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Prepublished online as a Blood First Edition Paper on March 13, 2003; DOI 10.1182/blood-2002-11-3479.
NEOPLASIA
From the Departments of Genetics and Pathology and of
Oncology, Radiology and Clinical Immunology, Uppsala University;
Departments of Oncology, Pathology, and Hematology, Lund University
Hospital; Department of Medical Biosciences, Pathology, Umeå
University; Departments of Medicine and of Microbiology, Pathology and
Immunology, Division of Pathology, Karolinska Institutet at Huddinge
University Hospital, Stockholm; Department of Medicine, Falun Hospital;
and the Departments of Molecular Medicine, Hematology, and Pathology,
Karolinska Hospital and Institutet, Stockholm, Sweden.
Mantle cell lymphoma (MCL) is believed to originate from a naive B
cell. However, we recently demonstrated that a subset of MCL displayed
mutated VH genes. We also reported restricted use of
certain VH genes. To assess the prognostic impact of these new findings, we performed VH gene analysis of 110 patients, revealing that 18 (16%) patients had mutated and 92 (84%)
patients had unmutated VH genes. Because the mutation rate
was low in the mutated group (2.2%-6.7%), further investigation of
the germline VH gene in T cells from 5 patients with
mutated VH genes was carried out; results showed that the
unrearranged VH gene was identical to the published
sequence. These data confirm that the base pair substitutions within
the rearranged VH genes represent hypermutations, and
indicate germinal center exposure. However, VH gene
mutation status did not correlate with prognosis because there was no
difference in clinical outcome between the unmutated and mutated
groups. The most frequently used VH genes were
VH3-21 (21 patients) and VH4-34 (19 patients).
A novel finding was that VH3-21+ MCL almost
exclusively expressed Mantle cell lymphoma (MCL) is generally an
aggressive and incurable malignant lymphoma with a median survival time
of 3 to 4 years.1,2 Most patients are diagnosed with
widespread disease with disseminated lymphadenopathy and frequent
involvement of the bone marrow (BM), spleen, and gastrointestinal (GI)
tract.1,2 Tumor cells typically express CD5, CD19, and
CD20 but lack CD23 expression.3 Almost all MCLs exhibit
the (11;14) translocation, leading to rearrangement of the
BCL1 gene and overexpression of cyclin
D1.4,5 The up-regulation of this cell cycle regulatory protein contributes to the pathogenesis of MCL, but alone it is insufficient for tumor progression.6 Despite this
characteristic diagnostic marker, the disease entity includes a
heterogeneous group of patients with various clinical courses. Few
known markers can further define prognostic subgroups within the
disease, with the exceptions of the morphologically distinct blastoid
variant of MCL (MCL-BV) and the high proliferation index that have been reported to identify patients with aggressive disease.1,7 Thus, the identification of more predictors of outcome will be the
first step in revealing biologic differences in the disease, which may
ultimately be exploited to enable improved treatment strategies.
