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CHARACTERIZING B-CELL LYMPHOCYTOSIS
IDENTIFIED IN ATSDR STUDIES:
SYNOPSIS OF A PROTOCOL
BA SLADE, DC MIDDLETON, MS MILLER AND RF VOGT
This synopsis has been extracted from a protocol
developed by agencies of the United States Public Health Service (1) in
response to a recommendation from the workshop.
BACKGROUND
Over the last 5 years, the Agency for Toxic Substances
and Disease Registry (ATSDR) and the Centers for Disease Control and Prevention
(CDC) has used a standardized panel of immune biomarkers in 10 epidemiologic
studies (see Attachment A). One of the goals in performing these tests
was to compare the prevalence of abnormal immune parameters in people living
near hazardous waste sites (target populations) to the prevalence in people
from demo-graphically-matched comparison populations.
Abnormal Immunophenotypes
Laboratory immune testing included immunophenotyping
(IPT) by flow cytometric analysis to identify the major types of lymphocytes
(T-cells, B-cells, NK-cells) and certain sub-populations of these lymphocyte
types. Over time, the laboratory investigators began to repeatedly note
IPT findings classically associated with early B-cell chronic lymphocytic
leukemia. These identifications were made subjectively, based upon the
analysts' knowledge and experience.
These characteristics included:
(1) an unusually high percentage of B-lymphocytes among all lymphocytes;
(2) reduced (dim) staining for certain B-lymphocyte cell surface markers
(eg, CD20,
normally present on all B-cells; and, CD45, normally present on all mature
white
blood cells); and,
(3) the abnormal presence of CD5 (a T-cell marker) on the surface of the
B-lymphocytes.
Some or all of these features were noted during
IPT analysis in 11 of 2006 study participants age 40 and older, including
8 of 1176 from target areas and 3 of 830 from comparison areas. These 11
participants with abnormal immunophenotypes will be referred to as the
“original 11 cases” throughout this protocol. The prevalence ratio of these
cases (target vs. comparison areas) was 1.9.
Monoclonality
B-CLL results from the unchecked proliferation of
a monoclonal cell line arising from a common mutated progenitor cell. Specialized
laboratory testing for evidence of monoclonality was positive in 3 of 3
participants tested. Telephone contact with the remaining 8 cases revealed
that a fourth individual had already been diagnosed with B-CLL. The possibility
that these 11 cases (and possibly other participants not yet identified)
might be at increased risk for B-CLL or other B-cell lymphoproliferative
disorders could not be ignored.
Workshop on B-cell Lymphoproliferative Disorders
In June 1995, ATSDR sponsored a workshop on B-cell
lymphoproliferative disorders and invited experts in epidemiology, hematopathology,
and immunology to review and assess these findings. These experts found
the 11 cases described above to be of sufficient interest to warrant further
study, but noted not all markers were assessed in all participants (eg,
CD5) and that a rigorous case-definition was lacking. This protocol was
developed in response to workshop recommendations to re-test participants
as necessary using a rigorous case definition for B-cell monoclonal lymphocytosis
(BCML).
Case Definition
The case definition of BCML will be based on standard
laboratory criteria, including kappa/lambda surface immunoglobulin (sIg)
analysis and/or immunoglobulin gene rearrangement. Additional blood samples
will be requested from the original 11 cases and from other selected study
participants. Additional participants will be selected by age (40 or greater)
and by applying specific screening criteria to the previous laboratory
results.
OBJECTIVES
The objectives of this study are as follows:
(1) determine if previously detected B-cell abnormalities have persisted
in individual
participants;
(2) determine if these B-cell abnormalities are useful in screening for
B-cell
monoclonality (positive predictive value) (important for future studies);
(3) compare the prevalence of B-cell monoclonality among target populations
to the
prevalence among comparison populations; and,
(4) advise individual participants with persistent BCML (considered at
increased risk for
B-cell lymphoproliferative disorders) to seek appropriate medical evaluation
and
care.
METHODS
ATSDR Multisite Data
While lymphocyte immunophenotyping by flow cytometry
was performed as part of the ATSDR immune panel at the ten study sites,
the interpretation of flow cytometry results was based somewhat on the
judgment and experience of the laboratory personnel. As the EHLS/CDC became
more experienced over time, there may have been some variability in interpretation.
In addition, CD19/CD5 staining was added to the original test panel late
in the study period; only 39% of the participants (over the age of 40)
were tested for this marker. 7/11 of the “original 11” cases were identified
after implementation of CD5/CD19 testing. Thus, it seemed unlikely that
all participants at risk for BCML were identified in the “original 11”
cases.
