Leukemia Research 38 (2014) 509–515
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Analysis of the IGHV region in Burkitt’s lymphomas supports a germinal center origin and a role for superantigens in lymphomagenesis Maria Joao Baptista a,1 , Eva Calpe a , Eva Fernandez b , Lluis Colomo b , Teresa Marta Cardesa-Salzmann c , Pau Abrisqueta a , Francesc Bosch a,∗,2 , Marta Crespo a,2 a Laboratory of Experimental Hematology, Department of Hematology, Vall d’Hebron University Hospital, Universitat Autonòma de Barcelona, Barcelona, Spain b Department of Pathology, Hospital Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain c Department of Pediatric Oncology, Hospital Sant Joan de Déu, University of Barcelona, Barcelona, Spain
a r t i c l e
i n f o
Article history: Received 7 October 2013 Received in revised form 20 December 2013 Accepted 1 January 2014 Available online 13 January 2014 Keywords: BL IGHV SHM Superantigens Intraclonal diversity Glycosylation
a b s t r a c t The analysis of immunoglobulin heavy chain variable (IGHV) region may disclose the influence of antigens in Burkitt’s lymphomas (BL). IGHV sequences from 38 patients and 35 cell lines were analyzed. IGHV3 subset genes were the most used and IGHV4-34 gene was overrepresented. IGHV genes were mutated in 98.6% of the cases, 36% acquired potential glycosylation sites, and in 52% somatic-hypermutation-process was ongoing. Binding motifs for superantigens like Staphylococcal protein A and carbohydrate I/i were preserved in 89% of the cases. IGHV analysis of BL cells supports a germinal center origin and points toward a role for superantigens in lymphomagenesis. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Burkitt’s lymphoma (BL) is a B-cell lymphoproliferative disorder classified into three clinical variants according to the geographic distribution, the infection of the Epstein–Barr virus (EBV), and the association with human immunodeficiency virus (HIV). The clinical variants are endemic BL (eBL), sporadic BL (sBL) and HIV-associated BL (HIV-BL) [1]. On the basis of morphology, phenotype, and genetics, BL is currently regarded as a germinal center (GC) derived neoplasm, however in the WHO classification a different origin for the endemic and sporadic forms has been suggested due to differences in the expression of specific EBV-related molecules and in somatic hypermutation (SHM) patterns [1].
∗ Corresponding author at: Department of Hematology, University Hospital Vall d’Hebron, Pg de la Vall d’Hebron 119-129, 08035 Barcelona, Spain. Tel.: +34 4893806. E-mail address: [email protected] (F. Bosch). 1 Current address: Josep Carreras Leukaemia Research Institute, Department of Hematology, ICO-Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain. 2 These authors contributed equally to this manuscript. 0145-2126/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2014.01.001
The molecular analysis of the immunoglobulin heavy chain variable (IGHV) region provides insight into the stage of B-cell development at which clonal expansion occurs in B-cell tumors and the possible involvement of antigen selection. A biased use of certain IGHV genes and/or common heavy complementary determining region 3 (HCDR3) has been found in several leukemia and lymphomas and suggests a potential role for antigens in the proliferation of tumor B-cells [2–4]. The presence of mutations in the IGHV genes has been used as a marker of the passage of B-cells through the GC and the presence of intraclonal diversity has been used to identify tumor clones derived from GC [5]. Moreover, the acquisition of potential glycosylation sites in IGHV genes has been described in GC derived tumors but has been reported to be uncommon in normal B-cells [6,7]. These sites can be responsible for interactions with elements in the GC microenvironment and their acquisition can be linked to ongoing SHM activity [6]. Interestingly, recent works have suggested that autoantigens and superantigens may be also implicated in the development of B-cell tumors, like the possible role for carbohydrate I/i and for Staphylococcal protein A (SpA) in chronic lymphocytic leukemia (CLL) development by stimulating proliferation of B-cells that express particular subsets of immunoglobulin genes [4,8].
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The aim of this work was to perform a comprehensive analysis exploring the features of the IGHV region in a large series of BLs in order to give insights into BL lymphomagenesis. For that, IGHV region genes were studied focusing on the IGHV mutational status, the presence of intraclonal diversity, the acquisition of potential glycosylation sites, and the presence of superantigenic-binding motifs.
stages, 2 at stage I, 5 at II, 5 at III and 2 at IV. According to the 3 clinical variants of BL, one case was an eBL (sample 24), 4 were HIV-BL (samples 5–7 and 10) and the remaining 21 were sBL. EBV infection was studied in 14 cases. Infection was detected in 27.3% of the sBL (3 out of 11) and in 33.3% of HIV-BL (1 out of 3).
2. Materials and methods
In this study we conducted a comprehensive analysis of the IGHV regions of BL cells including a total of 73 cases. IGHV-D-J rearrangements were identified in 65 cases, being 93.8% (61 out of 65) functionally productive and 6.2% (4 out of 65) potentially productive (Supplementary Table S1). Genes from the IGHV3 family were the most prevalently used (47.9%), followed by IGHV4 (35.6%), IGHV1 (9.6%), IGHV2 (4.1%), and IGHV5 (2.7%) families. Overall, the usage of individual IGHV genes was significantly different from that of the CD5(−) normal B-cells (P = 0.044) (Supplementary Fig. S1) [21]. When individual genes were compared, the frequency of the IGHV4-34 gene was significantly higher in BL than in normal B-cells (17.8% vs. 4.3%; P < 0.001). Neither a bias in the usage of IGHD and IGHJ genes nor a preferential combination of V-D-J genes was found. HCDR3 lengths were very heterogeneous and the HCDR3 amino acid sequences revealed no stereotyped subsets according to previously established criteria [2].
2.1. Patients and samples A total of 73 BL cases were studied: 26 patients diagnosed in our institution and 12 patients and 35 cell lines whose IGHV-D-J sequences were retrieved from public databases (For more details please see Supplementary Table S1). Accession numbers for the sequences retrieved from public databases are the following: Z46269, Z46284–Z46288 [9]; AF006517–AF006528 [10]; X87438, X87440, X87442, X87444, X87447, X87449, X87451, X87453, X87459 [11]; Z74663, Z74665, Z74668, Z74671, Z74672, Z74693, Z74695 [12]; AF176233–AF176239 [13]; L29115, L29116 [14]; X82081, X82083, X82085 [15]; and AF003377 [16]. BL cells from the cases diagnosed in our institution were obtained from tumor biopsy (N = 17), bone marrow (N = 5), peripheral blood (N = 2), pleural fluid (N = 1), or ascitic fluid (N = 1) before treatment was initiated. The IGHV-D-J sequences have been submitted to GenBank, and accession numbers are listed in Supplementary Table S1. 2.2. Analysis of IGHV-D-J rearrangements IGHV-D-J rearrangements were identified by PCR amplification and subcloning procedures. For details of PCR amplification please see Supplementary Materials and methods. The analysis of the nucleotide sequences was performed with the use of IMGT/VQUEST and ClustalW/EMBL tools [17,18].
3.2. BL have biased IGHV gene usage
3.3. Mutational profile of IGHV in the majority of BL is consistent with an antigen-driven SHM process
2.3. Potential glycosylation sites analysis The IGHV amino acid sequences from the 73 BL cases were studied for the presence of potential inherent or acquired glycosylation sites. Thus, sequences were scanned from heavy chain framework region 1 (HFR1) to HFR3 to find the N-X-S/T N-glycosylation motif, where X can be any amino acid except for P, D, or E [6]. 2.4. Superantigen binding sites analysis The presence of binding motifs for SpA and carbohydrate I/i superantigens was analyzed. SpA binding motif is restricted to the IGHV3 subgroup. It consists of 13 amino acids and only one non-conservative amino acid change is allowed to retain SpA activity. The inversion of charge from K to E in position 65 is considered deleterious because it induces electrostatic repulsion [19]. Carbohydrate I/i HFR1 hydrophobic patch consists of a W at position 7 on -strand A and of A-V-Y at positions 24–26 on -strand B of IGHV4-34 gene transcripts. In order to maintain binding affinity no non-conservative amino acid change is admitted [20]. 2.5. Statistical analysis Fisher’s exact test and 2 -test, with Bonferroni adjustment for multiple comparisons, were used to estimate differences in nominal variables. Quantitative variables comparisons were done with Student t-test and with ANOVA or with Mann–Whitney and Kruskal–Wallis and a significance level of P = 0.05 was set. Analyses were performed with the use of SPSS v18.0 (IBM, Somer, NY).
