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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1413-1419
NEOPLASIA
From the Hematopathology Section, Laboratory of Pathology, National
Cancer Institute, National Institute of Health, and Flow and Image
Cytometry Section, Laboratory of Medical and Molecular Genetics,
Division of Cell and Gene Therapies, Center for Biologics Research and
Evaluation, Food and Drug Administration, Bethesda; Genetic
Epidemiology Branch, National Cancer Institute, National Institute of
Health, Rockville; and NIH Intramural Sequencing Center and National
Human Genome Research Institute, National Institute of Health,
Gaithersburg, MD.
In this study, we wished to determine whether familial chronic
lymphocytic leukemia of B-cell phenotype (CLL) shares with sporadic
B-CLL the same immunoglobulin (Ig) heavy chain variable region (VH)
gene usage and occurrence of somatic mutation, to gain insight into the
pathogenetic relatedness of these epidemiologically distinct forms of
CLL. We therefore analyzed the expressed Ig heavy chain genes in 23 cases (11 families) of familial CLL, and compared these results with
data previously reported for sporadic CLL. In addition, we assessed the
relationship of the occurrence of somatic mutation to several clinical
and phenotypic features. The distribution of V genes among these cases
was similar to that observed in sporadic CLL: VH3 > VH1 > VH4.
Thirteen of the 23 cases (57%) showed germ line VH gene sequences,
whereas somatic mutations were detected in 10 cases (43%). The average
mutation frequency of these latter 10 cases of was 6.7% (ranging from
1.7% to 8.8%), and evidence of antigen selection was noted in 6. Intraclonal variation, followed by clonal evolution and the appearance
of a second clone over a 20-year period was observed in 1 case,
suggesting that mutations can continue to accumulate after neoplastic
transformation. The presence of somatic mutations correlated with age
at presentation, low white blood cell (WBC) count, and low fluorescence
intensity of surface CD5, and the potential significance of these
relationships is discussed. Our data indicate that familial and
sporadic B-CLL display a similar pattern of immunoglobulin gene usage
and frequency of somatic mutation, and are consistent with a common
ontogeny and immunogenetic origin for these 2 epidemiologically
distinct forms of CLL.
(Blood. 2000;95:1413-1419)
Chronic lymphocytic leukemia of B-cell phenotype (CLL)
is the most common leukemia in the adult population in the United
States and Europe.1 This is a clinically indolent neoplasm
and results from an accumulation of CD5+ neoplastic B-cells
with a low proliferative rate. The normal counterpart of B-CLL is
believed to be the B-1a cell,2 which accounts for 5% to
30% of the normal circulating B-cell population, and is characterized
by surface expression of IgM/IgD and CD5, and the ability to produce
autoantibodies.3 CLL has been reported to show nonrandom
usage of VH region families and genes, an observation hypothesized to
be related to the B-cell subset of origin, and to the peculiar
antibody-mediated autoimmunity sometimes accompanying this
leukemia.3,4
Traditionally, CLL has been viewed as a homogeneous disease, with its
characteristic immunophenotype and indolent clinical course. However,
more recent cytogenetic and immunogenetic studies have challenged this
viewpoint. Several different cytogenetic abnormalities have been
reported, each with somewhat different accompanying clinical and/or
phenotypic features.5,6 Trisomy 12 and 11q23 deletions have
been associated with a shorter median survival,7-12 whereas
cases with 13q14 deletions appear to have a more favorable
outcome.12
Further evidence for heterogeneity in CLL comes from investigations of
the expressed immunoglobulin genes. Although early studies of the IgH
gene in CLL failed to demonstrate somatic mutation,13-16 more recent analyses have shown that a substantial (20%-50%)
percentage of cases show somatic mutations, suggesting that CLL may
arise from either pregerminal center naive B-cells, or germinal center exposed B-cells.3,17-20 These studies also suggest that the
presence or frequency of somatic mutation correlates with
immunophenotype,20 the usage of variable region gene (VH)
families18 and the presence of specific chromosomal
abnormalities.19 Fais et al18 reported somatic
mutation in 50% of the IgM+ expressing cases, with the highest
incidence noted in the VH3 family, compared with VH1 and VH4. Oscier et
al19 noted that cases with trisomy 12 lacked somatic
mutation, whereas cases with 13q14 deletion showed significant levels
of somatic mutation. The existence of CLL lymphocytes having undergone
somatic mutation suggests that a subset of CLL is derived from memory B
cells that have passed through the germinal center stage of B-cell
differentiation. Taken together, the above observations indicate that
CLL is more heterogeneous than previously thought and may develop
either from ontogenically naive B lymphocytes or from a more mature
antigen-exposed memory B cells.