Analysis of somatic hypermutation of the immunoglobulin variable heavy
chain (IgVH) genes has provided valuable insight into the
origin of B-cell lymphomas. This process occurs during a
T-cell-dependent immune response in the germinal center (GC) of
secondary lymphoid follicles.8 Therefore, a mutated
VH gene in a B-cell lymphoma indicates GC or post-GC B-cell
origin, whereas an unmutated VH gene indicates derivation
from a pre-GC B cell that has proliferated independently of T-cell
help. In MCL, the postulated cell of origin is a B cell deriving from
the mantle zone of a lymphoid follicle that has not entered the GC and,
therefore, lacks hypermutation.9 This fact has
been questioned because MCL patients with mutated VH genes
have been reported by our group and others.10-13 Recently, we investigated 51 MCL patients and showed that approximately 20% of
them displayed hypermutated VH genes (more than 2%
sequence deviation from the corresponding germline gene was defined as mutated); the remaining had unmutated VH
genes.13 This led us to suggest that MCL may consist of 2 subsets VH gene use has been of great interest in B-cell
malignancies In the present study, we investigated the prognostic impact of somatic
hypermutation and restricted VH gene use in a large cohort
of MCL patients. We could confirm our recent finding that MCL comprises
a mutated (16%) and an unmutated (84%) group, but no significant
difference in survival was found between the 2 subsets even when
choosing different mutation cut-off levels (1%-5%). Analysis of the
corresponding unrearranged germline gene was performed in T cells from
5 mutated cases and showed 100% homology to the published sequence,
verifying that the incorporated mutations in the rearranged
VH genes corresponded to hypermutations. A novel and
interesting finding was that VH3-21+ MCL
patients showed almost exclusive use of the V Patients and materials
PCR amplification and nucleotide sequence analysis
In most samples (n = 78), clonal products from the VH gene PCR were sequenced directly using the BigDye Terminator Cycle Sequencing Reaction Kit version 2.0 (Perkin-Elmer, ABI, Foster City, CA). Cloning of the VH gene PCR products was performed in 32 patients as previously described,37 and 3 to 10 colonies from each PCR product were sequenced. To investigate the presence of ongoing mutations in patients with mutated VH genes, further subcloning of the amplified rearrangements was performed in 5 patients using the Zero Blunt TOPO PCR Cloning kit, which includes the proofreading enzyme Pfu (Invitrogen, Paisley, United Kingdom). At least 10 colonies were subsequently sequenced from each PCR product. This cloning kit was also used for the analysis of germline VH gene sequences amplified from T-cell DNA. VL gene PCR products of VH3-21+ patients were cloned using the previously described method37 or the Zero Blunt TOPO PCR Cloning kit. All sequences were analyzed using an automated DNA sequencer (ABI 377 or ABI 3700; Applied Biosystems, Foster City, CA). Sequencing of the unrearranged germline gene from T cells of 5 patients T cells were isolated from frozen tumor samples from 5 patients with mutated VH genes (from PB in 3 patients, from lymph node or spleen in the other 2 patients) using a CD4 Positive Isolation Kit (Dynal ASA, Oslo, Norway). Briefly, magnetic beads coated with mouse anti-CD4 IgM monoclonal antibody were incubated with the sample, which allowed the CD4+ T cells in the sample to bind to the magnetic beads. Subsequent washing with the use of a magnet enabled isolation of the bound cells.Primers were designed to amplify the unrearranged VH germline gene for VH3-23, VH3-72, VH3-74, VH4-34, and VH4-59 using the Primer3 software program.38 Forward primers hybridized to a region upstream of framework region (FR)1, and the reverse primer hybridized to a region downstream of FR3, hence excluding a rearranged germline VH gene from being amplified. Primer sequences (5' to 3') were: VH3-23 forward, AGTTTGGGCTGAGCTGGCTTTTTC; VH3-23 reverse, AATCTGCATTTGGTGCGTGTGAG; VH3-72 forward, CCCCAGAATTCCCAGGTGTTTTC; VH3-72 reverse, AAGGCTCCAGAGTCCTGCAAAAA; VH3-74 forward, TTGCTGATCAGGACTGCACACAG; VH3-74 reverse, ACTTGATGAATCAGCCCCAGATG; VH4-34 forward, ATGGACCTCCTGCACAAGAACAT; VH4-34 reverse, CAGACCACTGAGCTGTTGAGGAA; VH4-59 forward, CACCTCTCCATACAAAGGCACCAC; VH4-59 reverse, GGCGCTGAGCAGCACCTG. The PCR reaction was carried out using the same conditions as for VH gene family-specific PCR except that annealing temperatures of 65°C for VH3-23, VH3-72, VH4-59 and 64°C for VH3-74 and VH4-34 were used. PCR products were cloned, and at least 10 colonies were sequenced per sample. Analyses of immunoglobulin sequences The obtained sequences were aligned to immunoglobulin sequence databases Ig BLAST (National Center for Biotechnology Information, USA), V-BASE (MRC Centre for Protein Engineering, Cambridge, United Kingdom), and International ImMunoGeneTics (IMGT).39
VH gene sequences containing mutations resulting in less
than 98% homology to the germline gene were defined as mutated. The
presence of mutations in hotspot regions was evaluated by analyzing the
number of mutations that occurred in the germline sequence motif
RGYW/WRCY.40 Ongoing somatic hypermutation was defined as
the presence of a replacement mutation in at least 2 of 10 sequences analyzed.