3. Results 3.1. Characteristics of the patients The study group comprises previously reported and characterized BL cases (N = 47) as well as de novo BL cases diagnosed at our institution (N = 26). The main clinical and biological characteristics of the patients from our institution are summarized in Supplementary Table S2. Briefly, there were 21 male patients and 5 female. Fourteen patients were considered pediatric, since they were younger than 16 years old, and the remaining 12 were adults. The median age at diagnosis was 39 years old for adults (range, 27–69) and 6 years old for children (range, 3–16). Adult patients were mainly diagnosed at stage IV (N = 11) with only 1 patient at stage II. Pediatric patients were diagnosed at all four different
The analysis of the IGHV mutational profile of the series revealed a high mutational load (mean % germline identity (GI) ± standard deviation (SD): 94.7 ± 3.7). In this BL series, 80.8% (59 out of 73) of the cases were considered to express mutated IGHV genes whereas 17.8% (13 out of 73) had borderline/minimally mutated IGHV genes (Supplementary Table S1). We considered that all but one of the cases had undergone SHM (For details about the analysis of IGHV mutational load see Supplementary Materials and methods). The mutational load of sBL cases (N = 49) was lower than the one observed in eBL cases (N = 14) (mean %GI ± SD: 96.1 ± 2.6 vs. 92.2 ± 4.1; P = 0.001) and than the one observed in HIV-BL (N = 10) (mean %GI ± SD: 96.1 ± 2.6 vs. 91.2 ± 3.7; P < 0.001). Concerning EBV infection, there was a trend for higher mutational load in EBVinfected cases but this difference was not statistically significant (mean %GI ± SD: 93.2 ± 3.9 vs. 95.1 ± 3.8; P = 0.052). The immunoglobulin class mainly used was IgM (IgM: 58 out of 68; IgG: 8 out of 68; and IgA: 2 out of 68). No preferential light chain use was detected (kappa: 29 out of 54 and lambda: 25 out of 54). No association between heavy or light chain usage and clinical variants, EBV status, IGHV mutational status, or %GI was found. In CLL the absence of SHM is related to adverse prognosis and correlates with high expression of ZAP-70 protein [22], however whether levels of ZAP-70 also correlate with IGHV mutational status in BL has not been ascertained yet. In our series, 23.1% of BL cases (6 out of 26) were positive for ZAP-70 (for details about ZAP70 analysis please see Supplementary Materials and methods) but no correlation was found between ZAP-70 expression and IGHV mutational load. 3.4. BL cases show intraclonal diversity and acquire potential glycosylation sites To study intraclonal diversity in the BL cases diagnosed at our institution, at least 8 subclones with identical or nearly identical HCDR3 sequences from each case were compared to the corresponding germline sequence (Table 1). The presence of a
M.J. Baptista et al. / Leukemia Research 38 (2014) 509–515 Table 1 Analysis of intraclonal diversity in BL. Sample number
Clinical variant
Number of subclones
IGHV gene
CM
UCM
ID
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 24 25 26
sBL sBL sBL sBL HIV-BL HIV-BL HIV-BL sBL sBL HIV-BL sBL sBL sBL sBL sBL sBL sBL sBL sBL sBL sBL sBL eBL sBL sBL
10 9 9 10 11 10 10 11 24 10 12 12 12 14 14 14 12 11 15 12 12 11 8 11 12
IGHV5-a IGHV3-30-3 IGHV4-59 IGHV4-59 IGHV3-33 IGHV3-48 IGHV4-39 IGHV4-39 IGHV2-5 IGHV3-30 IGHV3-74 IGHV1-8 IGHV4-39 IGHV4-59 IGHV3-30 IGHV3-11 IGHV4-34 IGHV4-59 IGHV2-70 IGHV3-30 IGHV4-34 IGHV3-21 IGHV3-21 IGHV1-24 IGHV3-21
0 0 2 0 7 1 4 0 0 4 7 1 1 1 0 0 2 0 0 0 1 1 0 3 0
1 2 0 5 12 10 6 2 7 10 2 4 16 14 27 11 23 10 5 4 10 10 5 12 8
No No Yes No Yes Yes Yes No No Yes Yes Yes Yes Yes No No Yes No No No Yes Yes No Yes No
Abbreviations: CM, confirmed mutation (a mutation observed in more than one subclonal sequence but not in all sequences); UCM, unconfirmed mutation (a mutation observed only in one subclonal sequence); ID, intraclonal diversity; sBL, sporadic Burkitt lymphoma; eBL, endemic Burkitt lymphoma; HIV-BL, HIV-associated Burkitt lymphoma.
confirmed mutation (a mutation observed in more than one subclonal sequence but not in all sequences) was considered indicative of ongoing SHM. Based on this criterion, intraclonal diversity was detected in 52% of cases (13 out of 25). The presence of glycosylation sites in the IGHV genes allows Bcells to interact with stromal elements present in the GC potentially facilitating the retention of cells in these structures [6,23]. In follicular lymphoma (FL), the glycosylation of surface immunoglobulins creates a functional bridge between FL cells and microenvironmental lectins that can replace the need for antigen stimulation for B-cell survival in the GC [23]. When studying the whole series herein described, we found that 26 out of 72 cases with mutated IGHV genes (36.1%) acquired new potential glycosylation sites (Table 2). Cases with novel potential glycosylation sites presented higher mutational loads than those without them (mean %GI ± SD: 92.5 ± 3.8 vs. 95.9 ± 3.0; P < 0.001). The incidence of BL cases with novel glycosylation sites was statistically different between the different clinical variants (eBL: 64.3%, sBL: 25.0%, HIV-BL: 50.0%; P = 0.016); less sBL acquired novel glycosylation sites when compared to eBL (P = 0.010) but not with HIV-BL. EBV infected cases showed similar incidence of novel glycosylation sites (45.8%) than not-infected cases (34.5%). Finally, 72% of the cases (18 out of 25) analyzed for both the presence of glycosylation sites and intraclonal diversity showed at least one of the two features. Altogether, these findings suggest that BL cells derive from the GC. 3.5. Superantigenic-binding motifs are not affected by the SHM process in BL The presence of binding motifs for superantigens has not been extensively studied in leukemia/lymphoma; however, some subsets of CLL cases have been found to retain those motifs, suggesting a putative role for superantigens in the selection of the clonogenic progenitor [4,8]. SpA and carbohydrate I/i are superantigens
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whose binding motifs are present in the germline sequence of the genes from the IGHV3 subgroup and in the IGHV4-34 gene (HFR1 hydrophobic patch) respectively [19,20]. Interestingly, the IGHV3 subgroup was the most used by BL cells and the IGHV4-34 gene usage was biased. Altogether, genes from the IGHV3 subgroup and the IGHV4-34 gene accounted for 65.7% of the IGHV genes expressed in this BL series. We scanned the amino acid sequences from IGHV3 subgroup for the presence of SpA binding sites considering only those cases with a known nucleotide sequence from HFR1 to HFR3 (N = 26) (Table 3). We observed that in 24 out of the 26 IGHV3 subgroup cases, the SpA binding motif was conserved. In addition, no mutation leading to loss of activity was found in any of the 8 BL cases with ongoing SHM. The presence of mutations in the hydrophobic patch for carbohydrate I/i in HFR1 was investigated in all cases expressing the IGHV4-34 gene except in case 29 because its nucleotide sequence for HFR1 was unknown. Most of the BL cases kept the binding motif for carbohydrate I/i intact (10 out of 12) (Table 4). Moreover, in none of the 2 BL cases that had ongoing SHM and used the IGHV4-34, the binding motif was affected. SpA and carbohydrate I/i binding motifs were preserved even in those BL cases highly targeted by SHM, pointing toward a role for these superantigens in the pathogenesis of BL.
