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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 1848-1854
Unmutated Ig VH Genes Are Associated With a More
Aggressive Form of Chronic Lymphocytic Leukemia
By
Terry J. Hamblin,
Zadie Davis,
Anne Gardiner,
David G. Oscier, and
Freda K. Stevenson
From the Department of Haematology, Royal Bournemouth Hospital,
Bournemouth, UK; and the Molecular Immunology Department, Tenovus
Research Laboratory, Southampton General Hospital, Southampton,
UK.
 |
ABSTRACT |
Despite having several characteristics of naïve B cells,
chronic lymphocytic leukemia (CLL) cells have been shown in some cases
to have somatically mutated Ig variable region genes, indicating that
the cell of origin has passed through the germinal center. A previous
study of patients with CLL found an association between lack of somatic
mutation and trisomy 12 and, therefore, possibly with a less favorable
prognosis. We have sequenced the Ig VH genes of the tumor
cells of 84 patients with CLL and correlated our findings with clinical
features. A total of 38 cases (45.2%) showed  98% sequence
homology with the nearest germline VH gene; 46 cases
(54.8%) showed >2% somatic mutation. Unmutated VH genes were significantly associated with V1-69 and D3-3 usage, with atypical
morphology; isolated trisomy 12, advanced stage and progressive disease. Survival was significantly worse for patients with unmutated VH genes irrespective of stage. Median survival for stage A
patients with unmutated VH genes was 95 months compared
with 293 months for patients whose tumors had mutated VH
genes (P = .0008). The simplest explanation is that
CLL comprises 2 different diseases with different clinical courses.
One, arising from a memory B cell, has a benign course, the other,
arising from a naïve B cell, is more malignant.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CHRONIC LYMPHOCYTIC leukemia (CLL) is
characterized by the relentless accumulation of monoclonal B
cells with the appearance of small mature lymphocytes and
with a characteristic immunophenotype. Typically, they are
positive for CD5, CD23, and CD19 and negative for surface CD22 and
FMC7.1 Surface Ig (usually IgM plus IgD) is sparse
and the immunoglobulin-associated molecule, CD79b, is low or
absent.2 Most cells are in the G0 phase of
the cell cycle and are unresponsive to mitogenic stimuli.3
The cells overexpress the Bcl-2 gene product and are resistant to
apoptosis.4
Because CD5 positive B cells are found in the fetal spleen5
and surface IgD is a feature of cells that have not yet met antigen in
the germinal center,6 it has been suggested that CLL is a
tumor of naïve B cells possibly arising in the follicular mantle zone.7
A further means of establishing the stage of differentiation of a
B lymphocyte is provided by examining the Ig variable domain genes. Although the selection and recombination of VH,
D, and JH genes and the insertion of nontemplated
nucleotides at the VH-D and D-JH junctions are
early events occurring in the bone marrow, the process of somatic
mutation, which tends to focus amino acid changes in the first
and second complementarity determining regions (CDR1 and
CDR2) of the molecule, occurs in the germinal center
environment.8
Early sequences of the VH genes of tumor cells from
patients with CLL found them to be in germline
configuration,9-11 tending to confirm their origin from a
naïve B cell. However, reports began to appear in the
literature detailing cases with evidence of somatic mutation
culminating in 1994 with a review of the literature by Schroeder and
Dighiero,12 which found that 36 of 75 reported cases had
VH genes with less than 98% sequence homology to the appropriate germline gene. The figure of 98% was chosen because polymorphisms, which are quite common in VH genes, can
account for that degree of disparity.13 Schroeder and
Dighiero suspected that CLL might be a heterogeneous disorder, but were
unable to cull from the literature the comprehensive clinical detail
needed to establish this. They did report, however, that some of the cases with mutated VH genes were CD5 negative and therefore
not cases of classical CLL.
More recently, a multicenter study of 64 patients with surface
IgM+, CD5+ CLL also found 2 groups of roughly
equal numbers with respectively mutated and unmutated VH
genes.14 Although no clinical detail was available
otherwise to distinguish the 2 subsets, the investigators were able to
confirm the observation of Schroeder and Dighiero that the presence or
absence of somatic mutations was associated with the use of particular
VH genes.
In 1997, our group examined the VH genes of 22 patients
with classical B-cell CLL segregated according to karyotype. Tumors with trisomy 12 had unmutated VH genes, but those with
13q14 abnormalities detected by conventional cytogenetics had evidence
of somatic mutations.15 Because it has been previously
shown that CLL patients with trisomy 12 have a poorer survival than
those with abnormalities at 13q14,16 this pointed to an
association between clinical status and degree of somatic mutation.