Statistical analyses Survival was calculated from the date of diagnosis until last follow-up or death. Kaplan-Meier survival analysis and log-rank analysis were performed to study the prognostic impact of various parameters in our MCL cohort, and P was calculated as 2-sided. Cox proportional hazards analysis was used to study the interrelationship between univariate significant parameters. Fisher exact test with 1-tailed P and McNemar 2 test
were used to calculate the significance of differences in VH gene use between MCL and healthy B cells. The
Mann-Whitney U test was applied to calculate the
significance of differences in distribution of age at diagnosis between
groups. Statistica 6.0 software (StatSoft, Tulsa, OK) was used for all calculations.
Clinical data We investigated 110 patients; 90 were men and 20 women. Eighty-four patients were diagnosed with classical MCL and 26 with MCL-BV. Median age at diagnosis was 68 years (range, 34-85 years). At follow-up, 56 patients had died and 53 were alive. Median follow-up time (n = 109) was 23 months (range, 0-162 months), and median survival time was 38 months (range, 2-162 months). Adequate staging was performed for 102 patients, revealing that 9 were in stage I-II and 93 were in stage III-IV. One hundred patients had nodal disease; only 6 did not have any nodal involvement (information from 4 patients lacking). Of these 6 patients without nodal involvement, 3 had BM/blood involvement as the sole site. Seventy-four patients had BM involvement, 24 had splenic involvement, 39 patients had extranodal involvement, and 32 had leukemic disease (of which only 8 had lymphocyte counts exceeding 20 × 109/L). The distribution of IPI scores (n = 89) was as follows: 0 to 2, 44%; 3 to 5, 56%. The median s-LDH value (n = 93) was 6.6 µkatal (µkat)/L (range, 3.8-29 µkat/L; normal upper limit, 6.7 µkat/L). Treatment was recorded for 106 patients; 4 patients were untreated, 2 received radiotherapy alone, and the rest received 1 or more chemotherapy regimens or antibody treatment. Most patients received CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone)/CNOP (cyclophosphamide, mitoxantrone, vincristine, prednisone) treatment (n = 78), but chlorambucil/COP (cyclophosphamide, vincristine, prednisone) (n = 18), rituximab (n = 36), and nucleoside analogues (n = 40) were also used. Nineteen patients underwent high-dose chemotherapy with autologous stem cell transplantation (ASCT). Major clinical factors are summarized in Table 1.
IgH rearrangements One hundred seventeen IgH gene rearrangements were amplified and sequenced from 110 patients, including 7 patients with double rearrangements. Sequence results of the first 51 patients have been published.13 Of the 117 sequences, most had in-frame rearrangements, whereas 9 sequences had a stop codon or an out-of-frame rearrangement. Four of these nonfunctional sequences were detected in patients who had a double rearrangement though the other rearranged allele was in-frame. Of the other 5 nonfunctional sequences, 2 were VH3-21+ rearrangements. Percentages of VH gene family use of the 117 sequences were as follows: VH1, 14%; VH2, 3%; VH3, 50%; VH4, 25%; VH5, 7%; VH6, 2%. The most frequently used VH genes were VH3-21 (18%; n = 21), VH4-34 (16%; n = 19), VH3-23 (8%; n = 9), VH5-51 (7%; n = 8), and VH1-8 (6%; n = 7). In Table 2, we compared the percentage use of these VH genes with the use reported in CD5+ B cells from the PB of 2 healthy elderly donors41 and in PB B cells from 5 healthy elderly donors.42 This comparison revealed a higher frequency of the VH3-21 gene and the VH4-34 gene in MCL than in controls (P < .001). Use of the 2 most frequent VH genes is referred to in "Discussion" in terms of percentage of 110 patients19% for VH3-21 and 17% for VH4-34.
