold to HLA-A*0201 (predicted half-life for binding greater than or equal to 1 min in bimas and a score greater or equal 15 in syfpeithi). Most peptides displayed a binding half-life or score in the low or intermediate affinity range (95% in a range of half life 1 120 min in bimas and 98% in a score range 15 25 in syfpeithi). Only five of 354 CDR-derived and three of 369 FR-derived nonameric peptides had a binding half-life of greater than 4 h in bimas and one of 451 CDRderived and 19 of 616 FR-derived peptides showed a binding score greater than 26 in syfpeithi (Tables II and III, respectively). The majority of peptides were FR-rather than CDR-derived (nonameric peptides 616 vs. 451 in syfpeithi, 504 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 499 510

VDJ usage and immunogenicity in CLL Fig 4. (A) Kaplan Meier plot comparing time from diagnosis to treatment between mutated and unmutated V H genes. (B) Kaplan Meier plot comparing time from diagnosis to treatment between V H 1 and V H 2toV H 6 family genes. (C) Kaplan Meier plot comparing time from diagnosis to treatment between V H 1 (excluding V H 1-69) and V H 2toV H 6 family genes. (D) Kaplan Meier plot comparing time from diagnosis to treatment between unmutated rearrangements using V H 1-69, D H 3-3, J H 6 with marked sequence homologies and unmutated V H 1-69 rearrangements utilizing other D H and J H rearrangements. Table II. Number (%) of HLA-A*0201-binding CDR- and FR-derived peptides 9 and 10 amino acids in length in relation to predicted binding half-life analysed by the bimas database. Number of epitopes 1 60 min 61 120 min 121 240 min >240 min Nonamer bimas Total (72 patients) 723 (100) 648 (90) 41 (6) 26 (4) 8 (1) FR-derived 369 (51) 310 (84) 36 (10) 20 (5) 3 (1) CDR-derived 354 (49) 338 (95) 5 (1) 6 (2) 5 (1) (36 patients) 384 (53) 348 (91) 16 (4) 13 (3) 7 (2) (36 patients) 339 (47) 300 (89) 25 (7) 13 (4) 1 (1) Decamer bimas Total (72 patients) 710 (100) 677 (95) 16 (2) 9 (1) 8 (1) FR-derived 458 (65) 438 (96) 10 (2) 6 (1) 4 (1) CDR-derived 252 (35) 239 (95) 6 (2) 3 (1) 4 (2) (36 patients) 375 (53) 353 (94) 8 (2) 6 (2) 8 (2) (36 patients) 335 (47) 324 (97) 8 (2) 3 (1) 0 (0) respectively), with two-thirds in the CDR2-FR3-CDR3 geographic region of the IgV H and fewer peptides within the flanking FR1-CDR1-FR2 regions. We defined a peptide as being derived from the CDR if one or more amino acids of the peptide were located within the CDR (Trojan et al, 2000). None of the patient sequences reported here were included in the previously published studies (Trojan et al, 2000). Of note, there was no statistically significant difference in predicted ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 499 510 505

K. Mauerer et al Table III. Number (%) of HLA-A*0201-binding CDR- and FRderived peptides 9 and 10 amino acids in length in relation to predicted Score analysed by the syfpeithi database A BIMAS score-nonamer binding affinity of nonameric or decameric peptides to HLA- A*0201 in mutated and unmutated CLL patients (P ¼ 0Æ41), as illustrated in Fig 5A,B. However, we observed an increased number of FR1 region peptides in mutated patients (ratio FR1/ CDR3 2Æ1), whereas more peptides were CDR3-derived (ratio FR1/CDR3 0Æ7) in the unmutated patients. Also, the overall number of epitopes derived from mutated patients was consistently higher than those from unmutated patients. Although the algorithms for predicted binding affinity for MHC class II molecules are less well developed, the frequency of MHC class II binding peptides were similar in mutated and unmutated sequences, providing no evidence of selection pressure against specific class II binding epitopes. A limitation of idiotypic vaccinations is the need to develop patient-specific reagents. We observed that 30% of FR peptides were shared among patients with only 80 different HLA- A*0201 binding peptide sequences identified, and four peptides shared among 10 or more patients. We also identified low-scoring CDR-derived peptides that were identical in several patients and therefore not unique for the clonal B cells. In addition, we found no increase in the number or in the scores of nonameric or decameric peptides predicted to bind to HLA-A2 in Ig sequences from HLA-A2-negative patients (P ¼ 0Æ29), as shown in Fig 6A and B, indicating no selection pressure against the development of sequences that could be recognized by cytotoxic T lymphocytes (CTLs). Discussion Total number of epitopes 15 16 17 25 >26 Nonamer syfpeithi Total (72 patients) 1067 (100) 406 (38) 641 (60) 20 (2) FR-derived 616 (58) 177 (29) 420 (68) 19 (3) CDR-derived 451 (42) 229 (51) 221 (49) 1 (0Æ2) (36 patients) 545 (51) 221 (41) 309 (57) 15 (3) (36 patients) 522 (49) 185 (35) 332 (64) 5 (1) Decamer syfpeithi Total (72 patients) 816 (100) 434 (53) 376 (46) 6 (1) FR-derived 559 (69) 275 (49) 278 (50) 6 (1) CDR-derived 257 (31) 159 (62) 98 (38) 0 (0) (36 patients) 437 (54) 218 (50) 215 (49) 4 (1) (36 patients) 379 (46) 216 (57) 161 (43) 2 (1) In B-CLL there is distinct V H,D H, and J H gene segments usage patterns compared with healthy B cells or other B cell malignancies (Rettig et al, 1996; Fais et al, 1998; Widhopf & Kipps, 2001; Camacho et al, 2003; Duke et al, 2003; Potter et al, 2003). The Ig mutational status in this disease is associated with distinct cytogenetics and clinical outcome and is also associated with differences in V H,D H, and J H gene Binding affinity (score) B Binding affinity (score) BIMAS score-decamer Fig 5. Predicted binding affinity to HLA*A0201 in mutated and unmutated CLL patients. The top line in the box plot represents the 75% level, the bottom line the 25% level, and the middle line shows the median or 50% level. The Graph comprises the bulk of peptides with a bimas Score less than 60 min. (A) No statistically significant difference in predicted binding affinity of nonameric peptides to HLA*A0201 using the bimas algorithm in mutated and unmutated patients. (B) No statistically significant difference in predicted binding affinity of decameric peptides to HLA*A0201 using the bimas algorithm in mutated and unmutated patients. segments usage (Fais et al, 1998; Hamblin et al, 1999; Duke et al, 2003; Degan et al, 2004). The present study confirmed and extended these studies and, in particular, examined whether this preferential gene usage was determined by chromosomal location. In B-lineage ALL there is an association between gene location and V H D H J H gene usage that is probably associated with the ontogenetically more immature phenotype of B-lineage ALL cells compared with healthy B cells (Mortuza et al, 2001; Li et al, 2004). No such spatial relationship was observed in the present study for mutated compared with unmutated CLL cases, suggesting that molecular mechanisms rather than gene location regulate gene segments selection in B-CLL (Oettinger et al, 1990; McBlane et al, 1995). We also examined whether Ig SHM increased immunogenicity in Ig mutated cases and, surprisingly, found no evidence to support this hypothesis. B-cells with receptors that have been selected by antigen exhibit a higher frequency of amino acid replacement (R) 506 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 499 510

VDJ usage and immunogenicity in CLL A Binding affinity (score) B Binding affinity (score) HLA-A*0201 positive BIMAS score - nonamer HLA-A*0201 positive BIMAS score - decamer HLA-A*0201 negative HLA-A*0201 negative Fig 6. Predicted binding affinity for HLA*A0201 in HLA-A2 positive and negative CLL patients. The top line in the box plot represents the 75% level, the bottom line the 25% level, and the middle line shows the median or 50% level. The Graph comprises the bulk of peptides with a bimas Score less than 60 min. (A) No statistically significant difference in predicted binding affinity of nonameric peptides to HLA*A0201 using the bimas algorithm in HLA-A2 positive and negative CLL patients. (B) No statistically significant difference in predicted binding affinity of decameric peptides to HLA*A0201 using the bimas algorithm in HLA-A2 positive and negative CLL patients. mutations in the sequences encoding the antigen-contacting CDRs than in those encoding the structural FRs (Kocks & Rajewsky, 1989). A recent study demonstrated that the targeting preference for RGYW motifs, base charge bias for transitions, and focusing of R mutations in CDRs, away from FRs were consistent with the hypothesis that V H mutations in B-CLL result from the classical SHM process in response to antigen stimulation (Messmer et al, 2004a). We observed a similar pattern with an increased R/S ratio in CDRs and a lowered R/S ratio in FRs. Of note, cases with less than 2% mutations from the most similar germline gene, normally classified within the unmutated group, also demonstrated a similar distribution in keeping with the canonical SHM process. Further evidence for antigen selection as a promoting factor in CLL development is the identification of CLL subsets, largely within the unmutated patients, with highly restricted HCDR3 features, including identical V H,D H, and J H usage, HCDR3 length, and shared