Familial aggregation of CLL (familial CLL) has been observed more
frequently than in any other type of leukemia, and immunogenetic factors related to HLA type have been implicated in its
development.21-23 Besides familial and genetic
predisposition, environmental factors may also play a role in this
clustering of cases. Although familial CLL is indistinguishable from
sporadic CLL in its morphology and immunophenotype, the pathogenetic
relationship between these epidemiologically distinct forms of CLL has
not been clearly established. There are virtually no cytogenetic or
molecular genetic studies comparing familial CLL with sporadic CLL.
Likewise, studies of immunoglobulin gene usage and somatic mutation of
immunoglobulin genes are limited in familial CLL, for comparison to
sporadic CLL.24,25 To gain insight into the pathogenetic
relationship between familial CLL and sporadic CLL, we wished to study
the spectrum of immunoglobulin heavy chain gene usage and the presence
or absence of somatic mutation in familial CLL. Because of the
genetic overlay to familial CLL, we hypothesized that it may be a more
restricted disease with regard to its immunoglobulin gene usage
or ontogenic development, as reflected by presence or absence of
somatic mutation.
We therefore investigated the immunoglobulin gene usage and pattern of
somatic mutation in 23 patients from 11 families with familial CLL and
compared these findings with those reported in sporadic CLL.
Furthermore, we examined the relationship between the presence or
absence of somatic mutation in the expressed IgH genes, and several
clinical and immunophenotypic features. We found a pattern of
immunoglobulin gene usage and somatic mutation similar to that reported
for sporadic CLL, suggesting that familial CLL displays a similar
heterogeneity. We also found an unexplained inverse correlation between
CD5 antigen density and the presence of somatic mutation.
Patients/samples
Immunophenotypic analysis
Isolation of RNA and first-strand cDNA Total cellular RNA was isolated from cryo-preserved mononuclear cell suspensions using TRIZOL reagent (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions. After DNase I (GIBCO BRL, Gaithersburg, MD) treatment, 1 to 5 µg total RNA were incubated with 50 µmol of random hexamer primers (PE, Foster City, CA) for 10 minutes at 70°C. After cooling on ice, the reaction mixture was added to a final volume of 20 µL containing 200 U of Super Script II, 1 × First Strand Buffer (50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl2), 10 mmol/L of DTT, 20 U of RNase inhibitor, and 0.5 mmol/L of each dNTP (GIBCO BRL, Gaithersburg, MD). The reaction was performed for 10 minutes at 25°C followed by 50 minutes at 42°C. Subsequently, the enzyme was inactivated for 15 minutes at 70°C.Polymerase chain reactions One microliter of cDNA was amplified using GeneAmp System 2400 (PE, Foster City, CA) with a 50 pmol specific upstream primer corresponding to 1 of the 6 human VH family leader sequences (VHL1, 5'-CCATGGACTGGACCTGGAGG-3'; VHL2, 5'-ATGGACATACTTTGTTCCAGC-3'; VHL3, 5'-CCATGGAGTTTGGGCTGAGC-3'; VHL4, 5'-ATGAAACACCTGTGGT TCTT-3'; VHL5, 5'-ATGGGGTCAACCGCCATCCT-3'; VHL6, 5'-ATGTCTGTCT CCTTCCTCAT-3')26 and a 50 pmol downstream primer (JHa, 5'-ACCTGAGGAGACGGTGACC-3')27 corresponding to a consensus sequence at the 3' end of the J region in a 50 µL volume. The polymerase chain reaction (PCR) contained 1 × PCR buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl), 1.5 mmol/L MgCl2 (for VHL1, VHL3, and VHL5) or 2.0 mmol/L MgCl2 (for VHL2, VHL4, and VHL6), 0.2 mmol/L of each dNTP, 1.25 U Taq polymerase (PE, Foster City, CA) mixed with TaqStartTM antibody (Clontech, Palo Alto, CA). First, 1 cycle of amplification was performed under the following conditions: denaturation at 95°C for 7 minutes, annealing at 65°C for 4 minutes, and extension at 72°C for 1 minute. The next 40 cycles of amplification were at 95°C for 1 minute, 61°C for 30 seconds, and at 72°C for 1 minute. The reaction was completed with extension at 72°C for 6 minutes. The PCR products were analyzed on a 3% NuSieve GTG agarose gel (FMC, Rockland, ME) and visualized by staining with ethidium bromide.Cloning and sequencing of PCR products After excision of the PCR products from an agarose gel, DNA was isolated using a QIAEX DNA extraction kit (Qiagen, Santa Clarita, CA). The recovered DNA was ligated into the pCR2.1 vector, following the manufacturer's