4. Discussion The molecular analysis of the IGHV region may offer some clues into the pathogenesis of B-cell derived lymphomas and may also provide evidence for interactions of the malignant clones with the microenvironment [5]. The analysis of the IGHV gene repertoire conducted in this series showed that IGHV3 was the predominant subgroup used by BL cells, followed by IGHV4, and revealed that the IGHV repertoire used by BL cells was different from the one used by normal B-cells. Moreover, the IGHV4-34 gene was found overrepresented, confirming the trend that was previously observed in several smaller series [10,24]. Interestingly, an over-usage of this gene has also been reported in other lymphomas/leukemias [8,25–27]. The IGHV4-34 gene encodes for intrinsically autoreactive antibodies and it’s underrepresented in GC and memory B-cells from healthy individuals in order to prevent plasma cells from producing auto-antibodies [28]. The high prevalence of BL cells that express IGHV4-34 suggests that autoreactive antigens can be involved in driving the expansion of BL. Likewise, during acute EBV and Cytomegalovirus infections increased titers of IGHV4-34 antibodies are detected, and patients with CLL infected with EBV also have IGHV4-34 gene overrepresentation [29]. In our BL series, however, those cases infected with EBV did not show a bias in IGHV4-34 usage. Notwithstanding, since EBV infection is strongly associated with BL and EBV could act in a hit and run fashion leaving little trace of its presence in the cells, a role for EBV in the selection of the tumor cells should not be discarded. The mutational load of the IGHV genes of this BL series was high (mean %GI = 94.7) and mutations were found in all but one of the cases. These results indicate that BL cells have been targeted by the SHM machinery reinforcing their putative GC or post-GC origin [1]. Moreover, eBL cases were found to have higher mutational loads (mean %GI = 92.2) than sBL cases (mean %GI = 96.1) whereas HIV-BL and eBL cases showed similar mutation loads, which corroborates previous reports on the different mutational load in IGHV of BL clinical variants [11,24,30]. We observed a trend for higher mutational load in EBV-infected cases (mean %GI = 93.2 vs. 95.1), but this did not reach statistical significance, in line with previously published
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Table 2 Potential glycosylation sites in the IGHV genes of BL cases. Sample number
Sample type
IGHV gene
Constitutive glycosylation site Position
43 66 25 47 34 12 32 9 19 16 28 42 64 23 24 26 30 35 37 48 49 54 69 10 15 20 38 61 65 67 2 5 31 6 40 46 60 63 68 45 50 33 11 51 55 17 21 29 39 44 53 57 58 59 70 71 72 73 7 8 13 36 52 56 41 3 4 14 18 27 62 1
Cell line Cell line Primary cell Cell line Primary cell Primary cell Primary cell Primary cell Primary cell Primary cell Primary cell Cell line Cell line Primary cell Primary cell Primary cell Primary cell Primary cell Primary cell Cell line Cell line Cell line Cell line Primary cell Primary cell Primary cell Primary cell Cell line Cell line Cell line Primary cell Primary cell Primary cell Primary cell Cell line Cell line Cell line Cell line Cell line Cell line Cell line Primary cell Primary cell Cell line Cell line Primary cell Primary cell Primary cell Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line Primary cell Primary cell Primary cell Primary cell Cell line Cell line Cell line Primary cell Primary cell Primary cell Primary cell Primary cell Cell line Primary cell
IGHV1-18 IGHV1-2 IGHV1-24 IGHV1-46 IGHV1-69 IGHV1-8 IGHV1-8 IGHV2-5 IGHV2-70 IGHV3-11 IGHV3-11 IGHV3-15 IGHV3-15 IGHV3-21 IGHV3-21 IGHV3-21 IGHV3-21 IGHV3-23 IGHV3-23 IGHV3-23 IGHV3-23 IGHV3-23 IGHV3-23 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30-3 IGHV3-33 IGHV3-43 IGHV3-48 IGHV3-48 IGHV3-48 IGHV3-48 IGHV3-48 IGHV3-53 IGHV3-66 IGHV3-66 IGHV3-7 IGHV3-74 IGHV3-74 IGHV4-31 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-39 IGHV4-39 IGHV4-39 IGHV4-39 IGHV4-39 IGHV4-39 IGHV4-4 IGHV4-59 IGHV4-59 IGHV4-59 IGHV4-59 IGHV4-59 IGHV5-51 IGHV5-a
HFR3-81 HFR3-81
HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57
HFR3-66
aa seq
NTS NTS
NHS NHS NHS NHS NHS NHS NHS NHS NHS NHS NHS NHS NHS
NYS
Novel glycosylation site Status
Position
aa seq
Status
No No No No No Mantained Mantained No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No Mantained Mantained Mantained Lost Lost Mantained Mantained Mantained Mantained Lost Mantained Mantained Lost No No No No No No No No No No No No No Lost
HFR3-66
NYS
HCDR2-63
NYT
HCDR2-60
NNS
HCDR1-36 HCDR1-36
NYT NYS
HCDR2-58
NSS
HCDR1-29
NFS
HFR3-84
NNT
HFR3-84 HFR3-84 HFR3-77
NNT NNT NIS
HCDR1-29 + HCDR2-59 HCDR2-63 HFR3-84
NFS + NNT NTT NNS
HCDR1-36
NYS
HFR3-84 HFR1- 24
NNT NVS
HFR3-90
NLT
HFR3-68 + HFR3-90 HFR3-68
NSS + NLS NAS
HFR3-66 HCDR1-31
NYT NST
HFR1-24 + HCDR1-34
NVS + NSS
HFR1-24 HCDR2-59
NVS NGT
HCDR1-29 + HFR3-95
NFT + NAS
Yes No No No No No No No No Yes No Yes No Yes Yes No Yes No No Yes No No No No No No Yes No Yes Yes Yes No No Yes Yes Yes No Yes No No No No No Yes Yes No Yes No Yes Yes No No No No No No No Yes Yes No No Yes No No No No No No Yes Yes No Yes
Notes: Amino acid changes leading to the acquisition of novel potential glycosylation sites are underlined.
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Table 3 Presence of the SpA binding site in BL cases using the IGHV3. Sample number
Sample type
IGHV gene
HFR1 16
16 42 64 48 49 54 69 15 10 20 38 61 65 67 2 5 6 40 46 60 63 68 45 50 11 51
Primary cell Cell line Cell line Cell line Cell line Cell line Cell line Primary cell Primary cell Primary cell Primary cell Cell line Cell line Cell line Primary cell Primary cell Primary cell Cell line Cell line Cell line Cell line Cell line Cell line Cell line Primary cell Cell line
18
IGHV3-11 IGHV3-15 IGHV3-15 IGHV3-23 IGHV3-23 IGHV3-23 IGHV3-23 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30-3 IGHV3-33 IGHV3-48 IGHV3-48 IGHV3-48 IGHV3-48 IGHV3-48 IGHV3-53 IGHV3-66 IGHV3-66 IGHV3-74 IGHV3-74
SpA binding motif
20
HCDR2
HFR3
65
67
SPA binding motif presence 72
74
75
77
79
90
92
93
R I S
E F
Q
S E M
C
D N
N T G G
R A G
S
R/K
S
G
N/G
S
L
K/I/T
Y
K
G
R
T
S
Q
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Notes: Amino acids are represented by a single letter code, non-conservative amino acid replacements are underlined, and the inversion of amino acid charge in HCDR2-65 position is shown in shadow.
data [24], although differences were reported by others [10,30]. Thus, our results could not rule out whether EBV can influence the mutational load of the IGHV genes. Previous studies in lymphoproliferative diseases derived from GC B-cells have showed that the majority of FL and eBL cases, and nearly half of the sBL and diffuse large B-cell lymphoma cases, acquire potential glycosylation sites in the IGHV genes, contrary to what is found in normal B-cells and other lymphoproliferatives diseases like CLL and multiple myeloma [6,7]. In the BL series herein analyzed, 36.1% of the cases acquired new potential glycosylation sites, and the acquisition of these motifs was correlated with higher mutational load. Even though the former association was not observed in a previous work [7], our results are in line with the notion that glycosylation sites help B-cells to stay in the GC and thus higher mutation loads can be achieved. In addition, we
found differences in the acquisition of glycosylation sites among the different clinical variants. In this sense, the variant associated with higher IGHV mutational loads, eBL, presented the higher incidence of acquisition of glycosylation sites. Notwithstanding, EBV-infection seemed not to be related to the acquisition of glycosylation sites since the incidence of their acquisition was similar in EBV-infected and non-infected cases. The analysis for both the presence of intraclonal diversity and glycosylation sites showed that 72% of the cases analyzed had at least one of the two features, which link cells to the GC microenvironment. Moreover, in all cases analyzed for intraclonal diversity, no novel potential glycosylation site was removed due to ongoing SHM. Also, in 2 out of 14 cases with no previous potential glycosylation sites, a subclonal mutation generating a novel potential glycosylation site was detected. Glycosylation sites may
Table 4 Presence of the carbohydrate I/i HFR1 hydrophobic patch in BL cases using IGHV4-34. Sample number
Sample type
HFR1 7
17 21 39 44 53 57 58 59 70 71 72 73 Carbohydrate I/i HFR1 hydrophobic patch
Primary cell Primary cell Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line
Carbohydrate I/i hydrophobic patch presence 24
25
26
G S S G V
I W
A
V
Y
Notes: Amino acids are represented by a single letter code and non-conservative amino acid replacements are underlined.
Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes
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therefore represent an advantage for tumor survival and expansion since they were not removed but acquired by some cases during the ongoing SHM process. These results are in agreement with other characteristics from BL cells, such as the expression of CD10 and BCL6, which indicate that a mature B-cell experiencing the GC reaction would be the normal counterpart for BL. The analysis for the binding sites for superantigens carbohydrate I/i and SpA in IGHV4-34 gene and IGHV3 subgroup genes, respectively, showed that these sites were preserved in the majority of cases (89.5%). Moreover, the ongoing SHM process found in cases with intraclonal diversity did not introduce mutations in these binding motifs in any subclone. Thus, SpA and carbohydrate I/i binding motifs were preserved even in those BL cases highly targeted by SHM suggesting that superantigens may be contributing to clonal selection and expansion of BL cells. Finally, patients included in the present study were both pediatric and adult and, importantly, samples were obtained from a variety of tissues, which may have influenced the results as a consequence of the interaction with different microenvironments. In conclusion, the detection of ongoing SHM and the presence of glycosylation sites in a high percentage of BL cases herein analyzed reinforce the hypothesis of a mature B-lymphocyte experiencing the GC reaction being the normal counterpart for BL. Moreover, the biased usage of IGHV3 family and IGHV4-34 genes and the fact that SHM does not affect superantigenic binding motifs suggest that superantigens may have a role in the pathogenesis of BL. These results contribute to a better knowledge of BL cell of origin and suggest a possible role for superantigens as players in BL lymphomagenesis process. Funding This work was supported by grants from Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias FIS 08/0211 and FIS PI11/00792, and from Fundació Marató de TV3 05/1810. M.J.B. was a recipient of a PhD fellowship (SFRH/BD/28698/2006) granted by Ministério da Ciência, Tecnologia e Ensino Superior, Portugal. M.C. holds a contract from Ministerio de Economía y Competitividad (MINECO) (RYC-2012-12018), Spain. Author’s contributions M.J.B., M.C. and F.B. designed the study; M.J.B., E.C. and E.F. performed the laboratory work; L.C., T.M.C.S., and P.A. recruited the patients; M.J.B. and M.C. analyzed and interpreted the data; M.J.B. performed statistical analysis; M.J.B., M.C., and F.B. wrote the manuscript. All authors approved final version of the manuscript. Conflicts of interest statement The authors reported no potential conflicts of interest. Acknowledgments The authors would like to thank Professor Freda K. Stevenson (University of Southampton Faculty of Medicine, Southampton, United Kingdom) for critical reading of the manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.leukres.2014. 01.001.
References [1] Swerdlow SH, Campo E, Harris LN, Jaffe ES, Pileri SA, Stein H, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC press; 2008. [2] Stamatopoulos K, Belessi C, Moreno C, Boudjograh M, Guida G, Smilevska T, et al. Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: pathogenetic implications and clinical correlations. Blood 2007;109:259–70. [3] Zibellini S, Capello D, Forconi F, Marcatili P, Rossi D, Rattotti S, et al. Stereotyped patterns of B-cell receptor in splenic marginal zone lymphoma. Haematologica 2010;95:1792–6. [4] Bomben R, Dal-Bo M, Benedetti D, Capello D, Forconi F, Marconi D, et al. Expression of mutated IGHV3-23 genes in chronic lymphocytic leukemia identifies a disease subset with peculiar clinical and biological features. Clin Cancer Res 2010;16:620–8. [5] Stevenson F, Sahota S, Zhu D, Ottensmeier C, Chapman C, Oscier D, et al. Insight into the origin and clonal history of B-cell tumors as revealed by analysis of immunoglobulin variable region genes. Immunol Rev 1998;162:247–59. [6] Zhu D, McCarthy H, Ottensmeier CH, Johnson P, Hamblin TJ, Stevenson FK. Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma. Blood 2002;99:2562–8. [7] Zhu D, Ottensmeier CH, Du M-Q, McCarthy H, Stevenson FK. Incidence of potential glycosylation sites in immunoglobulin variable regions distinguishes between subsets of Burkitt’s lymphoma and mucosa-associated lymphoid tissue lymphoma. Br J Haematol 2003;120:217–22. [8] Murray F, Darzentas N, Hadzidimitriou A, Tobin G, Boudjogra M, Scielzo C, et al. Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis. Blood 2008;111:1524–33. [9] Chapman CJ, Mockridge CI, Rowe M, Rickinson AB, Stevenson FK. Analysis of VH genes used by neoplastic B cells in endemic Burkitt’s lymphoma shows somatic hypermutation and intraclonal heterogeneity. Blood 1995;85:2176–81. [10] Riboldi P, Ikematsu W, Brambilla B, Caprani C, Gerosa M, Casali P. Diversity and somatic hypermutation of the Ig VHDJH, V kappa J kappa, and V lambda J lambda gene segments in lymphoma B cells: relevance to the origin of the neoplastic B cell clone. Hum Immunol 2003;64:69–81. [11] Klein U, Klein G, Ehlin-Henriksson B, Rajewsky K, Küppers R. Burkitt’s lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes. Mol Med 1995;1:495–505. [12] Chapman CJ, Zhou JX, Gregory C, Rickinson AB, Stevenson FK. VH and VL gene analysis in sporadic Burkitt’s lymphoma shows somatic hypermutation, intraclonal heterogeneity, and a role for antigen selection. Blood 1996;88:3562–8. [13] Isobe K, Tamaru J-I, Nakamura S, Harigaya K, Mikata A, Ito H. VH gene analysis in sporadic Burkitt’s lymphoma: somatic mutation and intraclonal diversity with special reference to the tumor cells involving germinal center. Leuk Lymphoma 2002;43:159–64. [14] Riboldi P, Gaidano G, Schettino EW, Steger TG, Knowles DM, Dalla-Favera R, et al. Two acquired immunodeficiency syndrome-associated Burkitt’s lymphomas produce specific anti-i IgM cold agglutinins using somatically mutated VH4-21 segments. Blood 1994;83:2952–61. [15] Eclache V, Magnac C, Pritsch O, Delecluse HJ, Davi F, Raphaël M, et al. Complete nucleotide sequence of Ig V genes in three cases of Burkitt lymphoma associated with AIDS. Leuk Lymphoma 1996;20:281–90. [16] Ikematsu H, Cerutti A, Zan H, Knowles DM, Ikematsu W, Casali P. Ongoing hypermutation in the Ig V(D)J gene segments and c-myc proto-oncogene of an AIDS lymphoma segregates with neoplastic B cells at different sites: implications for clonal evolution. Hum Immunol 2000;61:1242–53. [17] Lefranc M-P, Giudicelli V, Ginestoux C, Jabado-Michaloud J, Folch G, Bellahcene F, et al. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res 2009;37:D1006–12. [18] Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007;23:2947–8. [19] Graille M, Stura EA, Corper AL, Sutton BJ, Taussig MJ, Charbonnier JB, et al. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity. Proc Natl Acad Sci USA 2000;97: 5399–404. [20] Potter KN, Hobby P, Klijn S, Stevenson FK, Sutton BJ. Evidence for involvement of a hydrophobic patch in framework region 1 of human V4-34-encoded Igs in recognition of the red blood cell I antigen. J Immunol 2002;169:3777–82. [21] Brezinschek HP, Foster SJ, Brezinschek RI, Dörner T, Domiati-Saad R, Lipsky PE. Analysis of the human VH gene repertoire. Differential effects of selection and somatic hypermutation on human peripheral CD5(+)/IgM+ and CD5(−)/IgM+ B cells. J Clin Invest 1997;99:2488–501. [22] Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, et al. ZAP70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003;348:1764–75. [23] Coelho V, Krysov S, Ghaemmaghami AM, Emara M, Potter KN, Johnson P, et al. Glycosylation of surface Ig creates a functional bridge between human follicular lymphoma and microenvironmental lectins. Proc Natl Acad Sci USA 2010;107:18587–92. [24] Chapman CJ, Wright D, Stevenson FK. Insight into Burkitt’s lymphoma from immunoglobulin variable region gene analysis. Leuk Lymphoma 1998;30:257–67.
M.J. Baptista et al. / Leukemia Research 38 (2014) 509–515 [25] Capello D, Martini M, Gloghini A, Cerri M, Rasi S, Deambrogi C, et al. Molecular analysis of immunoglobulin variable genes in human immunodeficiency virus-related non-Hodgkin’s lymphoma reveals implications for disease pathogenesis and histogenesis. Haematologica 2008;93: 1178–85. [26] Sebastián E, Alcoceba M, Balanzategui A, Marín L, Montes-Moreno S, Flores T, et al. Molecular characterization of immunoglobulin gene rearrangements in diffuse large B-cell lymphoma: antigen-driven origin and IGHV4-34 as a particular subgroup of the non-GCB subtype. Am J Pathol 2012;181: 1879–88. [27] McCann KJ, Ashton-Key M, Smith K, Stevenson FK, Ottensmeier CH. Primary central nervous system lymphoma: tumor-related clones exist in the
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blood and bone marrow with evidence for separate development. Blood 2009;113:4677–80. [28] Pugh-Bernard AE, Silverman GJ, Cappione AJ, Villano ME, Ryan DH, Insel RA, et al. Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance. J Clin Invest 2001;108:1061–70. [29] Kostareli E, Hadzidimitriou A, Stavroyianni N, Darzentas N, Athanasiadou A, Gounari M, et al. Molecular evidence for EBV and CMV persistence in a subset of patients with chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors. Leukemia 2009;23:919–24. [30] Bellan C, Lazzi S, Hummel M, Palummo N, de Santi M, Amato T, et al. Immunoglobulin gene analysis reveals 2 distinct cells of origin for EBV-positive and EBV-negative Burkitt lymphomas. Blood 2005;106:1031–6.
