1
‘CLL-LIKE’ B-CELL PHENOTYPES
DETECTED IN SUPERFUND STUDIES:
EPIDEMIOLOGIC METHODS AND FINDINGS
SM SARASUA, RF VOGT, DC MIDDLETON, BA SLADE,
MA MCGEEHIN, JA LYBARGER
INTRODUCTION
The Agency for Toxic Substances and Disease Registry (ATSDR) conducts
health investigations in communities near hazardous waste sites to evaluate
the relationship between hazardous substances in the environment and adverse
effects on human health. Some of these are cross-sectional, symptom and
disease-prevalence studies that compare the health status of populations
living near hazardous waste sites (target area residents) to populations
with no
site-related exposure (comparison area residents). These studies utilize
standardized questionnaires and panels of biomedical tests (biomarkers)
to evaluate the function of target organ systems.
In ten cross-sectional studies conducted since 1991,
ATSDR has employed a standardized panel of immune tests to determine the
prevalence of abnormal immune parameters among study participants. The
results of these studies have been combined into a single database with
over 50,000 immune test results on about 6000 individuals. The size of
this combined dataset permits
the investigation of abnormal immune parameters with prevalences in
the 1-10% range.
As results accumulated from these studies, investigators
recognized a recurring pattern of laboratory findings which may be associated
with early B-cell chronic lymphocytic leukemia (B-CLL). Over the course
of the ten studies, eleven participants were identified as having such
“B-CLL-like” phenotypes. This paper will describe the epidemiologic
methods used in these
studies, provide a brief overview of the results, and discuss the implications
of these findings.
Methods
Design and Test Selection
These cross-sectional studies used a stratified
random sample from target and comparison areas. Each study included a questionnaire
interview and collection of a blood sample for biomedical testing. A standardized
questionnaire was used to evaluate the frequency of self-reported symptoms
and illnesses in each community.
The biomedical tests included a panel of immune tests
established by ATSDR. The tests selected were based on recommendations
from a workshop on immune biomarkers in environmental health studies (1).
This panel included the following tests: total white blood cell count,
total lymphocyte count, lymphocyte phenotyping, and serum immunoglobulin
levels. A
detailed discussion of the immune tests used in these studies is presented
in Chapter 2.
Samples from three of the participants identified
with B-CLL-like phenotypes were further analyzed for B-cell monoclonality
using kappa/lambda light chain phenotyping (Chapter 3).
Study Populations
The target population for each study consisted of
residents living in a well-defined area located close to a hazardous waste
site. The selection of each target area was based on environmental sampling
data that identified contaminated soil, groundwater, surface water, or
sediment. Since individual exposure data were not typically available,
residence in a target area does not imply that every study participant
was exposed to hazardous waste; but this was the only surrogate of exposure
available.
The comparison areas were located more than 5 miles
from the site of interest and were not near any other hazardous waste sites.
The comparison area for each study was matched to the target area with
regard to demographic and socioeconomic factors. Some of the socioeconomic
factors considered were: style and age of housing; household income; and
location in or near an
urban population center. Selections were typically made after evaluation
of U.S. census data, discussions with local officials, and site visits
by the study teams.
Participant Selection
A door-to-door census was conducted in the target
and comparison areas. Information was collected on the number of
people in each household, the age and sex of each resident, and the length
of time they had lived at the address. The census served as the sampling
frame for selecting study participants. Typically, participants were required
to have lived in the area for at least one full year to be eligible for
the study. A stratified, random sampling scheme based on census data was
used to select participants from age and sex categories. This also
ensured that the age- and sex-distributions for target and comparison area
study participants were similar.
Data Collection and Analysis
Selected study participants were initially contacted
by telephone and requested to come to a local facility for an in-person
interview and specimen collection.
The ATSDR core symptom and illness prevalence questionnaire
was administered to study participants during face-to-face interviews.
This questionnaire collects information on the participant’s health and
the household environment.