instruction (TA cloning kit; Invitrogen, San Diego, CA). Three to 5 colonies were picked at random and grown overnight in 3 mL LB (Quality Biological, Gaithersburg, MD) medium. Recombinant plasmids were purified by DNA affinity columns (Qiagen, Valencia, CA) and selected by restriction analysis. The double-stranded plasmid was sequenced in an automatic DNA sequencer (PE Applied Biosystems 377 × l Automated DNA; PE Applied Biosystems, Foster City, CA) using the BigDyeTM Terminator Cycle Sequencing Ready Reaction kit (PE, Foster, CA), following manufacturer's instruction.Analysis of VH gene usage and mutations Sequences obtained from each sample were compared with germ line sequences in the V Base sequence directory (I.M. Tomlinson, MRC Center for Protein Engineering, Cambridge, UK) using MacVector 6.0 sequence analysis software (Oxford Molecular Group, Campbell, CA), and the closest sequence was assigned. Attribution of the D segments was based on the identification of at least 6 consecutive bases without mismatches. The nomenclature proposed by Corbett et al28 was adopted. We considered a VH gene sequence to be mutated if it had equal or more than 1.5% sequence alterations when compared with the published germ line sequence. Mutated cases had between 5 and 26 nucleotide differences. Nonmutated cases had 0 or 1 nucleotide differences from published germ line VH gene sequences.
Statistical analysis The relationship between age, gender, WBC count, Rai stage, duration of disease from diagnosis, the intensity of surface antigens, and the presence of somatic mutations was examined using a variety of approaches, including Mann-Whitney U test (nonparametric) and independent sample t tests. Multiple linear regression was used to examine the effects of several independent variables, but this analysis was limited by the small number of subjects. The relationship between the presence of somatic mutation and the fluorescence intensity of CD5, CD20, and CD45 on the leukemia cells was analyzed by similar methods. A 2-sided P value of <.05 was considered significant.
Clinical features The clinical features of 23 cases of familial B-CLL are summarized in Table 1. The age, WBC count, Rai stage, and duration of disease correspond to the date when the patients were referred to NIH. One individual (2a) was evaluated at 2 time points with a 10-year interval and the Rai stage remained unchanged. Except for 1 family (no 4) with 3 affected individuals, there were 2 affected individuals in each family. In all families but 1, the patients were siblings. Thirteen of the 23 cases were male, with an age ranging from 40 to 75 years (median, 61 years). The WBC count ranged from 6.1 to 148.8 × 103 /µL (median, 45.2 × 103 /µL), and Rai stage at presentation are listed in Table 1. The duration of disease ranged from less than 1 year to 20 years.
Immunophenotypic analysis and clonality All cases had a typical immunophenotype characterized by monotypic surface immunoglobulin, CD5, CD19, and CD23 expression. Two- color FACS analysis revealed coexpression of CD5 in more than 99% of the CD19+ B cells, except for cases 5b, 7b, and 11b, in which there were some residual normal B cells (1.0%, 4% and 1.1%, respectively). All neoplastic B cells expressed CD23, or  .
Clonality was verified by PCR using cDNA generated from PBMC and the VH
family specific primers. PCR products from all cases showed a single
predominant band corresponding to 1 of 6 VH families. The 3 cases
containing 1% or more normal Bcells, 5b, 7b, and 11b, showed some
additional faint bands presumably originating from this population
(data not shown).
VH, D, and JH gene usage by familial B-CLL A summary of the data are shown in Table 2. All VH families, except VH6, were observed at least once in these cases of familial B-CLL. VH3, VH1, and VH4 families were expressed at the highest frequency (37.5%, 33.3%, and 20.8%, respectively), with VH2, VH4, VH5, and VH7 each occurring only once. In 4 families, identical usage of the VH family was recognized in at least 2 cases, ie, VH3 in family nos 3, 4, and 7, VH1 in family no 6. However, only the 2 cases of family no 6 used the same VH gene (VH1-69/DP-10). In the other cases, different VH genes were used. Comparison with the germ line JH gene segments showed the preferential usage of JH4b, followed by JH6b and JH5b (43.5%, 30.4%, and 13.0%, respectively). D segments were attributed in 70% of cases.