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Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Analysis of the IGHV region in Burkitt’s lymphomas supports a germinal center origin and a role for superantigens in lymphomagenesis Maria Joao Baptista a,1 , Eva Calpe a , Eva Fernandez b , Lluis Colomo b , Teresa Marta Cardesa-Salzmann c , Pau Abrisqueta a , Francesc Bosch a,∗,2 , Marta Crespo a,2 a Laboratory of Experimental Hematology, Department of Hematology, Vall d’Hebron University Hospital, Universitat Autonòma de Barcelona, Barcelona, Spain b Department of Pathology, Hospital Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain c Department of Pediatric Oncology, Hospital Sant Joan de Déu, University of Barcelona, Barcelona, Spain
a r t i c l e
i n f o
Article history: Received 7 October 2013 Received in revised form 20 December 2013 Accepted 1 January 2014 Available online 13 January 2014 Keywords: BL IGHV SHM Superantigens Intraclonal diversity Glycosylation
a b s t r a c t The analysis of immunoglobulin heavy chain variable (IGHV) region may disclose the influence of antigens in Burkitt’s lymphomas (BL). IGHV sequences from 38 patients and 35 cell lines were analyzed. IGHV3 subset genes were the most used and IGHV4-34 gene was overrepresented. IGHV genes were mutated in 98.6% of the cases, 36% acquired potential glycosylation sites, and in 52% somatic-hypermutation-process was ongoing. Binding motifs for superantigens like Staphylococcal protein A and carbohydrate I/i were preserved in 89% of the cases. IGHV analysis of BL cells supports a germinal center origin and points toward a role for superantigens in lymphomagenesis. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Burkitt’s lymphoma (BL) is a B-cell lymphoproliferative disorder classified into three clinical variants according to the geographic distribution, the infection of the Epstein–Barr virus (EBV), and the association with human immunodeficiency virus (HIV). The clinical variants are endemic BL (eBL), sporadic BL (sBL) and HIV-associated BL (HIV-BL) [1]. On the basis of morphology, phenotype, and genetics, BL is currently regarded as a germinal center (GC) derived neoplasm, however in the WHO classification a different origin for the endemic and sporadic forms has been suggested due to differences in the expression of specific EBV-related molecules and in somatic hypermutation (SHM) patterns [1].
∗ Corresponding author at: Department of Hematology, University Hospital Vall d’Hebron, Pg de la Vall d’Hebron 119-129, 08035 Barcelona, Spain. Tel.: +34 4893806. E-mail address: [email protected] (F. Bosch). 1 Current address: Josep Carreras Leukaemia Research Institute, Department of Hematology, ICO-Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain. 2 These authors contributed equally to this manuscript. 0145-2126/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2014.01.001
The molecular analysis of the immunoglobulin heavy chain variable (IGHV) region provides insight into the stage of B-cell development at which clonal expansion occurs in B-cell tumors and the possible involvement of antigen selection. A biased use of certain IGHV genes and/or common heavy complementary determining region 3 (HCDR3) has been found in several leukemia and lymphomas and suggests a potential role for antigens in the proliferation of tumor B-cells [2–4]. The presence of mutations in the IGHV genes has been used as a marker of the passage of B-cells through the GC and the presence of intraclonal diversity has been used to identify tumor clones derived from GC [5]. Moreover, the acquisition of potential glycosylation sites in IGHV genes has been described in GC derived tumors but has been reported to be uncommon in normal B-cells [6,7]. These sites can be responsible for interactions with elements in the GC microenvironment and their acquisition can be linked to ongoing SHM activity [6]. Interestingly, recent works have suggested that autoantigens and superantigens may be also implicated in the development of B-cell tumors, like the possible role for carbohydrate I/i and for Staphylococcal protein A (SpA) in chronic lymphocytic leukemia (CLL) development by stimulating proliferation of B-cells that express particular subsets of immunoglobulin genes [4,8].
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The aim of this work was to perform a comprehensive analysis exploring the features of the IGHV region in a large series of BLs in order to give insights into BL lymphomagenesis. For that, IGHV region genes were studied focusing on the IGHV mutational status, the presence of intraclonal diversity, the acquisition of potential glycosylation sites, and the presence of superantigenic-binding motifs.
stages, 2 at stage I, 5 at II, 5 at III and 2 at IV. According to the 3 clinical variants of BL, one case was an eBL (sample 24), 4 were HIV-BL (samples 5–7 and 10) and the remaining 21 were sBL. EBV infection was studied in 14 cases. Infection was detected in 27.3% of the sBL (3 out of 11) and in 33.3% of HIV-BL (1 out of 3).
2. Materials and methods
In this study we conducted a comprehensive analysis of the IGHV regions of BL cells including a total of 73 cases. IGHV-D-J rearrangements were identified in 65 cases, being 93.8% (61 out of 65) functionally productive and 6.2% (4 out of 65) potentially productive (Supplementary Table S1). Genes from the IGHV3 family were the most prevalently used (47.9%), followed by IGHV4 (35.6%), IGHV1 (9.6%), IGHV2 (4.1%), and IGHV5 (2.7%) families. Overall, the usage of individual IGHV genes was significantly different from that of the CD5(−) normal B-cells (P = 0.044) (Supplementary Fig. S1) [21]. When individual genes were compared, the frequency of the IGHV4-34 gene was significantly higher in BL than in normal B-cells (17.8% vs. 4.3%; P < 0.001). Neither a bias in the usage of IGHD and IGHJ genes nor a preferential combination of V-D-J genes was found. HCDR3 lengths were very heterogeneous and the HCDR3 amino acid sequences revealed no stereotyped subsets according to previously established criteria [2].
2.1. Patients and samples A total of 73 BL cases were studied: 26 patients diagnosed in our institution and 12 patients and 35 cell lines whose IGHV-D-J sequences were retrieved from public databases (For more details please see Supplementary Table S1). Accession numbers for the sequences retrieved from public databases are the following: Z46269, Z46284–Z46288 [9]; AF006517–AF006528 [10]; X87438, X87440, X87442, X87444, X87447, X87449, X87451, X87453, X87459 [11]; Z74663, Z74665, Z74668, Z74671, Z74672, Z74693, Z74695 [12]; AF176233–AF176239 [13]; L29115, L29116 [14]; X82081, X82083, X82085 [15]; and AF003377 [16]. BL cells from the cases diagnosed in our institution were obtained from tumor biopsy (N = 17), bone marrow (N = 5), peripheral blood (N = 2), pleural fluid (N = 1), or ascitic fluid (N = 1) before treatment was initiated. The IGHV-D-J sequences have been submitted to GenBank, and accession numbers are listed in Supplementary Table S1. 2.2. Analysis of IGHV-D-J rearrangements IGHV-D-J rearrangements were identified by PCR amplification and subcloning procedures. For details of PCR amplification please see Supplementary Materials and methods. The analysis of the nucleotide sequences was performed with the use of IMGT/VQUEST and ClustalW/EMBL tools [17,18].
3.2. BL have biased IGHV gene usage
3.3. Mutational profile of IGHV in the majority of BL is consistent with an antigen-driven SHM process
2.3. Potential glycosylation sites analysis The IGHV amino acid sequences from the 73 BL cases were studied for the presence of potential inherent or acquired glycosylation sites. Thus, sequences were scanned from heavy chain framework region 1 (HFR1) to HFR3 to find the N-X-S/T N-glycosylation motif, where X can be any amino acid except for P, D, or E [6]. 2.4. Superantigen binding sites analysis The presence of binding motifs for SpA and carbohydrate I/i superantigens was analyzed. SpA binding motif is restricted to the IGHV3 subgroup. It consists of 13 amino acids and only one non-conservative amino acid change is allowed to retain SpA activity. The inversion of charge from K to E in position 65 is considered deleterious because it induces electrostatic repulsion [19]. Carbohydrate I/i HFR1 hydrophobic patch consists of a W at position 7 on -strand A and of A-V-Y at positions 24–26 on -strand B of IGHV4-34 gene transcripts. In order to maintain binding affinity no non-conservative amino acid change is admitted [20]. 2.5. Statistical analysis Fisher’s exact test and 2 -test, with Bonferroni adjustment for multiple comparisons, were used to estimate differences in nominal variables. Quantitative variables comparisons were done with Student t-test and with ANOVA or with Mann–Whitney and Kruskal–Wallis and a significance level of P = 0.05 was set. Analyses were performed with the use of SPSS v18.0 (IBM, Somer, NY).
