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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 398-404
PLENARY PAPER
Detection of differentially expressed genes in lymphomas using
cDNA arrays: identification of clusterin as a new diagnostic marker for
anaplastic large-cell lymphomas
Axel Wellmann,
Catherine Thieblemont,
Stefania Pittaluga,
Akira Sakai,
Elaine S. Jaffe,
Paul Siebert, and
Mark Raffeld
From the Hematopathology Section, Laboratory of Pathology, National
Cancer Institute, National Institutes of Health, Bethesda, MD; and Gene
Cloning & Analysis, Clontech Laboratories, Palo Alto, CA.
 |
Abstract |
This study reports the first use of gene array technology for the
identification of a tumor-specific marker in lymphoid neoplasms. The
differential gene expression of 31 hematopoietic cell lines, representing most major lymphoma subgroups of B- and T-cell origin, was
assessed by hybridizing labeled complementary DNA to Atlas human
expression arrays containing 588 genes. Genes known to be specific for
B, T, or myelomonocytic lineages were appropriately identified in the
arrays, validating the general utility of this approach. One gene,
clusterin, not previously known to be expressed in lymphoid
neoplasms, was specifically found in all 4 anaplastic large-cell
lymphoma (ALCL) cell lines, but not in any of the 27 remaining tumor
lines. Using a monoclonal antibody against clusterin, its differential
expression was confirmed by Western blotting and immunohistochemistry.
A total of 198 primary lymphomas (representing most major lymphoma
subtypes), including 36 cases of systemic ALCL, were surveyed for
clusterin expression by immunohistochemistry and Western blotting. All
of the 36 ALCL cases marked for clusterin, with most cases showing
moderate to strong staining in the majority of neoplastic cells.
Clusterin expression was not related to expression of anaplastic
lymphoma kinase-1. With 2 exceptions, none of the remaining 162 non-ALCL cases marked with the clusterin antibody, including Hodgkin
disease and primary cutaneous ALCL. In reactive lymphoid tissues, only
follicular dendritic cells and fibroblastic reticular cells exhibited
staining. Clusterin is a highly conserved glycoprotein implicated in
intercellular and cell matrix interactions, regulation of the
complement system, lipid transport, stress responses, and apoptosis.
Although its function in ALCL is unknown, the unique expression of
clusterin within this category of lymphoma provides an additional
marker for the diagnosis of ALCL. This study illustrates the enormous
potential of gene array technologies for diagnostic marker discovery.
(Blood. 2000;96:398-404)
© 2000 by The American Society of Hematology.
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Introduction |
The identification of tumor-specific targets for
diagnostic or therapeutic use has been a principal goal for both
clinicians and biomedical researchers. Traditionally, investigators
have accumulated this information through comparative expression
studies of individual gene products that have been identified during
the course of biochemical, physiologic, or molecular biologic studies of various neoplasms. In this way, a series of diagnostic and prognostic markers have been developed that can distinguish among many
types of cancers and provide useful clinical information.
Recent advances in DNA sequencing technology and the development of
gene expression arrays have provided investigators with a powerful tool
to study the expression of thousands of genes in
parallel.1-3 This methodology promises to revolutionize the search for tumor-specific markers. Gene expression arrays are created
by depositing unique complementary DNA (cDNA) fragments on a solid
matrix that can be either a glass slide or a nylon filter. The slide or
filter is then hybridized with labeled cDNA from a tissue of interest.
The readout is performed by high throughput fluorescence scanners for
fluorescent probe hybridizations or by traditional phosphoimagers when
radioactively labeled probes are used. Large format macroarrays
containing hundreds of genes as well as high-density microarrays with
more than 20 000 different known genes and expressed sequence tags
(ESTs) can be generated using high-speed robotic
printers.4-6 For the analysis of the hybridization signals
on high-density microarrays, it is absolutely essential to have
computer assistance, and sophisticated algorithms for this purpose have
been developed.7-9
Array technology has broad applications, and its power is being
directed toward the study of global gene expression changes in both
physiologic and pathologic states. Such diverse physiologic conditions
as aging in mice,10 gene expression during sleep cycles,11 serum stimulation of fibroblasts,12
irradiation-induced stress,13 yeast
sporulation,14 and the diauxic shift from aerobic to
anaerobic metabolism in yeast3 have already been put under
the microscope of this technology. Various pathologic conditions are
also being scrutinized, from inflammatory diseases15 to
neoplasia.8,16-21 Array technology is particularly well
suited for studying gene expression changes between normal tissues and their corresponding cancers, and there have already been several studies of gene expression in colon cancer,8,16 renal cell carcinoma,17 rhabdomyosarcoma,18
leukemias,19 cervical cancer,20 and ovarian
carcinoma.21 Most of these latter studies have focused on
global differences between normal and cancer in an attempt to better
understand the pathogenesis of the particular neoplasm. Validation of
array results is generally performed by assessing RNA expression of the
sample by traditional Northern blot or reverse transcriptase-polymerase
chain reaction because the identification of tumor-specific markers has
not been a primary goal of most of these studies.