N-sequences. The probability of the same V H D H J H rearrangement occurring twice is in the order of one in 7650 (1/51 1/25 1/6), representing the probability of each particular gene relative to the total number of functional genes in each group (Widhopf et al, 2004). However, subsets of cases with CLL have been described that share distinct IgV region genes (Tobin et al, 2003, 2004; Ghiotto et al, 2004; Messmer et al, 2004b, Widhopf et al, 2004). In the present series, we identified 21 of the 375 cases using V H 1-69 with D H 3-3 and J H 6, among which four cases had identical heavy chains with another 16 cases having marked HCDR3 homology. In addition, we identified identical HCDR3 regions in three patients using V H 3-11, D H 3-10 and J H 1 and in two patients using V H 5-51, D H 3-3 and J H 4, strongly indicative of antigen selection in subsets of CLL. Therefore, these findings are unlikely to be a random phenomenon. These specific HCDR3 features implicate that CLL tumours are expressing highly similar Ig structures on their surfaces, pointing to restricted antigen recognition sites and possibly involvement of specific antigen in CLL development, however the identity of such an antigen is currently unknown. The recently identified V H 4-39/VkO2-utilizing IgG + CLL subgroup has been shown to display comparable antigen binding sites to monoclonal antibodies reacting towards bacterial carbohydrates and certain autoantigens (Ghiotto et al, 2004). In addition, an antibody termed SMI, which utilizes the V H 1-69/ VkA27 genes and has been isolated from a CLL patient, shows reactivity to a variety of self antigens, such as human Ig, myoglobulin, thyroglobulin, actin and single-stranded DNA (Martin et al, 1992). Interestingly, the restriction of HCDR3s found in our subgroups was primarily confined to the unmutated cases with poor prognosis. Considering the recent data showing more B-cell receptor (BCR) signalling, as measured by syk phosphorylation, in unmutated cases than in mutated cases (Lanham et al, 2003), this may indicate that a limited number of antigens may signal through the BCR and play a more important role in tumour progression of unmutated cases, calling for further studies of the binding sites of such restricted BCRs. Taken together, these findings, as well as the increased R/S ratio in HCDRs in the mutated and minimally mutated cases, are in keeping with an antigendriven process in all CLL patients, including both mutated and unmutated cases, which is compliant with the gene expression profiling data, suggesting a common gene expression signature irrespective of mutational status in CLL (Klein et al, 2001; Rosenwald et al, 2001). However, we did observe additional differences in mutated compared with unmutated cases with respect to HCDR3 length and charge. cases had significantly shorter HCDR3 length with less negatively charged amino acids and higher estimated pi compared with unmutated cases. However, this might reflect selection for specific structural motifs that facilitate antigen binding, since antibodies with a shorter HCDR3 length and more space in the antibody-binding pocket may bind to antigens with a higher affinity (Rosner et al, 2001). ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 499 510 507

K. Mauerer et al On analysis of the impact of V H gene usage by their position on chromosome 14 on prognosis a significantly higher probability of earlier requirement for treatment in patients using D H distal V H genes was noted. However, this was accounted for entirely by the poor outcome of patients using V H 1-69, found predominantly in the unmutated patients [66 of 202 (33%) unmutated cases but only three of 173 (2%) mutated cases]. The prognostic role of V H gene SHM status in CLL is well established (Damle et al, 1999; Hamblin et al, 1999) and in the present study the median time from diagnosis to treatment for unmutated patients was significantly shorter than that of mutated patients. Of interest, the subset of 14 unmutated patients with marked HCDR3 amino acid sequence homologies utilizing V H 1-69 in combination with D H 3-3 and J H 6 had an even worse outcome than the remaining unmutated patients utilizing V H 1-69 with other D H, and J H rearrangements, not only suggesting an antigen-driven cellular mechanism but potentially directly implicating specific antigens in the modulation of clinical disease. In addition, we observed a significantly shorter time to treatment for all V H 1 family members, even when the much over-used V H 1-69 gene was excluded from