The suspicion that the clinical heterogeneity of CLL might have a
biological basis led us to extend this study. We have now examined the
Ig VH gene sequences in a series of 84 patients with classical B-cell CLL attending our hematology clinic and compared our
results with various clinical characteristics of the patients and their
survival. The striking finding to emerge is that the presence of
VH gene mutations places the CLL patient in a disease group
with a clearly better prognosis.
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MATERIALS AND METHODS |
Patients.
Patients were selected for study from over 600 cases of CLL who have
attended our hematology clinic in recent years. An attempt was made to
include a representative selection of karyotypic disorders, but most
patients were chosen simply because they happened to be attending the
outpatient clinic for routine follow-up. Twenty-two of the patients
were included in the series linking trisomy 12 with the absence of
somatic mutation that we reported recently.15 Almost all of
the patients studied came from the local area and included many whose
diagnosis was made incidentally from a blood count ordered for another
purpose and whose CLL has remained entirely asymptomatic. It has been
our practice, nevertheless, to continue to observe such asymptomatic
patients once or twice a year. They were staged at diagnosis according
to the Binet Classification.17 Immunophenotyping was
performed afresh during the course of the current investigation. All
cases scored 4 or 5 according to the Royal Marsden scoring system for
CLL,1 were CD23 and CD5 positive, and had monotypic
expression of weak surface Ig.
All patients have been followed for at least a year since diagnosis,
the longest follow-up being 25 years. Patients were designated as
having stable or progressive disease on the basis of the following criteria: lymphocyte count doubling time of less than 1 year; progression to a more advanced Binet stage; development of systemic symptoms; development of Richter's syndrome; downward trend of hemoglobin (Hb) or platelet count to below the normal range (Hb < 13.5 g/dL for males and < 11.5 g/dL for females; platelet count < 150 × 109/L) even when not meeting the criteria for
stage C disease (Hb < 10 g/dL; platelet count < 100 × 109/L). Possession of one of these characteristics was
sufficient to qualify as progressive disease. Patients with progressive
disease have generally been treated conventionally with chlorambucil as first line therapy and with fludarabine for those with resistant disease. Patients with stable disease were not offered chemotherapy.
Lymphocyte morphology was assessed on blood films stained with Jenner
Geimsa. Atypical morphology was defined according to the criteria of
Matutes et al18 as greater than 10% prolymphocytes or
greater than 15% cells with cleaved nuclei and/or lymphoplasmacytoid cells in the blood of patients whose predominant cell type was a small
lymphocyte with coarsely clumped chromatin.
Cytogenetics.
Whole peripheral blood was cultured at 37°C in the presence of
tetradecanoyl phorbol 12-myristate 13-acetate (TPA) (0.05 µg/mL) for
3 to 5 days. Cells were treated with colcemid (0.1 µg/mL) for 60 minutes at 37°C before harvesting. After hypotonic treatment (KCL
0.075 mol/L for 10 minutes at 37°C), cells were resuspended in
fixative (methanol: glacial acetic acid 3:1). Standard cytogenetic preparations were made, G banded, and karyotyped according to the
International System for Cytogenetic Nomenclature (1995).
Preparation of cDNA and DNA.
Blood samples for testing were taken during the past 5 years. Some were
tested immediately, while for others, the lymphocytes were
cryopreserved and tested later. Because the VH gene
signature is believed not to change during the clinical course of CLL,
it was deemed satisfactory to determine this at any stage of the disease whether treated or not. The preferred source material was RNA,
as this reduces the possibility of amplifying an aberrantly rearranged
VH gene; cDNA was synthesized by reverse transcription using an oligo(dT) primer as previously described.19 Where
RNA was not available, genomic DNA was extracted using the QIAmp blood kit (Qiagen, Hilden, Germany).
Amplification of VH genes.