We have begun to apply this technology for identification of
tumor-specific markers in lymphomas. As a first approach we have compared the gene expression profiles of a series of lymphoma cell
lines derived from most of the common lymphoma subtypes, using
commercial nylon filter macroarrays from Clontech Laboratories. These
arrays are composed of 588 known genes grouped into several functional
categories. Our initial goal was to validate the use of these arrays by
an analysis of genes contained on the filters already known to be
differentially expressed among the selected hematopoietic cell
lines. After successful validation, we then searched for the presence
of previously unknown differentially expressed genes that could
potentially represent new diagnostic markers. Differential gene
expression was then validated at the protein level on selected cell
lines, and finally on a series of primary lymphoma tissues, using a
combination of immunoblotting and immunohistochemistry.
We now report a general approach for the identification of
tumor-specific markers using gene macroarray technology and describe a
new marker, clusterin (Apo J), that is specifically expressed in a
subtype of large-cell lymphoma, anaplastic large-cell lymphoma (ALCL).
| |
Materials and methods |
Cell lines and growth conditions
Thirty-one hematopoietic cell lines, comprised of 19 B-cell, 10 T-cell, and 2 myeloid cell lines, were selected for comparative gene
expression studies. The B- and T-cell lines were chosen to represent a
broad range of primary lymphoid neoplasms, and within each neoplastic
subtype we attempted to obtain a minimum of 2 examples (Table
1).
All cell lines were cultured in RPMI 1640, supplemented with 10%
heat-inactivated fetal calf serum, L-glutamine, and
antibiotics. To limit the effect of cell culture conditions on gene
expression as much as possible, all cell lines were harvested during
their exponential growth phase (0.3-0.7 × 106/mL).
Centrifugations were carried out at 4°C and total RNA was extracted
immediately thereafter.
cDNA arrays
Atlas cDNA expression arrays from Clontech Laboratories (Palo Alto,
CA) were chosen for these studies. These arrays consist of 588 human
cDNA fragments, organized into broad functional groups. A complete list
of the genes included on the membranes is available on the Clontech Web
site (http://www.clontech.com). All cDNAs used for printing on the
array have been sequence verified by the company.
RNA extraction, labeling, and hybridization of Atlas human cDNA
expression arrays
Total RNA was extracted from the cell lines using a guanidium
isothiocyanate-based method (Ultraspec II RNA isolation system, Biotex
Lab, Houston, TX) according to the manufacturer's instructions. Subsequently, polyA + messenger RNA (mRNA) was isolated from 500 µg
of total RNA using 2 rounds of Oligotex beads purification (Qiagen,
Valencia, CA), according to the manufacturer's instructions. The polyA + mRNA was then subjected to DNase I treatment to minimize genomic DNA
contamination. The quality of the mRNA was assessed by gel
electrophoresis, as well as by OD 260/280 ratios.
cDNA labeling, hybridization, and washing of the cDNA Atlas array
membranes were carried out according to the instructions accompanying
the macroarrays. Briefly, 1 to 2 µg polyA+ RNA was used as template
for cDNA synthesis, which was done in the presence of  -P-32-labeled
deoxyadenosine triphosphate (dATP) (Amersham Pharmacia Biotech,
Piscataway, NJ). The labeled probes were purified by spin column
centrifugation (Chroma Spin-200, Clontech Laboratories), and
hybridization was carried out at 68°C in a rotation hybridization oven (Robbins Scientific, Sunnyvale, CA) using
1 × 106 cpm/mL (Cherenkov) of radioactive probe.
The membranes were then washed at 68°C (4 times with 2 × standard sodium citrate [SSC], 1% sodium dodecyl sulfate [SDS],
followed by 2 times 1 × SSC, 0.1% SDS) and exposed for 1 to 3 days and analyzed by a phosphoimaging system (Molecular Dynamics,
Sunnyvale, CA). Stripping of the membranes was carried out
according to the manufacturer's instructions and they were re-used up
to 3 times.
Array exposures on the phosphoimager were normalized by equalizing the
intensity of the signals from a set of housekeeping genes provided on
the arrays. Following this normalization step, the array images were
printed and the hybridization signals of all 588 genes were scored
visually on a 0 to 3 scale, with 0 being no signal, and 1 to 3 representing increasing signal intensities. However, to simplify
interpretation in our initial analysis reported here, all positive
signal intensities (1-3) were grouped together. Therefore,
each gene on the array was scored as either positive or negative.