analysis. Our data suggest that location of V H gene segments does not influence outcome and therefore other mechanisms rather than gene localization appear to be responsible for the poorer outcome of patients using specific V H gene segments. Despite much effort, the biologic basis for the difference in clinical outcome between mutated and unmutated patients remain elusive, although gene expression and, as mentioned above, differential BCR signalling have been described (Klein et al, 2001; Rosenwald et al, 2001; Lanham et al, 2003). Our group and others have previously shown that Ig-derived peptides can be presented in a MHC-dependent manner and induce CTL responses (Trojan et al, 2000; Harig et al, 2001). The strength of CTL responses depends upon the binding affinity of the target peptide to HLA class I (Sette et al, 1994). Therefore, potential differences in the binding affinity of such Ig-derived peptides between mutated and unmutated patients were an intriguing hypothetical correlate for the distinct clinical outcome. Although we have not formally assessed immunogenicity for all cases in the present analysis, we have previously demonstrated an association between binding affinity of Ig-derived peptides and immunogenicity (Gricks & Gribben, 2003). Surprisingly, our analysis using bioinformatics of mutated and unmutated patients revealed no statistically significant difference of the predicted binding affinity of peptides to specific MHC class I and II alleles. Therefore, we found little evidence that the improved clinical outcome of mutated patients is associated with an increased immune response to such peptides in mutated B-CLL, unless one assumes that the consistently higher number of epitopes found in all subsets of mutated versus unmutated patients plays a cumulative role, thus initiating more immune responses. On the other hand our data indicate that both unmutated and mutated patients may equally profit from immunotherapeutic strategies, especially in the light of the more recent data that low binding peptides may also induce T cell-mediated cytotoxicity (Gross et al, 2004; Hernandez et al, 2004). A limitation of idiotypic immunotherapy is the need to develop patient-specific vaccines. In that context it should be noted that 30% of the FR-derived peptides analysed were shared among patients. Given the IgV H sequence homologies observed in the present and other studies, this is not unexpected and may introduce to a new era of vaccination therapy (Lou et al, 2004). In summary, the non-random use of gene segments, the targeting preference of replacement mutations in the CDR region and the expression of highly homologous HCDR3 structures among CLL subsets provides further evidence of antigen selection as a promoting factor in CLL development. Our data suggest that V H D H J H recombination is not dependent on the physical localization on chromosome 14q32, but more likely to be governed by molecular mechanisms. In addition, we found that the position of the V H gene segments does not influence clinical outcome, thus other mechanisms rather than the location are likely to be responsible for the poorer clinical outcome of CLL patients using V H 1-69. Finally there was no significant difference in the binding affinity of HLA epitopes of the Ig of mutated and unmutated patients, suggesting that both patient groups may profit from immunotherapy targeting such peptides. However, the identification of a set of commonly expressed epitopes may further lead to a more widespread use of this approach, which is currently under investigation in preclinical in vivo models. Acknowledgements The present study was supported by grants from the Deutsche Krebshilfe and Dr Mildred Scheel Stiftung (to K.M. and S.A.) and by a grant CA81538 to the CLL Research Consortium from the National Cancer Institute (to J.G.G.). References Alt, F.W., Blackwell, T.K. & Yancopoulos, G.D. (1987) Development of the primary antibody repertoire. Science, 238, 1079 1087. Brezinschek, H.P., Brezinschek, R.I. & Lipsky, P.E. (1995) Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction. Journal of Immunology, 155, 190 202. Brezinschek, H.P., Foster, S.J., Brezinschek, R.I., Dorner, T., Domiati- Saad, R. & Lipsky, P.E. (1997) Analysis of the human VH gene repertoire. Differential effects of selection and somatic hypermutation on human peripheral CD5(+)/IgM+ and CD5(-)/IgM+ B cells. Journal of Clinical Investigation, 99, 2488 2501. Caligaris-Cappio, F., Gobbi, M., Bofill, M. & Janossy, G. 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