One fifth to one third of a sample of cDNA was amplified by polymerase
chain reaction (PCR) using a mixture of oligonucleotide 5'
primers specific for each leader sequence of the VH1 to
VH6 families,20 together with either mixed
3' primers complementary to the germ line JH
regions21 or 3' primers complementary to the constant
region.14 When there was failure to amplify, an alternative
mixture of 5' primers specific for framework 1 region of
VH1 to VH6 was substituted.21 In
our hands, the VH1 leader primer also amplifies sequences
from the closely related VH7 family. In all cases, PCR was
performed in a final volume of 50 µL with 20 pmol of each primer, 50 µmol deoxyribonucleotide triphosphates (dNTPs), and 2.5 U
Taq DNA polymerase with reaction buffer (Boehringer, Lewes, E Sussex,
UK). Amplification consisted of an initial denaturation step of 3 minutes at 94°C followed by 30 cycles of 94°C, 56°C and
72°C for 45 seconds each, with a final extension step of 10 minutes
at 72°C. All PCR reactions were performed in duplicate. For each
PCR, a control with no added template was used to check for contamination.
Sequencing and cloning of PCR products.
Clonal sequences were determined by sequencing amplicons from at least
2 independent PCR reactions. The majority of samples were sequenced
directly using an automated DNA sequencer (Applied Biosystems, Foster
City, CA). However, for the first 35 cases, cloning of gel-purified
products into pGEM-TA vector was performed.19,21 After
transformation of JM109 competent cells, clones found to contain an
insert of appropriate size by restriction analysis of plasmid DNA were
sequenced.19,21 A minimum of 5 bacterial colonies
were analyzed. In addition, in 3 later cases, direct sequencing was
unsuccessful and the sequence was determined by cloning.
Analysis of Ig gene sequences.
Nucleotide sequences were aligned to EMBL/GenBank and current databases
(V-BASE sequence directory,22 using MacVector 4.0 sequence
analysis software; International Biotechnologies Inc, New Haven, CT).
Where there was >2% deviation from a germline VH
sequence, the Chang and Casali formula23 was used to
determine whether the replacement mutations had undergone antigenic
selection. We have followed the criteria of Corbett at al24
in assigning membership of the 2 longer D gene families (D2 and D3),
but the requirement for 10 consecutive nucleotides of identity are
probably too stringent for the shorter D gene families, and we have
followed Fais et al14 in requiring only 7 consecutive
nucleotides with no more than 2 differences. We have eliminated DIR
segments and "minor" D segments from analysis.24
Statistical analysis.
The significance of associations between characteristics was determined
using Fisher's exact test. Survival curves from date of diagnosis were
plotted using GraphPad Prism (version 2) software (San Diego, CA). This
program calculates survival fractions using the Kaplan-Meier method and
compares survival curves using the log-rank test.
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RESULTS |
Clinical and cytogenetic features of patients studied.
We studied 35 women and 49 men with classical B-cell CLL. The mean age
at diagnosis was 63.3 years (median, 65; range, 33 to 92). A total of
62 were stage A, 9 stage B, and 13 stage C. A total of 34 had
progressive disease and 50 stable disease. Lymphocyte morphology was
typical in 58 cases and atypical in 26 cases.
Karyotypes are shown in Tables 1 and
2. Trisomy 12 was found as a single
abnormality in 11 patients and in combination with other abnormalities
in a further 15 patients. Nine patients had translocations
or deletions at 13q14 as single abnormalities, and 7 had these in
association with other abnormalities. Ten had translocations involving
chromosomal regions close to Ig genes on chromosomes 14 and 22, mainly
in association with other abnormalities. Deletions involving 11q23 were
found in 8 patients. Twenty-three patients had a normal karyotype. In 2 patients, chromosomal analysis was not attempted, and in 2, no
metaphases were obtained.
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Table 1.
Demographics and Clinical and Laboratory Findings in
Patients With CLL Whose VH Genes Showed 98%
Sequence Homolgy With the Nearest Germline VH Gene
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Table 2.
Demographics and Clinical and Laboratory Findings in
Patients With CLL Whose VH Genes Showed <98%
Sequence Homology With the Nearest Germline VH Gene
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Use of VH, D, and JH genes.
In common with several other series,9,12,14 we found an
overuse of the V1-69 gene. Twenty-eight of the 51 translatable VH genes were used in this series. Six genes accounted for
57% of cases. V1-69, V3-23, and V4-34 were each used on 10 occasions (each 11.9%) and V1-02, V3-30.3, and V3-07 were each used on 6 occasions (each 7.1%). However, the normal antibody repertoire does
not use VH genes randomly, and V3-23 (13.9%), V3-07
(5.6%), and V3-30.3 (8.3%) are the VH genes most commonly
used by normal CD5+ B cells.25 In addition, we
have previously shown that the V4-34 gene product is used by between
2.9% to 10.8% of normal B cells.26 On the other hand,
V1-69 is used by only 1.6% of normal B cells27 and
therefore appears overrepresented in this series.