Preferential lineage expression was arbitrarily defined as any gene
overrepresented in the cell lines of 1 lineage (B, T, or myeloid) by a
ratio of 3:1 or greater, at any signal intensity.
Lymphoma subtype-restricted expression was defined as any gene
expressed at any signal intensity in all or all but 1 member of a
specific subtype of lymphoma, but not in more than 1 example of the
other lymphoma cell line subtypes.
Primary lymphomas
A group of 198 well-characterized lymphomas including 90 T-cell
lymphomas, 78 B-cell lymphomas, and 30 cases of Hodgkin lymphoma (Table
2) were retrieved from the files of the
Hematopathology Section, Laboratory of Pathology, National Cancer
Institute, Bethesda, MD. These lymphomas were chosen to represent all
major lymphoma subtypes and included 36 cases of primary ALCL and 9 cases of cutaneous ALCL. Among the 78 B-cell non-Hodgkin lymphomas were 12 cases that showed weak or focal CD30 expression. In addition, 30 representative examples of nonneoplastic lymphoid tissues including tonsil, lymph node, and spleen were studied to determine the occurrence and distribution of clusterin in normal lymphoid tissues. All diagnoses
were confirmed by immunohistochemistry performed on paraffin-embedded
tissue sections, using selected members of a panel of monoclonal
antibodies comprised of CD20, CD3, CD4, CD8, CD5, CD10, CD15, CD30,
CD43, CD23, ALK-1, TIA-1, perforin, CD56, cyclin D1, and Bcl-2,
performed during the diagnostic workup. Anaplastic lymphoma kinase-1
(ALK-1) (Dako Corp, Carpinteria, CA) immunohistochemistry was performed
on all cases of ALCL.
Analysis of clusterin expression
To verify clusterin expression, all cases and cell lines were
evaluated using a monoclonal anticlusterin antibody (Upstate Biotechnologies, Lake Placid, NY). This antibody specifically recognizes the human subunit of the clusterin heterodimer.
Immunohistochemistry with anticlusterin was performed as follows:
5-µm paraffin sections of primary cases and paraffin-embedded cell
line clots were mounted on Fisherbrand/plus Superfrost Precleaned
slides (Fisher Scientific, Pittsburgh, PA) and dried overnight at
58°C. After deparaffinization, the slides were placed in 10 mmol/L
citrate buffer pH 6.0 containing 0.1% Tween 20 and antigen retrieval
was performed in a microwavable pressure cooker for 8 minutes of
boiling time. The sections were rinsed in 0.05 mol/L Tris-HCl, pH 7.6 with 3% goat serum and incubated with the primary anticlusterin
antibody (50 ng/mL) overnight at room temperature. The remainder of the
procedure (secondary antibody application and avidin-biotin detection)
was carried out with an automated immunostainer (Ventana Medical
System, Tucson, AZ).
Western blot analysis
Total cell lysates were prepared by lysing cell line pellets
or 6-µm thick frozen sections in Laemmli sample buffer.22
The protein concentrations were determined using a BCA-200 protein assay kit (Pierce Chemical, Rockford, IL). Thirty micrograms of each
extract was separated on 12% SDS-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. After blocking with a
10-mm TRIS-saline solution (pH 7.4) containing 3% bovine serum
albumin, the membranes were incubated overnight with the anticlusterin
antibody at 1µg/mL. The blots were developed using an
131I-labeled goat antimouse immunoglobulin secondary
antibody, followed by overnight exposure on Kodak film or a
phosphoimager screen.
| |
Results |
Differential gene expression in hematopoietic cell lines
Macroarray hybridization data from all 31 hematopoietic cell lines
(Table 1) were collected and scored as described in "Materials and
methods." Of the 588 genes represented on the Atlas arrays, 380 (65%) genes showed detectable levels of expression, whereas 208 genes
were not expressed (35%). A total of 267 genes were expressed across
B-, T-, and myeloid cell lineages, whereas 113 genes were
preferentially expressed within a single lineage. Not unexpectedly, a
high percentage of genes expressed across all cell lineages were genes
involved in basic biologic processes such as cell proliferation and
signal transduction, stress response, and DNA repair.
Of the 113 genes that showed preferential expression within a single
restricted lineage, 74 were preferentially expressed in T-cell lines,
36 preferentially expressed in B-cell lines, and 3 in myeloid cell
lines. Several of these included genes known to be lineage restricted,
and these showed the expected restriction pattern. For example, CD19,
CD40, and CD27 were expressed exclusively in B-cell lines; CD4 and
CD100 (semaphorin) were confined to T-cell lines; and CD33 and
granulocyte colony-stimulating factor (G-CSF) receptor expression were
limited to the 2 myeloid leukemia cell lines (Table
3).