Mutation pattern in familial B-CLL The mutation analysis data are summarized in Table 2, and the deduced amino acid sequences of the IgH (VDJ) gene are depicted in Figure 1. The similarity of the VH genes to the closest germ line genes is shown as percentage identity and ranged from 91.2% to 100% (Table 2). Thirteen of the 23 cases showed 99% to 100% homology to their closest germ line genes. The others showed differences of more than 5 nucleotides (ranging from 5 to 26). It cannot be excluded that some of these nucleotide differences derive from PCR errors or represent germ line polymorphisms, especially, in those cases showing 1 nucleotide difference. However, since most, if not all, of VH germline gene segments have been identified,30 these nucleotide differences were considered to be the result of somatic mutations.
Intraclonal mutations in family 2 We were able to analyze 2 samples collected over a 10-year interval (1988 and 1998) from case 2a (Figure 1). Six independent colonies were screened from the 1988 sample. All displayed rearrangements of the same VH1 family (VH1-02/DP-75), and used the same D and JH region (D6-19 and JH5b, respectively). However, sequence variations were detected among the 6 colonies. Three clones formed 1 identical set (a/1), 2 formed a second identical set (a/2), and the last clone (a/3) had yet a third distinct, although related, sequence. The subsequent specimen, obtained 10 years later in 1998, contained 2 distinct sequences. 1 (a'/1) was nearly identical to the a/3 sequence variant from the 1988 sampling, with the exception of 2 additional mutations present in CDR2. The second sequence was a completely different rearrangement, using a VH7 family gene (VI-4), and D and JH region genes, D2-15 and JH4b, respectively. These 2 subclones were verified by sequencing multiple colonies from separate RT-PCR reactions. No additional intraclonal variations were detected. These findings are consistent with the occurrence of intraclonal variations in the 1988 biopsy and clonal evolution in 1998 (a'/1), and suggest the emergence of a new clone as shown by the a'/2 sequence. Consistent with the molecular data, flow analysis of the 1998 specimen suggested a bimodal distribution of CD5 positive B cells (data not shown). Because intraclonal variation is highly unusual in CLL, it is important to reemphasize that the morphologic and phenotypic features of this patient's disease were fully consistent with CLL (CD5+, CD23+).Correlation of somatic mutations with clinical and phenotypic parameters The age at presentation, gender, Rai stage, WBC count, and duration of disease were examined in relation to the presence or absence of somatic mutations. Subjects with somatic mutations tended to be older 66.8 versus 54.9, P = .005 and have lower WBC counts 25.1 versus 63.6, P = .015 in univariate analysis. The Rai stage, duration since presentation, and gender distributions by mutation status were similar. Small numbers limited the multivariate analysis.
B-cell chronic lymphocytic leukemia has traditionally been thought of as a homogeneous disease because of its rather uniform morphology and immunophenotype. Recent cytogenetic and molecular biologic analyses have challenged this view. It has become apparent that cases of CLL may contain distinct and recurring cytogenetic abnormalities with up to 30% of cases with trisomy 12,9,32 45% with del 13q24,11 and another 20% with deletions of 11q23.11 Moreover, analysis of the expressed immunoglobulin genes in CLL indicate that as many as 30% to 50% of cases show evidence of somatic mutation, with a high percentage of these showing evidence of having undergone antigen selection.18 These data suggest that sporadic CLL develops from either a CD5+ naive B cells or from a more mature CD5+ B cell that has undergone somatic mutation and antigen selection. This recently recognized heterogeneity in sporadic CLL led us to ask whether the familial variant of CLL would show the same heterogeneity with respect to the expressed immunoglobulin gene as that seen in sporadic CLL, or whether familial CLL would show a more restricted profile.
We thank Patricia H. Carter for sample preparation and immunophenotyping results and Laura Fontaine, RN, for invaluable assistance with patient management and specimen organization.
Submitted July 21, 1999; accepted October 15, 1999.
Reprints: Mark Raffeld, Laboratory of Pathology, National Cancer Institute, Building 10, Room 2N110, 9000 Rockville Pike, Bethesda, MD 20892; e-mail: mraff{at}box-m.nih.gov.
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.
The US government retains nonexclusive, royalty-free license to any copyright covering this article.
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Top
Abstract
Introduction
k)!]} × qk × (1 
k, where n is the total number
of observed mutations, k is the number of observed R
(replacement) mutations in the CDRs or framework regions (FRs), and
q is the probability that an R mutation will localize to the
CDRs or FRs (q = CDR rel × CDR Rf. or
FRrel × FR Rf). The inherent susceptibility
of R mutations of the CDRs and FRs (CDR Rf and FR Rf,
respectively) is calculated for each of the identified germ line genes
and it is based on any single nucleotide change that gives rise to an
amino acid replacement, whereas substitutions that result in stop
codons are excluded. CDR rel and FR rel
represented the relative sizes of the CDRs and FRs.