ATSDR and the Environmental Health Laboratory Sciences
(EHLS) division at the Centers for Disease Control and Prevention (CDC)
established standardized protocols for specimen collection, handling and
transportation, and for laboratory quality control procedures, which were
used throughout the four-year period. Specimens for laboratory analysis,
including the Basic
Immune Test Battery (BITB), were collected at the local facility. Whole
blood samples for lymphocyte phenotyping were shipped overnight to EHLS/CDC.
Serum samples were separated, frozen, and stored at the local facility,
then shipped later to EHLS/CDC. Complete blood counts were performed on
fresh blood samples by local hospital laboratories.
RESULTS
Study Sites and Participants
At the time of this workshop, immune tests had been
conducted in 10 health studies of populations residing near hazardous waste
sites in the US. Most of these sites had multiple contaminants; Table
1 lists the major contaminants identified at each site. Individual exposure
information was typically not available, and it is likely that some persons
in the target areas were not exposed to contaminants from the nearby hazardous
waste site.
Lymphocyte phenotypes were determined on a total
of 5868 participants in these 10 studies, 3812 from target areas and 2056
from comparison areas. Of these, 1499 participants were age 45 and
older: 891 from target areas and 608 from comparison areas. The demographics
of the target and comparison groups in this older cohort were very similar.
Most of these participants were white (88%); the remainder of the cohort
included Asians (7%), Blacks (3%), and other races (2%). The mean age of
the target group was 58.4 years (s.d.=8.9); the mean age of the comparison
group was 58.9 years (s.d.=8.7). Females comprised 51% of the target
group and 52% of the comparison group.
Laboratory Findings of “B-CLL-like” Lymphocyte Phenotypes
Analysts at CDC identified samples from 11 participants
with phenotypic characteristics similar to those seen in early B-cell chronic
lymphocytic leukemia (B-CLL) (Tables 2 and 3). At the time of identification,
the analysts were unaware of the total cell counts, demographic information
(except for age), and exposure category (target or comparison).
These phenotypic characteristics (termed “B-CLL-like”)
included some or all of the following features:
(1) an unusually high proportion of B-lymphocytes
among all lymphocytes (typically 50%
or more);
(2) reduced (dim) staining for the white blood cell
surface markers CD20 (normally present on
all B-cells) and/or
CD45 (normally present on all mature white blood cells); and/or,
(3) the abnormal presence of CD5 (a T-cell surface
marker) on the surface of mature
B-lymphocytes (identified
by the CD19 cell surface marker).
Participants with the B-CLL-like phenotypes were
contacted by ATSDR physicians; additional samples were obtained in 8 of
the 11 cases and the B-CLL-like findings were confirmed in each. Peripheral
blood samples from the last three participants with B-CLL-like phenotypes
were evaluated for monoclonality by analyzing cell surface kappa and lambda
immunoglobulin
light chains (Chapter 3). All three were found to have an excess proportion
of B-cells expressing kappa light chains, confirming the presence of a
monoclonal B-cell population.
Further details regarding the laboratory findings
may be found in Chapters 2 and 3.
Medical History and Demographic Characteristics in
Participants with B-CLL-like Phenotypes
During discussions with the 11 participants with B-CLL-like
phenotypes, one participant was found to already have a diagnosis of CLL.
Another participant was unwilling to provide additional medical information.
The remaining 9 said they were unaware of any medical condition related
to CLL or any other blood disorder.
The average age of the 11 persons with B-CLL-like
phenotypes was 63; the youngest was 47 and the two oldest were 72. Six
(54%) were males and all were white (Table 3).
B-CLL-like Phenotypes and the Population Distributions of
Leukocyte, Lymphocyte and B-cell Counts
The quantitative blood cell counts for each participant
with a B-CLL-like phenotype were compared with the distributions for all
participants age 45 or older (Table 4; Figures 1-5). The leukocyte count
(white blood cell count, or WBC) includes all peripheral blood leukocytes,
granulocytes, monocytes, and lymphocytes. The average percentile of leukocyte
counts for all B-CLL-like results was 81. In clinical terms, the leukocyte
count was moderately elevated
in the one participant with a diagnosis of CLL and modestly elevated
in the participant who was unwilling to provide additional information
to ATSDR. Two of the remaining 9 had leukocyte counts near the upper
limit of the reference range; 7 were well within the reference range. However,
one was below the 50th percentile.