3. Results 3.1. Characteristics of the patients The study group comprises previously reported and characterized BL cases (N = 47) as well as de novo BL cases diagnosed at our institution (N = 26). The main clinical and biological characteristics of the patients from our institution are summarized in Supplementary Table S2. Briefly, there were 21 male patients and 5 female. Fourteen patients were considered pediatric, since they were younger than 16 years old, and the remaining 12 were adults. The median age at diagnosis was 39 years old for adults (range, 27–69) and 6 years old for children (range, 3–16). Adult patients were mainly diagnosed at stage IV (N = 11) with only 1 patient at stage II. Pediatric patients were diagnosed at all four different
The analysis of the IGHV mutational profile of the series revealed a high mutational load (mean % germline identity (GI) ± standard deviation (SD): 94.7 ± 3.7). In this BL series, 80.8% (59 out of 73) of the cases were considered to express mutated IGHV genes whereas 17.8% (13 out of 73) had borderline/minimally mutated IGHV genes (Supplementary Table S1). We considered that all but one of the cases had undergone SHM (For details about the analysis of IGHV mutational load see Supplementary Materials and methods). The mutational load of sBL cases (N = 49) was lower than the one observed in eBL cases (N = 14) (mean %GI ± SD: 96.1 ± 2.6 vs. 92.2 ± 4.1; P = 0.001) and than the one observed in HIV-BL (N = 10) (mean %GI ± SD: 96.1 ± 2.6 vs. 91.2 ± 3.7; P < 0.001). Concerning EBV infection, there was a trend for higher mutational load in EBVinfected cases but this difference was not statistically significant (mean %GI ± SD: 93.2 ± 3.9 vs. 95.1 ± 3.8; P = 0.052). The immunoglobulin class mainly used was IgM (IgM: 58 out of 68; IgG: 8 out of 68; and IgA: 2 out of 68). No preferential light chain use was detected (kappa: 29 out of 54 and lambda: 25 out of 54). No association between heavy or light chain usage and clinical variants, EBV status, IGHV mutational status, or %GI was found. In CLL the absence of SHM is related to adverse prognosis and correlates with high expression of ZAP-70 protein [22], however whether levels of ZAP-70 also correlate with IGHV mutational status in BL has not been ascertained yet. In our series, 23.1% of BL cases (6 out of 26) were positive for ZAP-70 (for details about ZAP70 analysis please see Supplementary Materials and methods) but no correlation was found between ZAP-70 expression and IGHV mutational load. 3.4. BL cases show intraclonal diversity and acquire potential glycosylation sites To study intraclonal diversity in the BL cases diagnosed at our institution, at least 8 subclones with identical or nearly identical HCDR3 sequences from each case were compared to the corresponding germline sequence (Table 1). The presence of a
M.J. Baptista et al. / Leukemia Research 38 (2014) 509–515 Table 1 Analysis of intraclonal diversity in BL. Sample number
Clinical variant
Number of subclones
IGHV gene
CM
UCM
ID
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 24 25 26
sBL sBL sBL sBL HIV-BL HIV-BL HIV-BL sBL sBL HIV-BL sBL sBL sBL sBL sBL sBL sBL sBL sBL sBL sBL sBL eBL sBL sBL
10 9 9 10 11 10 10 11 24 10 12 12 12 14 14 14 12 11 15 12 12 11 8 11 12
IGHV5-a IGHV3-30-3 IGHV4-59 IGHV4-59 IGHV3-33 IGHV3-48 IGHV4-39 IGHV4-39 IGHV2-5 IGHV3-30 IGHV3-74 IGHV1-8 IGHV4-39 IGHV4-59 IGHV3-30 IGHV3-11 IGHV4-34 IGHV4-59 IGHV2-70 IGHV3-30 IGHV4-34 IGHV3-21 IGHV3-21 IGHV1-24 IGHV3-21
0 0 2 0 7 1 4 0 0 4 7 1 1 1 0 0 2 0 0 0 1 1 0 3 0
1 2 0 5 12 10 6 2 7 10 2 4 16 14 27 11 23 10 5 4 10 10 5 12 8
No No Yes No Yes Yes Yes No No Yes Yes Yes Yes Yes No No Yes No No No Yes Yes No Yes No
Abbreviations: CM, confirmed mutation (a mutation observed in more than one subclonal sequence but not in all sequences); UCM, unconfirmed mutation (a mutation observed only in one subclonal sequence); ID, intraclonal diversity; sBL, sporadic Burkitt lymphoma; eBL, endemic Burkitt lymphoma; HIV-BL, HIV-associated Burkitt lymphoma.
confirmed mutation (a mutation observed in more than one subclonal sequence but not in all sequences) was considered indicative of ongoing SHM. Based on this criterion, intraclonal diversity was detected in 52% of cases (13 out of 25). The presence of glycosylation sites in the IGHV genes allows Bcells to interact with stromal elements present in the GC potentially facilitating the retention of cells in these structures [6,23]. In follicular lymphoma (FL), the glycosylation of surface immunoglobulins creates a functional bridge between FL cells and microenvironmental lectins that can replace the need for antigen stimulation for B-cell survival in the GC [23]. When studying the whole series herein described, we found that 26 out of 72 cases with mutated IGHV genes (36.1%) acquired new potential glycosylation sites (Table 2). Cases with novel potential glycosylation sites presented higher mutational loads than those without them (mean %GI ± SD: 92.5 ± 3.8 vs. 95.9 ± 3.0; P < 0.001). The incidence of BL cases with novel glycosylation sites was statistically different between the different clinical variants (eBL: 64.3%, sBL: 25.0%, HIV-BL: 50.0%; P = 0.016); less sBL acquired novel glycosylation sites when compared to eBL (P = 0.010) but not with HIV-BL. EBV infected cases showed similar incidence of novel glycosylation sites (45.8%) than not-infected cases (34.5%). Finally, 72% of the cases (18 out of 25) analyzed for both the presence of glycosylation sites and intraclonal diversity showed at least one of the two features. Altogether, these findings suggest that BL cells derive from the GC. 3.5. Superantigenic-binding motifs are not affected by the SHM process in BL The presence of binding motifs for superantigens has not been extensively studied in leukemia/lymphoma; however, some subsets of CLL cases have been found to retain those motifs, suggesting a putative role for superantigens in the selection of the clonogenic progenitor [4,8]. SpA and carbohydrate I/i are superantigens
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whose binding motifs are present in the germline sequence of the genes from the IGHV3 subgroup and in the IGHV4-34 gene (HFR1 hydrophobic patch) respectively [19,20]. Interestingly, the IGHV3 subgroup was the most used by BL cells and the IGHV4-34 gene usage was biased. Altogether, genes from the IGHV3 subgroup and the IGHV4-34 gene accounted for 65.7% of the IGHV genes expressed in this BL series. We scanned the amino acid sequences from IGHV3 subgroup for the presence of SpA binding sites considering only those cases with a known nucleotide sequence from HFR1 to HFR3 (N = 26) (Table 3). We observed that in 24 out of the 26 IGHV3 subgroup cases, the SpA binding motif was conserved. In addition, no mutation leading to loss of activity was found in any of the 8 BL cases with ongoing SHM. The presence of mutations in the hydrophobic patch for carbohydrate I/i in HFR1 was investigated in all cases expressing the IGHV4-34 gene except in case 29 because its nucleotide sequence for HFR1 was unknown. Most of the BL cases kept the binding motif for carbohydrate I/i intact (10 out of 12) (Table 4). Moreover, in none of the 2 BL cases that had ongoing SHM and used the IGHV4-34, the binding motif was affected. SpA and carbohydrate I/i binding motifs were preserved even in those BL cases highly targeted by SHM, pointing toward a role for these superantigens in the pathogenesis of BL.