It was the recommendation of the Workshop that objective
laboratory criteria be established that could be applied across the entire
study population to identify additional candidates for the follow-up study.
The laboratory findings common to the “original 11” cases were reviewed
to establish sensitive screening criteria. The following unusual features
were noted in the flow cytometry results:
Unusual feature #1: Increased portion (%) of mature B-lymphocytes
(CD19+) expressing the CD5 surface marker
The presence of this marker is considered a hallmark
characteristic of B-CLL. This marker identified 7 of the “original
11” cases; the remaining 4 cases were not tested for CD19/CD5. This identification
was made subjectively at the time of data acquisition. In order to develop
objective criteria, the distribution of CD5+ B-lymphocytes for the study
population was examined. Consistent with accepted laboratory practice,
the upper 2.5% of this distribution (>/= 60%) will be considered elevated
(above the reference range) and will be used to select follow-up study
participants. Since this marker was not tested across the entire study
population, this marker could not be used as the sole screening criterion.
Unusual feature #2: High percentage of B-lymphocytes among all lymphocytes
An increased number (relative or absolute) of B-lymphocytes
is an essential characteristic of B-CLL and/or BCML. This characteristic
was used to identify 10 of the “original 11” cases. When these cases were
identified, CBC results were not available to the EHLS/CDC laboratory (needed
for calculation of the absolute count) and selection was based on an increased
relative portion of B-cells. The absolute count (see previous discussion)
is the more appropriate selection criteria for B-cell lymphocytosis. This
data is now available and the absolute B-lymphocyte count is now known
for the study population. In order to develop objective criteria, the distribution
of absolute B-lymphocyte counts for the study population was examined.
Consistent with accepted laboratory practice, the upper 2.5% of this distribution
(>/=830 cells/mm3) will be considered elevated (above the reference range)
and will be used to select follow-up study participants.
Unusual feature #3: decreased (“dim”) intensity of CD20 cell surface
reactivity
This laboratory finding was detected in 8 of the
“original 11” cases. Detection is based upon subjective analysis
made at the time of data acquisition. No objective criteria can be
established from the available data. All participants identified with “dim”
CD20 staining also show either feature #1, feature #2, or both.
Recruitment of Study Participants
Individuals (age 40 or above) will be selected for
the follow-up study based on results of the immune tests from previous
ATSDR health studies. Based on recommendations of the workshop and
analysis of the ATSDR Multisite Database, the selection criteria will include:
(1) 60%
or more of B-lymphocytes (CD19+) are also CD5+; or,
(2) an
absolute B-cell lymphocytosis (>/= 830 cells/mm3).
Using these criteria, there are 80 participants
(including the “original 11” cases) from previous ATSDR health studies
eligible for this follow-up study. Assuming a 75% participation rate,
specimens will be collected from approximately 60 individuals.
Specimen Collection and Shipping
For those willing to participate in the follow-up
study, arrangements will be made for the individual to read and sign an
informed consent form. Blood samples will be collected under conditions
established for field specimen collection in ATSDR and CDC studies.
A Complete Blood Count (CBC) with differential will
be performed at a local contractor hospital within 8 hours of collection.
Immunophenotyping will be performed at the EHLS/CDC laboratory. Specialized
tests to identify the presence of a B-cell monoclonal population will be
performed at the FDA/NCI laboratory. To insure the integrity and biosafety
of each specimen, collection tubes will be sent directly to each laboratory.
To complete all of the tests as prescribed, approximately 60 mls of blood
drawn in 8 different collection tubes will be required.
Laboratory Test Panel
The laboratory tests that will be used to evaluate
the immunologic/hematologic status of participants is described below.
ATSDR Basic Immune Test Battery (BITB) (2)
1. ATSDR BITB includes a complete blood count,
extended lymphocyte phenotypes and quantitative serum immunoglobulins performed
at CDC or contractor laboratories with quality control from CDC.
2. Serum protein electrophoresis with immunofixation
will be included in the BITB as a focused/reflex test.
Tests for Monoclonality
1. Co-expression of CD19 and CD5, performed
at EHLS/CDC.
2. Kappa-lambda analysis for monoclonality,
performed at FDA/NCI.
3. Polymerase chain reaction (“PCR”) analysis
of B-cell DNA using primers specific for the
variable, J H and
Cm regions, performed at FDA.
