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Prepublished online as a Blood First Edition Paper on June 14, 2002; DOI 10.1182/blood-2002-03-0892.
 Previous Article | Table of Contents | Next Article 
Blood, 1 October 2002, Vol. 100, No. 7, pp. 2289-2290
PLENARY PAPER
Inherited predisposition to CLL is detectable as subclinical
monoclonal B-lymphocyte expansion
Andy C. Rawstron,
Martin R. Yuille,
Julie Fuller,
Matthew Cullen,
Ben Kennedy,
Stephen J. Richards,
Andrew S. Jack,
Estella Matutes,
Daniel Catovsky,
Peter Hillmen, and
Richard S. Houlston
From the Academic Unit of Haematology and Oncology,
University of Leeds, HMDS, West Yorkshire; Academic Department of
Haematology and Cytogenetics, Institute of Cancer Research, Surrey;
Section of Cancer Genetics, Institute of Cancer Research, Surrey,
United Kingdom.
 |
Abstract |
Monoclonal chronic lymphocytic leukemia (CLL)-phenotype
cells are detectable in 3.5% of otherwise healthy persons using flow cytometric analysis of CD5/CD20/CD79b expression on CD19-gated B cells.
To determine whether detection of such CLL-phenotype cells is
indicative of an inherited predisposition, we examined 59 healthy,
first-degree relatives of patients from 21 families with CLL.
CLL-phenotype cells were detected in 8 of 59 (13.5%) relatives,
representing a highly significant increase in risk (P = .00002). CLL-phenotype cell levels were stable with
time and had the characteristics of indolent CLL. Indolent and
aggressive clinical forms were found in family members, suggesting that
initiation and proliferation involves distinct factors. The detection
of CLL-phenotype cells provides a surrogate marker of carrier status, potentially facilitating gene identification through mapping in families and direct analysis of isolated CLL-phenotype cells.
(Blood. 2002;100:2289-2290)
© 2002 by The American Society of Hematology.
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Introduction |
The etiology of chronic lymphocytic leukemia
(CLL) is largely unknown; however, several studies have reported
families with an increased risk for CLL and other non-CLL
lymphoproliferative disorders, indicative of an inherited
predisposition.1-4 The detection of a CLL-associated
marker in healthy relatives of patients with familial CLL may provide a
surrogate marker of inherited predisposition, assisting in the
identification of causative gene mutations. It is possible to identify
markers of some B-cell malignancies in healthy persons, including the
follicular lymphoma-associated t(14;18) translocation5,6
and the neoplastic plasma cells common to myeloma and monoclonal
gammopathy of undetermined significance (MGUS).7,8 We have
previously reported a flow cytometry technique for quantifying CLL
cells when they represent as few as 0.5% of B cells, using the higher
CD5 and lower CD20/CD79 expression by the neoplastic
cells.9 Application of this technique to 910 control
subjects with normal hematologic parameters and no evidence of
malignant disease detected CLL-phenotype cells in 3.5%.10 We report here the frequency of CLL-phenotype cells in 59 healthy, first-degree relatives of affected patients in 21 CLL families.
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Study design |
Twenty-one families with 2 or more members who had CLL were
ascertained through clinicians in the United Kingdom. Diagnoses of CLL
were based on standard criteria.11 Fifty-nine healthy, first-degree relatives of a family member with CLL were studied, with
repeat samples assessed in 38 of 59 relatives. Median age of relatives
was 47 years (range, 23-86 years). Samples were also examined from 23 healthy spouses whose median age was 45 years (range, 23-79 years). The
prevalence of CLL-phenotype cells in familial CLL relatives was
compared to a sample of 910 persons reported
previously.10 The age-adjusted odds ratio was derived from
logistic regression analysis, and distribution of continuous variables
was compared with Wilcoxon or Mann-Whitney U test using STATA version 6.0 (Stata, College Station, TX). Samples were
obtained with informed consent and Ethical Review Board
approval from the Royal Marsden National Health Service (NHS) Trust.
CLL-phenotype cells were enumerated as reported.10
Briefly, 106 leukocytes isolated from peripheral blood were
incubated with: (1) CD20 fluorescein isothiocyanate (FITC), CD79b
phycoerythrin (PE), CD19 Cy5/PE, and CD5
allophycocyanin (APC); (2) anti-kappa FITC, anti-lambda
PE, CD19 Cy5/PE, and CD5 APC. Total B lymphocytes were identified by
their CD19 and light-scatter characteristics. Samples containing cells
that represented more than 50 events in all 3 so-called CLL regions
were subjected to extended phenotyping. Cells were incubated with CD19
Cy5/PE, CD20 APC, CD5 PE or FITC, and either CD11a FITC, CD22 PE, CD23
PE, CD27 FITC, CD38 PE, kappa FITC, lambda PE, or FMC7 FITC. Antibodies
were either prepared in-house or were supplied by BD Biosciences
(Oxford, United Kingdom), Immunotech (Marseilles, France),
Serotec (Oxford, United Kingdom), or Chemicon (Harrow, United Kingdom).