The majority of the tumors used JH4 (35 of 84; 41.7%) or
JH6 (27 of 84; 37.1%), which is similar to their use in
normal CD5+ B cells (52% and 27%,
respectively).25
Because of extensive mutations or N additions or nucleotide loss, a D
segment gene could not be assigned to the sequences in 31 cases. The
most commonly used gene was D3-3 (DXP4), which was used in
8 cases, all of which had unmutated VH genes. In all, 17 of
the 27 different D-segment genes were used. D segments were not used
preferentially with any VH or JH gene, but
could not be assigned in the cases with the greatest numbers of somatic mutations.
Somatic mutations.
Whereas the leukemic cells of 38 patients (45.2%) had VH
genes with 98% sequence homology with the nearest germline gene, the remaining 46 cases (54.8%) showed evidence of somatic
hypermutation, dividing the series into 2 subsets.
There are clear differences between the 2 subsets. Patients with
unmutated VH genes (Table 1) had characteristics associated with a more malignant type of disease than those with evidence of
somatic mutations (Table 2). Those lacking mutations were significantly
more likely to have advanced stage disease, whereas those with
mutations were more likely to have stage A disease (P = .0009).
Lack of mutations was significantly associated with progressive disease and the presence of mutations with stable disease
(P < .0001). Atypical morphology was similarly associated with lack of mutations and typical morphology with their presence (P < .0001). Trisomy 12, as an isolated karyotypic
abnormality, was significantly associated with a lack of somatic
mutations (P = .0019), and deletions or translocations at 13q14
were significantly associated with their presence (P = .023).
There was a significant tendency for VH1 family genes
(P = .0142) and especially V1-69 (P = .0038) to be used
by the subset that lacked mutations. A similar tendency towards the use
of the D3-3 gene segment was found in this subset (P = .0058),
although there was no tendency for these gene segments to be used together.
For patients with unmutated VH genes, the mean age at
diagnosis was 64.9 years (median, 66; range, 36 to 88). There were 13 women and 25 men. For patients with mutated VH genes, the
mean age was slightly younger at 61.9 years (median, 63; range, 33 to
92), and the gender ratio was closer to unity with 22 women and 24 men,
although these differences did not reach statistical significance.
Antigenic selection.
Analysis of the distribution of replacement and silent
mutations23showed a clustering of replacement amino acids
in CDR1 and CDR2 in 17 of 46 (37.0%) of the cases with somatic
mutations and conservation of amino acid sequence in framework regions
in 28 of 46 (60.9%).
Intraclonal heterogeneity.
In 38 cases, at least 5 separate clones were analyzed. No intraclonal
heterogeneity was found. This is in contrast to the finding in
follicular lymphoma and implies that cells showing somatic mutations
were no longer under the influence of mutator enzymes and had therefore
passed through the germinal center. In 3 cases, a second blood sample
was analyzed respectively 5 years, 3 years, and 18 months after the
first. In each case, the clonal sequence was identical with the
original sequence (with respectively 0, 2, and 19 mutations).
Prognostic significance of VH gene mutations.
Survival curves were plotted according to the Kaplan-Meier method. Only
2 patients were censored as lost to follow-up. Graphs comparing the
survival of patients with mutated and unmutated VH genes
are plotted in Fig 1. The median survival
for patients with unmutated VH genes was 117 months and for
those with mutations, 293 months. The difference is very significant
(P = .001). Because the unmutated group contains more patients
with advanced stage disease, we plotted the survival curve for stage A
patients only (Fig 2). Median survival was
95 months for the patients without mutations and 293 months for those
with mutations. Again, the difference in survival was very significant
(P = .0008).
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| Fig 1.
Kaplan-Meier survival curve comparing CLL patients with
mutated and unmutated VH genes. Median survival for
unmutated CLL: 117 months; median survival for mutated CLL: 293 months.
The difference is significant at the P = .001 level (log-rank
test).
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| Fig 2.
Kaplan-Meier survival curve comparing stage A CLL
patients with mutated and unmutated VH genes. Median
survival for unmutated CLL: 95 months; median survival for mutated CLL:
293 months. The difference is significant at the P = .0008 level (log-rank test).