The most commonly used diversity (D) genes were D6-13 (12%), D2-2
(10%), and D3-3 (9%). Percentages of use of the joining
(JH) genes were JH1, 0%;
JH2, 3%; JH3, 8%; JH4,
34%; JH5, 10%, and JH6, 33%. The D and
JH genes could not be assigned for 8% and 12% of the
sequences, respectively.
Somatic hypermutation status Of the 110 patients, 18 (16%) had somatic hypermutations in their VH genes using the 2% cut-off level (Table 3), and 92 (84%) were unmutated. Three patients with a double rearrangement had 1 mutated and 1 unmutated sequence; hence, they were regarded as MCL patients with mutated VH genes (patients 41, 60, and 87). The number of mutations ranged from 5 to 15, corresponding to 97.8% to 93.3% homology to the germline gene, respectively. Five mutated VH gene rearrangements were subcloned to investigate the presence of ongoing somatic hypermutation, but none of the mutated sequences showed evidence of this. Mutated VH genes were also evaluated for the percentage of mutations that occurred in the known hypermutation hotspots, the RGYW/WRCY motifs. Of the total number of mutations (n = 121) in the 18 patients, 50% (n = 60) were in these known hotspots. In addition to the 18 MCL patients with more than 2% mutations, another 17 patients contained 1% to 2% mutations in their VH genes, corresponding to 3 or 4 mutations, which may also represent somatic hypermutation. None of the VH3-21+ MCL patients had mutated VH genes (Table 4).
VH3-21+ MCL light-chain analysis Immunophenotype data on the VH3-21+ MCL patients showed that all but one patient (patient 58) expressed light chains (Table 4).
VL analysis was performed on all
VH3-21+ MCL patients, and in-frame light-chain
rearrangements were detected and sequenced for 17 patients. Sixteen
patients used the V 3-19 gene, whereas 1 used
V9-49. For 2 V3-19+ patients,
a second rearrangement was also detected; both were out-of-frame
(patient 54, V9-49; patient 97, V2-11). Two out-of-frame rearrangements, V7-43 and
V2-23, were detected for patient 23. The most
frequently used J genes were J2/3
(sequence analysis did not distinguish between these genes in most
patients because they are homologous). Use of V3-19 in
combination with the J2/3 gene occurred in 11 patients. None of the V genes had more than 2% mutations.
Analysis of unrearranged germline VH gene In 5 patients with mutated VH genes displaying 96% to 97% homology to the published germline gene, we performed further analysis of the germline DNA from T cells obtained by cell sorting. In all 5 patients, VH gene sequences from the T cells were 100% identical to the germline gene. For example, 1 of the 5 patients had a clonal VH3-23 rearrangement in the MCL sample, and VH gene analysis showed 6 mutations between FR1 and FR3, whereas the unrearranged VH3-23 germline gene from the T cells of the same patient did not have any mutations. This demonstrated that the mutations in the rearranged VH gene represented true hypermutations.Survival analysis The impact of clinical and molecular parameters on survival in MCL was tested using the log-rank test (Table 5). Patients 68 years and younger at diagnosis (n = 53) had significantly better survival times than those older than 68 years (n = 56); median survival times were 49 months (range, 2-162 months) and 23 months (range, 2-109 months), respectively. Other factors found to be significant predictors of survival were IPI and s-LDH level, whereas no significant differences with reference to overall survival were recorded between sex, classical MCL versus MCL-BV, stages I-II versus III-IV, nodal disease versus nonnodal disease, or the involvement of tonsil/spleen/GI tract versus noninvolvement. No association was found between mutation status and overall survival when cut-off levels of 1%, 1.5%, 2%, 2.5%, and 5% for defining a mutated case were applied. Figure 1 shows the Kaplan-Meier curve comparing mutated and unmutated cases using the 2% cut-off level. Correlations were carried out between patients using specific VH genes and the remaining patients. Significantly longer overall survival was found in VH3-21+ patients compared with the rest of the material (log-rank test, P = .030); the VH3-21+ subset had a median survival time of 53 months (range, 5-146+ months) compared with 34 months (range, 2-162 months) for the remaining patients (Figure 2). This subset had a lower median age at diagnosis (median, 62 years; range, 44-85 years) compared with the rest of the MCL patients (median, 70 years; range, 34-84 years), but the Mann-Whitney U test did not reveal any significant difference regarding the ages of the subsets (P = .15). Median follow-up time for the VH3-21+ patients was 33 months (range, 0-146 months), whereas the median follow-up time for the remaining MCL patients was 20 months (range, 0-162 months). Detailed clinical characteristics of the VH3-21+ patients are shown in Table 6. There was no prognostic association with any of the other 4 frequently used VH genesVH4-34, VH3-23, VH5-51, and
VH1-8.
All parameters were tested in a univariate fashion using Cox proportional hazards analysis to determine the effect on prognosis. Age (P < .001), IPI (P = .008), and s-LDH level (P = .003) were all significant. When these factors were included in multivariate analysis, only age and s-LDH level retained their importance (P = .003 and P = .038, respectively).
For many years, it has been assumed that MCL harbors unmutated VH genes and therefore originates from naive B cells. However, in a recent report from our group investigating 51 MCL patients, we showed that approximately 20% of patients with MCL had somatic hypermutations (more than 2%) within their VH genes.13 In this follow-up study, we extended our cohort to 110 patients, and we demonstrate that MCL comprises a mutated (16%; n = 18) and an unmutated (84%; n = 92) group. These data shed light on the postulated cell of origin of MCL because the presence of somatic hypermutations in a subset of MCL implies that these lymphomas may originate from a B cell that has been exposed to the GC environment. To ensure that the observed mutations truly represent somatic hypermutations and not single nucleotide polymorphisms, we analyzed the unrearranged VH gene in T cells from 5 selected patients whose corresponding rearranged clonal VH gene carried mutations in the range 2.2% to 3.6%. For each of these 5 patients, the unrearranged VH gene was 100% homologous to the published germline sequence, confirming that the mutations corresponded to hypermutations in these patients. Additionally, analysis of the amount of mutations that occurred in the known hypermutation hotspot regions (RGYW/WRCY motifs) showed that 50% of the total number of mutations (n = 121) were in these sequence motifs, further substantiating the data. We also investigated whether there were ongoing somatic hypermutations but did not find evidence of this in the 5 mutated cases analyzed. This indicates that the clonal B cells in MCL patients with mutated VH genes are not under prolonged antigenic stimulation in the GC and probably derive from a post-GC B cell. We believe that most MCLs derive from pre-GC B cells residing in the follicular mantle but that a small subset has been exposed to the GC environment. An alternative explanation for mutated VH genes could be that the precursor lymphoma cells have undergone hypermutation outside the GC. Recently, it was reported that patients with X-linked hyper IgM syndrome, which is characterized by a mutation in the CD40L gene and the inability to form GCs, can still have a subset of B cells (CD27+IgM+IgD+) with mutated VH genes.43 This is suggestive of the existence of a second diversification pathway to acquire hypermutations without the classical interaction between B and T cells. The rate of somatic hypermutations in these CD27+IgM+IgD+ B cells was generally low (approximately 1%-2%) in patients with hyper IgM syndrome. However, little is known about this separate route, and it remains to be investigated whether different B-cell subsets, such as MCL precursors, acquire hypermutations by this process. In CLL, VH gene mutation status may be used to group patients into 2 prognostic subsets, in which patients with mutated VH genes have an overall survival time approximately twice as long as that of patients with unmutated VH genes.14-16 In