Samples were only classified as having a population of
CLL-phenotype cells if the phenotype was consistent with clinical
CLL for the antigens: CD5 (positive), CD20 (weak or negative),
CD79b (weak or negative), CD22 (weak), FMC7 (weak).12
| |
Results and discussion |
CLL-phenotype cells were detected in 8 of the 59 relatives
from 7 families (Table 1). Absolute
numbers were, on average, 1000-fold lower than the levels required for
a clinical diagnosis of CLL (median, 5 cells/µL; range, 3-127 cells/µL) and were similar to the levels detected in the outpatient
survey (median, 13 cells/µL; range, 2-1458 cells/µL;
P = .07).10 The observation that 13.5% of
relatives harbor CLL-phenotype cells translates to a 7-fold increase in
risk for subclinical disease (odds ratio [OR], 6.6; 95% confidence
interval [CI], 2.7-16.0; P = .00002, Figure
1). CLL-phenotype cells were only
detected in 1 of 23 unrelated family members, a prevalence not
significantly different from that in the outpatient group
(P > .1). This patient came from a different family than
the affected relatives. The highly significant increase in risk for
family members indicates that CLL-phenotype cells represent a surrogate
marker of carrier status in CLL families.
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| Figure 1.
Prevalence of monoclonal CLL-phenotype cells in
relatives of familial CLL index cases compared with the general
population.
(A) Highly significant overall difference (P is
age-adjusted). (B) Increase in prevalence in the familial relatives is
seen at all age groups, but it is not significant because of the
numbers of relatives available for assessment.
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|
Repeat analysis of most of the relatives was performed in a masked
fashion to examine whether aberrant cells were persistently detectable.
Median time between sampling was 18 weeks (range, 13-25 weeks). Six of
the 8 relatives with detectable CLL-phenotype cells were reassessed,
and all were positive at second assessment. The levels of CLL-phenotype
cells were not different between the 2 time points
(P = .1), with the second assessment level representing a
median 85% of the initial level (range, 30%-112%). Long-term follow-up is required to determine whether the relationship between the
CLL-phenotype cells and clinical disease follows a pattern similar to
that seen in MGUS and myeloma.13,14 However, the fact that
the levels are stable with time suggests that the generation of
CLL-phenotype B cells is governed by genetic mechanisms different from
their proliferative potential.
CLL-phenotype cells were not detectable in 38 of the relatives assessed
at both time points. However, in 2 relatives, CLL-phenotype cells were
detectable at either first or second assessment only. Levels were low
in each (1.4/µL and 1.9/µL, respectively), below the range found in
the outpatient study. As these relatives were not categorized as having
CLL-phenotype cells, our estimate of the prevalence of this phenotype
in relatives is likely to represent an underestimate of the true risk.
Indolent forms of clinical CLL are characterized by a high degree of
immunoglobulin H (IgH) hypermutation and a low level of CD38
expression.15-18 CLL-phenotype cells present in otherwise healthy persons show these characteristics.10 CD38
expression was also undetectable on the CLL-phenotype cells from the
affected relatives. CD38+ CLL-phenotype cells are
presumably not seen in subclinical form because such clones would
expand rapidly and present as clinical disease. However, aggressive
CD38+ and indolent CD38 forms of clinical
disease were present in the familial patients with clinical disease.
This also indicates that proliferative potential is regulated
separately from the oncogenic process and may have to do with the stage
of differentiation in the B cell undergoing neoplastic transformation.
Recent data have shown that CLL cells are most similar to memory B
cells.19 As in clinical disease, the CLL-phenotype cells
in healthy persons and familial CLL relatives all express CD27, which
is normally restricted to postgerminal-center B cells.20
In addition, the level of bcl-2 expression in the CLL-phenotype cells
is equivalent to that of normal circulating memory B cells, a level
approximately twice that of naive B cells (median, 1.9-fold higher;
P = .018). This supports the hypothesis that all
CLL-phenotype cells are derived from memory B cells. Additional studies
are warranted to determine whether clinical features relate to
particular memory B-cell subsets from which the neoplastic cells are derived.
In addition to detecting a CLL-phenotype in 8 relatives, a
non-CLL-phenotype monoclonal B-cell population was detected in a
relative from another family, with an extended phenotype suggestive of
marginal zone lymphoma. Non-CLL-phenotype monoclonal B-cell expansions
were also detected in 9 of 910 outpatients. The detection of
subclinical disease in myeloma, follicular lymphoma, and now CLL, as
well as other B-cell disorders, suggests that all common chronic
lymphoproliferative disorders have a premalignant counterpart. Furthermore, the finding that all these lymphoproliferative disorders appear within certain families at the clinical and the subclinical levels raises the possibility of a common genetic predisposition to the
development of B-cell malignancies. As in MGUS and myeloma, comparison
of cells from patients with subclinical, indolent, and progressive
disease should allow identification of some of the genetic factors
responsible for oncogenesis and disease progression.21-23 Application of our observation should facilitate identification of CLL
genes through mapping in families and direct analysis of isolated
CLL-phenotype populations.
| |
Footnotes |
Submitted April 2, 2002; accepted May 2, 2002.
Prepublished online as
Blood First Edition Paper, June 14, 2002; DOI
10.1182/blood-2002-03-0892.
Supported by grants from the Leukaemia Research Fund and
Yorkshire Cancer Research.
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 S. Houlston, Section of Cancer Genetics,
Institute of Cancer Research, Surrey, United Kingdom; e-mail:
r.houlston{at}icr.ac.uk.
| |
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Abstract
Introduction