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DISCUSSION |
Somatic mutation is believed to occur in germinal centers or similar
structures.8 Thus, the presence of such mutations in over
half of our cases of CLL and the statistical evidence supporting the
occurrence of antigenic selection in some of the cases argues strongly
that these are tumors of memory B cells and not of naïve B cells.
In any study of CLL, it is important to establish that the series is
not contaminated by other kinds of lymphoma. It is particularly important to exclude mantle cell lymphoma, which is also a tumor of
CD5+ B cells and which has a much worse prognosis than CLL.
Other diseases that must be excluded are splenic lymphoma with villous lymphocytes, of which a small proportion are reportedly
CD5+,28 and follicular lymphoma in leukemic
phase. In this series, we have tried very hard to exclude all such
cases. All had a Royal Marsden score of 4 or 5. No case
had t(11;14)(q13;q32) translocations. Although 7 had t(14;18)(q32;q21)
or t(18;22)(q21;q11) translocations, it is important to stress that
such structural abnormalities have been previously reported in CLL and
are not confined to follicular lymphoma.29 None of these
cases had cellular morphology or immunophenotype typical of follicular
lymphoma, and none showed the intraclonal variation of immunoglobulin
VH genes seen in that condition.30
Patients with unmutated VH genes had a distinctly more
malignant disease and a much shorter survival than those with somatic mutations. They were more likely to have atypical morphology, advanced
stage, and progressive disease. Such a distinct clinical course argues
in favor of CLL comprising 2 separate types of tumor arising at
different stages of B-cell maturation: 1 a pregerminal center
naïve B cell and 1 a postgerminal center memory B cell.
In this series of patients, the influence of karyotype on prognosis is
less than in our previous report,15 perhaps because we have
included here 5 patients with both trisomy 12 and abnormalities at
13q14. As more sensitive techniques such as fluorescent in situ
hybridization and comparative genomic hybridization are applied, it is
apparent that leukemic chromosomes are more complicated than has been
appreciated using conventional cytogenetics. It is clear that much more
detailed work is required to establish the influence of chromosomal
abnormalities on the 2 types of CLL.
We confirm in this study the biased use of certain VH genes
in CLL. Six genes accounted for over half of the cases. However, when
Brezinschek et al25 analyzed the normal B-cell
immunoglobulin repertoire by single cell PCR of DNA from B cells of 2 individuals, they found that many of the same genes were similarly
overused. An exception is V1-69, which apparently not overused by
normal B cells,27 yet has been reported consistently as the
most commonly used VH gene in CLL, usually in unmutated
form.9,14,31 V1-69 occurs in several polymorphic forms and
there is variation in the number of copies of V1-69 between different
individuals.32,33 It would be helpful, therefore, to
investigate the normal use of VH genes from a wider range
of normal individuals, including some from the same age range as
patients with CLL, before concluding that this gene is used more
commonly in CLL than in normals. Why the V1-69 gene is seen so commonly
in CLL in unmutated form remains unanswered. None of our patients used
the V4-39 gene, but this gene is also usually unmutated in the reports
in the literature, even in cases expressing surface
IgG.12,14 Explanations for these phenomena are
available,14 but lack certainty at present.
The suggestion that there might be 2 types of CLL pursuing distinct
clinical courses and definable by the stage of differentiation of the
cell of origin is independently supported by the work of Damle et
al.34 It raises many pathophysiological
questions, but clinicians will be interested in the prognostic
implications. Thirty-three of our 84 patients (39.3%) were diagnosed
below the age of 60 and might today be considered as candidates for
aggressive chemotherapy and stem cell autograft for this incurable
disease. Knowledge that 19 of them belonged to a subtype with a median survival of 25 years and that the other 14 belonged to a subtype with a
median survival of only 8 years would be valuable.
| |
ACKNOWLEDGMENT |
The authors acknowledge the help of Dr D. Zhu and A. Thompsett who
performed some of the gene sequences. Professor G.T. Stevenson and Dr
M. Glennie provided helpful discussions. Dr R. Chapman and Dr P. Thomas
gave statistical advice.
| |
FOOTNOTES |
Submitted March 11, 1999; accepted May 6, 1999.
Supported by a grant from Tenovus UK (T.J.H. and Z.D.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Terry J. Hamblin, MD,
Department of Haematology, Royal Bournemouth Hospital, Castle Lane
East, Bournemouth BH7 7DW, Dorset, UK; e-mail terjoha{at}aol.com.
| |
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