4. Peripheral blood smear for assessment of
cell morphology performed at FDA/NCI and/or
ATSDR.
5. DNA ploidy and cell cycle analysis to assess
proliferative activity of monoclonal cell line
performed at FDA.
Interpretation and Reporting of Test Results
Participants will be notified of their test results
by letter. Results of the CBC (with Differential White Blood Cell Count)
will be sent as soon as available from the contractor laboratory. If any
laboratory abnormality is identified that requires prompt medical attention,
the participant will be contacted immediately. The complete package of
immune test results with clinicopathologic
interpretation will be sent when all tests are completed. Since some
of these tests (eg, polymerase chain reaction) may require a significant
amount of time, notification may take up to 12 weeks after collection.
Participants will have the option of having the test results also sent
directly to their physicians.
FUTURE STUDIES
It is anticipated that once the cohort of study
participants with persistent monoclonal B-cell abnormalities has been identified,
these will be further characterized by various specialized laboratory tests
as discussed at the workshop. The following list provides some background
on tests under consideration for inclusion in these future studies.
Characterization of Monoclonal Populations
1. Loss of heterozygosity (LOH) in B-cell DNA
This assay detects changes
in DNA using 80 to 100 simple sequence length polymorphisms
(SSLP) that have been defined by the human genome
project. In many malignant conditions, a
mutation in one allele is associated with a deletion
of the other allele. This is termed “loss of
heterozygosity” (LOH).
2. V H -gene family characterization by anchored PCR (See Rassenti
and Kipps,
Chapter 9).
B-lymphocytes rearrange
gene segments to produce the light and heavy chain peptides that
make up the immunoglobulin (Ig) molecule. The heavy
chain variable region genes (“V H
-genes”) can be grouped into seven sub-families
based on similarities in DNA sequences. This
assay determines the relative contribution of different
Ig V H gene families to the B-cell
population. The V H gene sub-families can be detected
by the “anchored polymerase chain
reaction” technique. If a monoclonal population
is present, the distribution of V H families is
altered. Cells from certain V H families may be
more likely to transform into malignant clones.
3. Fluorescence in situ hybridization (FISH) for chromosomal abnormalities.
FISH utilizes chromosome-specific
probes labeled with fluorescent tags to detect
chromosomal abnormalities. The most common chromosomal
abnormality in B-CLL is trisomy
12; deletions in the short-arm of chromosome 13
are also seen in B-CLL. FISH is felt to be
more sensitive than conventional karyotyping and
thus may be of particular use in B-CLL
where the chromosomal abnormality is not necessarily
present in every cell.
Characterization of Ancillary Immune Processes
Related to Monoclonality
4. Common CLL Antigen (cCLLa) Surface Marker (See Faguet, Chapter
6).
Most cell surface markers
detected on B-CLL cells are differentiation antigens that may also be seen
on normal B-lymphocytes. The cCLLa marker is not a differentiation antigen
and thus is not expressed by normal lymphocytes. Furthermore, the cCLLa
antigen has been detected only in B-CLL and related leukemias (prolymphocytic
leukemia and hairy cell leukemia). While this cCLLa marker has been reported
to be specific for B-CLL, this investigative test not been applied in large
population studies.
5. Cytokine Profiles (See Fernandes et al., Chapter 15).
The secretion of and response
to certain cellular messengers (cytokines) have been used to characterize
abnormalities in B-CLL and may help to predict disease progression from
B-cell lymphocytosis.
6. Cytokine and Growth Factor Receptors (See Collins, Chapter 13).
The presence (or enhanced
expression) of certain cytokine receptors (eg, SCF, G-CSF, GM-CSF, IL-2,
IL-4, IL-6, THF-alpha) can be used to distinguish B-CLL cells from normal
resting or activated B-cells.
7. Characterization of T-cell Activation (See Donnenberg and Donnenberg,
Chapter 14).
This investigative test
has shown T-cell activation changes in patients with CLL. It is hypothesized
that T-cell/B-CLL interactions play a key role in the evolution of B-CLL.
REFERENCES
1. ATSDR. Protocol for Characterizing B-Cell Lymphoproliferative
Abnormalities Identified in
ATSDR Studies; July 1996.
2. Straight JM, Kipen HM, Vogt RF, Amler RW. Immune Function
Test Batteries for use in
Environmental Health Studies. U.S. Dept.
Health Human Services, Public Health Service,
Pub. No. PB94-204328.
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