contrast, we could not find any significant differences in median survival times when comparing mutated and unmutated MCL at the 2% cut-off level. This mutation border, which has been empirically derived for CLL to avoid counting Taq polymerase errors and polymorphisms as hypermutations,14 may, however, not be appropriate for defining a mutated case in MCL. There is a large difference in the percentage of mutated cases in each disease (approximately 40%-50% in CLL vs 16% in MCL), and the mean mutation percentage of the mutated group is lower in MCL than in CLL14 (3% vs approximately 6%). In addition to the 18 patients with mutated VH genes, another 17 patients had VH genes with a mutation rate of 1% to 2%, which may also represent mutated MCL. Therefore, we performed survival analysis using other percentage cut-off levels for defining a mutated case (1%-5%), but similar results were obtained using different borders. Hence, in our cohort of MCL patients, VH gene mutation status was not prognostically useful. Interestingly, it has been reported in an abstract by Garand et al44 that patients with nonnodal MCL (patients without clinical lymphadenopathy) have mutations more frequently (19 of 34 patients) than patients with nodal engagement (5 of 31 patients). They stated that survival did not differ significantly between mutated and unmutated MCL but that there was a tendency toward improved survival in the mutated subset. They also showed that patients with nonnodal MCL had a less aggressive disease than nodal patients.44 The proportion of MCL patients without lymphadenopathy seems generally small. Only 6 patients in our cohort had nonnodal disease, all of which were unmutated. Further analysis of this subset would be of interest to determine whether mutation status has a greater clinical impact within this rare entity. Biased use of individual VH genes has been demonstrated in
several entities of B-cell lymphoma, as mentioned. Restricted use of
the VH4-34 gene in MCL has been indicated through an
immunohistochemical detection method.21 This gene has also
been found overused in a small series of MCL patients.12
In our earlier study, we confirmed the overrepresentation of the
VH4-34 gene in MCL but could also show restricted use of
VH3-21.13 In this report, the 2 VH
genes were still overused, with VH3-21 representing 19% of
patients and VH4-34 representing 17% of patients. In
addition, our finding of skewed VH3-21 gene use was
recently supported by other groups, who have found increased use of
VH3-21 in MCL.45-47 Preferential use of the
VH4-34 gene has been found in several other B-cell malignancies, such as CLL, diffuse large B-cell lymphoma, and primary
central nervous system (CNS) lymphoma,18,21,23,24 but this
VH gene has also been found to be involved in autoimmunity, encoding antibodies to self-antigens31,32 and
nonself-antigens.21,28 The current hypothesis in
lymphoproliferative disease is that B cells using specific
VH genes encode immunoglobulin molecules that may be
stimulated by various antigens, resulting in an increased proliferation
rate that makes them more susceptible to
transformation.19,21,22,27 The VH3-21 gene has
also been associated with autoimmunity In contrast to CLL, VH3-21+ MCL patients
(n = 21) had unmutated VH genes and did not show any
specific common features within CDR3. In parallel with CLL, they showed
We thank Carin Backlin and Eva Pallin for their skillful technical assistance.
Submitted November 19, 2002; accepted February 28, 2003.
Prepublished online as Blood First Edition Paper, March 13, 2003; DOI 10.1182/blood-2002-11-3479.
Supported by grants from the Swedish Cancer Society, Lion's Cancer Research Foundation, Umeå University and Uppsala University, Gunnar Nilsson's Cancer Foundation, and the research foundation of the Department of Oncology at Uppsala University.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Richard Rosenquist, Department of Genetics and Pathology, Rudbeck Laboratory, Dag Hammarskjölds väg 20, Uppsala University, SE-751 85 Uppsala, Sweden; e-mail: richard.rosenquist{at}genpat.uu.se.
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Abstract
Introduction