The lymphocyte counts of the 11 persons with B-CLL-like
phenotypes were more consistently elevated. Four results were above the
99th percentile, and none was below the 80th. B-cell counts were also elevated:
ten of eleven were above the 98th percentile, and seven were above the
99th percentile.
Distribution of B-CLL-like Phenotypes and B-cell Counts
among Target and Comparison Populations
The overall prevalence of B-CLL-like phenotypes among
all participants age 45 or older was 11/1499 (0.7%). The prevalence of
B-CLL-like phenotypes among target area participants was 8/891 (0.9%);
the prevalence among comparison area participants was 3/608 (0.5%). The
prevalence ratio (PR) associating the B-CLL-like phenotype with residence
in a target area was: PR = 8/891 ¸ 3/608 = 1.8 (95% CI = 0.5-6.9).
The health studies at Sites 1-4 were based on contamination
by lead and other heavy metals and most study participants were children.
If Sites 1-4 are excluded, analysis of results from Sites 5-10 (which contained
predominantly VOCs and included more adults) reveal that among adults 45
and older:
(1) the prevalence of B-CLL-like phenotypes among
the target population was 8/827 (1.0%);
(2) the prevalence of B-CLL-like phenotypes among
the comparison population was 2/567
(0.4%); and,
(3) the prevalence ratio (target vs. comparison)
was:
PR = (8/827) / (2/567)
= 2.7 (95% CI = 0.6 – 12.9).
The overall mean values of laboratory parameters
did not differ significantly between target and comparison populations
(among all participants age 45 or older) with respect to total leukocyte
count, total lymphocyte count, B-cell count, or percentage of B-cells among
all lymphocytes.
DISCUSSION
CLL almost always arises from a monoclonal proliferation
of B-lymphocytes (B-CLL) (Chapter 6). CLL is often diagnosed by chance
when peripheral blood lymphocytosis is noted as an incidental finding during
routine laboratory testing. B-CLL is more fully characterized by lymphocyte
phenotyping, which reveals not only an increase in B-cells but also characteristic
patterns of B-cell surface markers (see Chapters 2 and 3). Lymphocyte phenotyping
from 11 study participants showed an increase in the relative proportion
of B-cells as well as cell surface marker changes seen in B-CLL (B-CLL-like
phenotypes) (detailed in Chapter 2). Three samples were tested for evidence
of monoclonality; all three had monoclonal populations of B-lymphocytes.
Also, one of the CLL-like cases had been previously diagnosed with CLL.
These observations strengthen the possibility of a relationship between
the B-CLL-like phenotype and disease endpoints (eg, B-CLL or other related
B-cell lymphoproliferative disorders).
B-CLL is the most common adult leukemia in the United
States today. The incidence of CLL rises sharply with age (Chapter
18). With an incidence of approximately 5/100,000 cases in persons over
the age of 50 and a mean survival of less than 10 years, the expected number
of CLL cases in the at-risk study population would be no more than one.
While the natural history of CLL is not well understood, investigators
were surprised to find 11 B-CLL-like pre-clinical cases (including one
case of diagnosed CLL, as was expected). This finding could indicate
an emerging epidemic of B-CLL or simply a slow and uncertain progression
from laboratory abnormalities to clinical disease.
While target area participants were more likely
(though not statistically significantly) to have the B-CLL-like findings
than were comparison area participants, it is not clear that environmental
exposures were the cause. Individual exposure data are not available and
some target area participants appear unlikely to have been exposed to hazardous
substances from the nearby site.
Nevertheless, this apparent increase in the prevalence of B-CLL-like
findings among target area participants cannot be ignored and warrants
further study.
REFERENCES
1. Straight JM, Kipen HM, Vogt RF, Amler RW. Immune Function Test Batteries
For Use In
Environmental Health Studies. US Department of Health
and Human Services, Public Health
Service; 1994. Publication Number PB94-204328.
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