4. Discussion The molecular analysis of the IGHV region may offer some clues into the pathogenesis of B-cell derived lymphomas and may also provide evidence for interactions of the malignant clones with the microenvironment [5]. The analysis of the IGHV gene repertoire conducted in this series showed that IGHV3 was the predominant subgroup used by BL cells, followed by IGHV4, and revealed that the IGHV repertoire used by BL cells was different from the one used by normal B-cells. Moreover, the IGHV4-34 gene was found overrepresented, confirming the trend that was previously observed in several smaller series [10,24]. Interestingly, an over-usage of this gene has also been reported in other lymphomas/leukemias [8,25–27]. The IGHV4-34 gene encodes for intrinsically autoreactive antibodies and it’s underrepresented in GC and memory B-cells from healthy individuals in order to prevent plasma cells from producing auto-antibodies [28]. The high prevalence of BL cells that express IGHV4-34 suggests that autoreactive antigens can be involved in driving the expansion of BL. Likewise, during acute EBV and Cytomegalovirus infections increased titers of IGHV4-34 antibodies are detected, and patients with CLL infected with EBV also have IGHV4-34 gene overrepresentation [29]. In our BL series, however, those cases infected with EBV did not show a bias in IGHV4-34 usage. Notwithstanding, since EBV infection is strongly associated with BL and EBV could act in a hit and run fashion leaving little trace of its presence in the cells, a role for EBV in the selection of the tumor cells should not be discarded. The mutational load of the IGHV genes of this BL series was high (mean %GI = 94.7) and mutations were found in all but one of the cases. These results indicate that BL cells have been targeted by the SHM machinery reinforcing their putative GC or post-GC origin [1]. Moreover, eBL cases were found to have higher mutational loads (mean %GI = 92.2) than sBL cases (mean %GI = 96.1) whereas HIV-BL and eBL cases showed similar mutation loads, which corroborates previous reports on the different mutational load in IGHV of BL clinical variants [11,24,30]. We observed a trend for higher mutational load in EBV-infected cases (mean %GI = 93.2 vs. 95.1), but this did not reach statistical significance, in line with previously published
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Table 2 Potential glycosylation sites in the IGHV genes of BL cases. Sample number
Sample type
IGHV gene
Constitutive glycosylation site Position
43 66 25 47 34 12 32 9 19 16 28 42 64 23 24 26 30 35 37 48 49 54 69 10 15 20 38 61 65 67 2 5 31 6 40 46 60 63 68 45 50 33 11 51 55 17 21 29 39 44 53 57 58 59 70 71 72 73 7 8 13 36 52 56 41 3 4 14 18 27 62 1
Cell line Cell line Primary cell Cell line Primary cell Primary cell Primary cell Primary cell Primary cell Primary cell Primary cell Cell line Cell line Primary cell Primary cell Primary cell Primary cell Primary cell Primary cell Cell line Cell line Cell line Cell line Primary cell Primary cell Primary cell Primary cell Cell line Cell line Cell line Primary cell Primary cell Primary cell Primary cell Cell line Cell line Cell line Cell line Cell line Cell line Cell line Primary cell Primary cell Cell line Cell line Primary cell Primary cell Primary cell Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line Primary cell Primary cell Primary cell Primary cell Cell line Cell line Cell line Primary cell Primary cell Primary cell Primary cell Primary cell Cell line Primary cell
IGHV1-18 IGHV1-2 IGHV1-24 IGHV1-46 IGHV1-69 IGHV1-8 IGHV1-8 IGHV2-5 IGHV2-70 IGHV3-11 IGHV3-11 IGHV3-15 IGHV3-15 IGHV3-21 IGHV3-21 IGHV3-21 IGHV3-21 IGHV3-23 IGHV3-23 IGHV3-23 IGHV3-23 IGHV3-23 IGHV3-23 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30-3 IGHV3-33 IGHV3-43 IGHV3-48 IGHV3-48 IGHV3-48 IGHV3-48 IGHV3-48 IGHV3-53 IGHV3-66 IGHV3-66 IGHV3-7 IGHV3-74 IGHV3-74 IGHV4-31 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-34 IGHV4-39 IGHV4-39 IGHV4-39 IGHV4-39 IGHV4-39 IGHV4-39 IGHV4-4 IGHV4-59 IGHV4-59 IGHV4-59 IGHV4-59 IGHV4-59 IGHV5-51 IGHV5-a
HFR3-81 HFR3-81
HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57 HCDR2-57
HFR3-66
aa seq
NTS NTS
NHS NHS NHS NHS NHS NHS NHS NHS NHS NHS NHS NHS NHS
NYS
Novel glycosylation site Status
Position
aa seq
Status
No No No No No Mantained Mantained No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No Mantained Mantained Mantained Lost Lost Mantained Mantained Mantained Mantained Lost Mantained Mantained Lost No No No No No No No No No No No No No Lost
HFR3-66
NYS
HCDR2-63
NYT
HCDR2-60
NNS
HCDR1-36 HCDR1-36
NYT NYS
HCDR2-58
NSS
HCDR1-29
NFS
HFR3-84
NNT
HFR3-84 HFR3-84 HFR3-77
NNT NNT NIS
HCDR1-29 + HCDR2-59 HCDR2-63 HFR3-84
NFS + NNT NTT NNS
HCDR1-36
NYS
HFR3-84 HFR1- 24
NNT NVS
HFR3-90
NLT
HFR3-68 + HFR3-90 HFR3-68
NSS + NLS NAS
HFR3-66 HCDR1-31
NYT NST
HFR1-24 + HCDR1-34
NVS + NSS
HFR1-24 HCDR2-59
NVS NGT
HCDR1-29 + HFR3-95
NFT + NAS
Yes No No No No No No No No Yes No Yes No Yes Yes No Yes No No Yes No No No No No No Yes No Yes Yes Yes No No Yes Yes Yes No Yes No No No No No Yes Yes No Yes No Yes Yes No No No No No No No Yes Yes No No Yes No No No No No No Yes Yes No Yes
Notes: Amino acid changes leading to the acquisition of novel potential glycosylation sites are underlined.
M.J. Baptista et al. / Leukemia Research 38 (2014) 509–515
513
Table 3 Presence of the SpA binding site in BL cases using the IGHV3. Sample number
Sample type
IGHV gene
HFR1 16
16 42 64 48 49 54 69 15 10 20 38 61 65 67 2 5 6 40 46 60 63 68 45 50 11 51
Primary cell Cell line Cell line Cell line Cell line Cell line Cell line Primary cell Primary cell Primary cell Primary cell Cell line Cell line Cell line Primary cell Primary cell Primary cell Cell line Cell line Cell line Cell line Cell line Cell line Cell line Primary cell Cell line
18
IGHV3-11 IGHV3-15 IGHV3-15 IGHV3-23 IGHV3-23 IGHV3-23 IGHV3-23 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30 IGHV3-30-3 IGHV3-33 IGHV3-48 IGHV3-48 IGHV3-48 IGHV3-48 IGHV3-48 IGHV3-53 IGHV3-66 IGHV3-66 IGHV3-74 IGHV3-74
SpA binding motif
20
HCDR2
HFR3
65
67
SPA binding motif presence 72
74
75
77
79
90
92
93
R I S
E F
Q
S E M
C
D N
N T G G
R A G
S
R/K
S
G
N/G
S
L
K/I/T
Y
K
G
R
T
S
Q
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Notes: Amino acids are represented by a single letter code, non-conservative amino acid replacements are underlined, and the inversion of amino acid charge in HCDR2-65 position is shown in shadow.
data [24], although differences were reported by others [10,30]. Thus, our results could not rule out whether EBV can influence the mutational load of the IGHV genes. Previous studies in lymphoproliferative diseases derived from GC B-cells have showed that the majority of FL and eBL cases, and nearly half of the sBL and diffuse large B-cell lymphoma cases, acquire potential glycosylation sites in the IGHV genes, contrary to what is found in normal B-cells and other lymphoproliferatives diseases like CLL and multiple myeloma [6,7]. In the BL series herein analyzed, 36.1% of the cases acquired new potential glycosylation sites, and the acquisition of these motifs was correlated with higher mutational load. Even though the former association was not observed in a previous work [7], our results are in line with the notion that glycosylation sites help B-cells to stay in the GC and thus higher mutation loads can be achieved. In addition, we
found differences in the acquisition of glycosylation sites among the different clinical variants. In this sense, the variant associated with higher IGHV mutational loads, eBL, presented the higher incidence of acquisition of glycosylation sites. Notwithstanding, EBV-infection seemed not to be related to the acquisition of glycosylation sites since the incidence of their acquisition was similar in EBV-infected and non-infected cases. The analysis for both the presence of intraclonal diversity and glycosylation sites showed that 72% of the cases analyzed had at least one of the two features, which link cells to the GC microenvironment. Moreover, in all cases analyzed for intraclonal diversity, no novel potential glycosylation site was removed due to ongoing SHM. Also, in 2 out of 14 cases with no previous potential glycosylation sites, a subclonal mutation generating a novel potential glycosylation site was detected. Glycosylation sites may
Table 4 Presence of the carbohydrate I/i HFR1 hydrophobic patch in BL cases using IGHV4-34. Sample number
Sample type
HFR1 7
17 21 39 44 53 57 58 59 70 71 72 73 Carbohydrate I/i HFR1 hydrophobic patch
Primary cell Primary cell Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line Cell line
Carbohydrate I/i hydrophobic patch presence 24
25
26
G S S G V
I W
A
V
Y
Notes: Amino acids are represented by a single letter code and non-conservative amino acid replacements are underlined.
Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes
514
M.J. Baptista et al. / Leukemia Research 38 (2014) 509–515
therefore represent an advantage for tumor survival and expansion since they were not removed but acquired by some cases during the ongoing SHM process. These results are in agreement with other characteristics from BL cells, such as the expression of CD10 and BCL6, which indicate that a mature B-cell experiencing the GC reaction would be the normal counterpart for BL. The analysis for the binding sites for superantigens carbohydrate I/i and SpA in IGHV4-34 gene and IGHV3 subgroup genes, respectively, showed that these sites were preserved in the majority of cases (89.5%). Moreover, the ongoing SHM process found in cases with intraclonal diversity did not introduce mutations in these binding motifs in any subclone. Thus, SpA and carbohydrate I/i binding motifs were preserved even in those BL cases highly targeted by SHM suggesting that superantigens may be contributing to clonal selection and expansion of BL cells. Finally, patients included in the present study were both pediatric and adult and, importantly, samples were obtained from a variety of tissues, which may have influenced the results as a consequence of the interaction with different microenvironments. In conclusion, the detection of ongoing SHM and the presence of glycosylation sites in a high percentage of BL cases herein analyzed reinforce the hypothesis of a mature B-lymphocyte experiencing the GC reaction being the normal counterpart for BL. Moreover, the biased usage of IGHV3 family and IGHV4-34 genes and the fact that SHM does not affect superantigenic binding motifs suggest that superantigens may have a role in the pathogenesis of BL. These results contribute to a better knowledge of BL cell of origin and suggest a possible role for superantigens as players in BL lymphomagenesis process. Funding This work was supported by grants from Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias FIS 08/0211 and FIS PI11/00792, and from Fundació Marató de TV3 05/1810. M.J.B. was a recipient of a PhD fellowship (SFRH/BD/28698/2006) granted by Ministério da Ciência, Tecnologia e Ensino Superior, Portugal. M.C. holds a contract from Ministerio de Economía y Competitividad (MINECO) (RYC-2012-12018), Spain. Author’s contributions M.J.B., M.C. and F.B. designed the study; M.J.B., E.C. and E.F. performed the laboratory work; L.C., T.M.C.S., and P.A. recruited the patients; M.J.B. and M.C. analyzed and interpreted the data; M.J.B. performed statistical analysis; M.J.B., M.C., and F.B. wrote the manuscript. All authors approved final version of the manuscript. Conflicts of interest statement The authors reported no potential conflicts of interest. Acknowledgments The authors would like to thank Professor Freda K. Stevenson (University of Southampton Faculty of Medicine, Southampton, United Kingdom) for critical reading of the manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.leukres.2014. 01.001.
References [1] Swerdlow SH, Campo E, Harris LN, Jaffe ES, Pileri SA, Stein H, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC press; 2008. [2] Stamatopoulos K, Belessi C, Moreno C, Boudjograh M, Guida G, Smilevska T, et al. Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: pathogenetic implications and clinical correlations. Blood 2007;109:259–70. [3] Zibellini S, Capello D, Forconi F, Marcatili P, Rossi D, Rattotti S, et al. Stereotyped patterns of B-cell receptor in splenic marginal zone lymphoma. Haematologica 2010;95:1792–6. [4] Bomben R, Dal-Bo M, Benedetti D, Capello D, Forconi F, Marconi D, et al. Expression of mutated IGHV3-23 genes in chronic lymphocytic leukemia identifies a disease subset with peculiar clinical and biological features. Clin Cancer Res 2010;16:620–8. [5] Stevenson F, Sahota S, Zhu D, Ottensmeier C, Chapman C, Oscier D, et al. Insight into the origin and clonal history of B-cell tumors as revealed by analysis of immunoglobulin variable region genes. Immunol Rev 1998;162:247–59. [6] Zhu D, McCarthy H, Ottensmeier CH, Johnson P, Hamblin TJ, Stevenson FK. Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma. Blood 2002;99:2562–8. [7] Zhu D, Ottensmeier CH, Du M-Q, McCarthy H, Stevenson FK. Incidence of potential glycosylation sites in immunoglobulin variable regions distinguishes between subsets of Burkitt’s lymphoma and mucosa-associated lymphoid tissue lymphoma. Br J Haematol 2003;120:217–22. [8] Murray F, Darzentas N, Hadzidimitriou A, Tobin G, Boudjogra M, Scielzo C, et al. Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis. Blood 2008;111:1524–33. [9] Chapman CJ, Mockridge CI, Rowe M, Rickinson AB, Stevenson FK. Analysis of VH genes used by neoplastic B cells in endemic Burkitt’s lymphoma shows somatic hypermutation and intraclonal heterogeneity. Blood 1995;85:2176–81. [10] Riboldi P, Ikematsu W, Brambilla B, Caprani C, Gerosa M, Casali P. Diversity and somatic hypermutation of the Ig VHDJH, V kappa J kappa, and V lambda J lambda gene segments in lymphoma B cells: relevance to the origin of the neoplastic B cell clone. Hum Immunol 2003;64:69–81. [11] Klein U, Klein G, Ehlin-Henriksson B, Rajewsky K, Küppers R. Burkitt’s lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes. Mol Med 1995;1:495–505. [12] Chapman CJ, Zhou JX, Gregory C, Rickinson AB, Stevenson FK. VH and VL gene analysis in sporadic Burkitt’s lymphoma shows somatic hypermutation, intraclonal heterogeneity, and a role for antigen selection. Blood 1996;88:3562–8. [13] Isobe K, Tamaru J-I, Nakamura S, Harigaya K, Mikata A, Ito H. VH gene analysis in sporadic Burkitt’s lymphoma: somatic mutation and intraclonal diversity with special reference to the tumor cells involving germinal center. Leuk Lymphoma 2002;43:159–64. [14] Riboldi P, Gaidano G, Schettino EW, Steger TG, Knowles DM, Dalla-Favera R, et al. Two acquired immunodeficiency syndrome-associated Burkitt’s lymphomas produce specific anti-i IgM cold agglutinins using somatically mutated VH4-21 segments. Blood 1994;83:2952–61. [15] Eclache V, Magnac C, Pritsch O, Delecluse HJ, Davi F, Raphaël M, et al. Complete nucleotide sequence of Ig V genes in three cases of Burkitt lymphoma associated with AIDS. Leuk Lymphoma 1996;20:281–90. [16] Ikematsu H, Cerutti A, Zan H, Knowles DM, Ikematsu W, Casali P. Ongoing hypermutation in the Ig V(D)J gene segments and c-myc proto-oncogene of an AIDS lymphoma segregates with neoplastic B cells at different sites: implications for clonal evolution. Hum Immunol 2000;61:1242–53. [17] Lefranc M-P, Giudicelli V, Ginestoux C, Jabado-Michaloud J, Folch G, Bellahcene F, et al. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res 2009;37:D1006–12. [18] Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007;23:2947–8. [19] Graille M, Stura EA, Corper AL, Sutton BJ, Taussig MJ, Charbonnier JB, et al. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity. Proc Natl Acad Sci USA 2000;97: 5399–404. [20] Potter KN, Hobby P, Klijn S, Stevenson FK, Sutton BJ. Evidence for involvement of a hydrophobic patch in framework region 1 of human V4-34-encoded Igs in recognition of the red blood cell I antigen. J Immunol 2002;169:3777–82. [21] Brezinschek HP, Foster SJ, Brezinschek RI, Dörner T, Domiati-Saad R, Lipsky PE. Analysis of the human VH gene repertoire. Differential effects of selection and somatic hypermutation on human peripheral CD5(+)/IgM+ and CD5(−)/IgM+ B cells. J Clin Invest 1997;99:2488–501. [22] Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, et al. ZAP70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003;348:1764–75. [23] Coelho V, Krysov S, Ghaemmaghami AM, Emara M, Potter KN, Johnson P, et al. Glycosylation of surface Ig creates a functional bridge between human follicular lymphoma and microenvironmental lectins. Proc Natl Acad Sci USA 2010;107:18587–92. [24] Chapman CJ, Wright D, Stevenson FK. Insight into Burkitt’s lymphoma from immunoglobulin variable region gene analysis. Leuk Lymphoma 1998;30:257–67.
M.J. Baptista et al. / Leukemia Research 38 (2014) 509–515 [25] Capello D, Martini M, Gloghini A, Cerri M, Rasi S, Deambrogi C, et al. Molecular analysis of immunoglobulin variable genes in human immunodeficiency virus-related non-Hodgkin’s lymphoma reveals implications for disease pathogenesis and histogenesis. Haematologica 2008;93: 1178–85. [26] Sebastián E, Alcoceba M, Balanzategui A, Marín L, Montes-Moreno S, Flores T, et al. Molecular characterization of immunoglobulin gene rearrangements in diffuse large B-cell lymphoma: antigen-driven origin and IGHV4-34 as a particular subgroup of the non-GCB subtype. Am J Pathol 2012;181: 1879–88. [27] McCann KJ, Ashton-Key M, Smith K, Stevenson FK, Ottensmeier CH. Primary central nervous system lymphoma: tumor-related clones exist in the
515
blood and bone marrow with evidence for separate development. Blood 2009;113:4677–80. [28] Pugh-Bernard AE, Silverman GJ, Cappione AJ, Villano ME, Ryan DH, Insel RA, et al. Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance. J Clin Invest 2001;108:1061–70. [29] Kostareli E, Hadzidimitriou A, Stavroyianni N, Darzentas N, Athanasiadou A, Gounari M, et al. Molecular evidence for EBV and CMV persistence in a subset of patients with chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors. Leukemia 2009;23:919–24. [30] Bellan C, Lazzi S, Hummel M, Palummo N, de Santi M, Amato T, et al. Immunoglobulin gene analysis reveals 2 distinct cells of origin for EBV-positive and EBV-negative Burkitt lymphomas. Blood 2005;106:1031–6.