A relatively small number of genes were specifically or preferentially
expressed in all of the representatives of a particular lymphoma subset
(Table 3). For example, cyclin D1 was expressed only in the 2 mantle
cell lymphoma lines and in 1 t(11;14)-bearing myeloma cell line, and
CD30 was expressed only in the 4 ALCL cell lines. N-Cadherin
was expressed in all 4 primary effusion lymphoma (PEL) cell lines and
in 3 other cell lines, each derived from a different lymphoma subtype.
Another example was clusterin, a gene included on the array because of
its putative role in apoptosis. Clusterin was specifically expressed in
all 4 ALCL cell lines and not in any of the other cell lines.
Representative portions of the Atlas arrays containing the clusterin
hybridization signal are shown in Figure 1.
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| Fig 1.
Representative Clontech human Atlas macroarray analyses.
The cDNA hybridization patterns from 2 ALCL cell lines (SR786 and
Karpas 299) and 2 non-ALCL cell lines (HL-60 and HSB) from 1 field of
the Atlas array are shown. The arrows identify the position of the
clusterin gene cDNA and show strong hybridization signals in
the 2 ALCL cell lines and no signals in the non-ALCL cell lines.
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Confirmation of clusterin as a differentially expressed gene in ALCL
cell lines
To confirm the differential expression of clusterin at the protein
level, we obtained a commercially available monoclonal antibody to
human clusterin and studied a subgroup of cell lines by Western blot
analysis. Clusterin is a heavily glycosylated protein that is
synthesized as a proprotein and cleaved into and  subunits that
remain covalently linked by disulfide bonds.23 The
clusterin antibody that we used reacts specifically with the 35- to
40-kd subunit.
Immunoblot analysis revealed clusterin expression only in the ALCL cell
lines (Figure 2). All ALCL cell lines, and
a human serum control for clusterin, showed a strong band of
approximately 40 to 42 kd consistent with the subunit of the
clusterin heterodimer. In addition, the primary ALCL cases, but not the
human serum control, showed a weaker high molecular weight band of
approximately 70 kd possibly representing a proprotein form of
clusterin containing the -subunit precursor. Two ALCL cell lines
(Karpas 299 and SR786) also displayed a 50- to 55-kd band of unknown
significance, and a smaller 30-kd band was present in the serum control
and in 1 ALCL cell line (Karpas 299). These weak reacting bands may
represent partially degraded or, in the case of the 50- to 55-kd band,
an alternatively glycosylated form of the chain. The identification of clusterin protein by immunoblot was also confirmed by
immunohistochemistry on paraffin-embedded cell line pellets, using the
same antibody (data not shown).
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| Fig 2.
Expression of clusterin in ALCL cell lines.
Western analysis of representative hematopoietic cell lines was
performed using a clusterin antibody specific to the subunit. A
major reaction product of approximately 40 kd is present in the 4 ALCL
cell lines (Karpas 299, KI-JK, SUDHL-1, and SR-786) and the serum
control, but not in 2 representative non-ALCL cell lines (KMS-12
[myeloma] and Molt-4 [T-lymphoblastic leukemia]).
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Clusterin specifically marks cases of classical ALCL
Because clusterin had not previously been reported to be
differentially expressed in lymphoid tissues, we were interested in
pursuing the possibility that it could be used as a diagnostic marker
for ALCL. For this purpose, we assembled a panel of 198 cases of
primary lymphomas, including 90 T-cell lymphomas, 78 B-cell lymphomas,
30 Hodgkin lymphomas (Table 2), and 30 representative examples of
nonneoplastic lymphoid tissues and stained them with the clusterin
antibody. Among the 90 T-cell lymphomas were 36 primary nodal ALCL
cases and 9 cutaneous ALCL cases.
In the reactive lymphoid tissues (tonsils, lymph node, and spleen),
clusterin was identified in follicular dendritic cells (FDC) and
fibroblastic reticular cells (Figure 3).
The FDCs showed strong cytoplasmic staining that extended into the cell
processes. Fibroblastic reticular cells showed a weaker but discernable
marking of their cytoplasmic processes. None of the lymphoid
elements displayed staining.
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| Fig 3.
Expression of clusterin in representative primary
lymphoid neoplasms.
Immunohistochemistry was performed using the anticlusterin antibody.
(A) and (B) Two anaplastic large cell lymphomas, positive for
clusterin. Note the strong characteristic Golgi staining (original
magnification × 400). (C) Peripheral T-cell lymphoma, negative
for clusterin (original magnification × 400). (D) Nasal NK/T
cell lymphoma, negative for clusterin (original magnification
× 400). (E) Nodular sclerosis Hodgkin disease (original
magnification × 200). Reed-Sternberg cells and variants are
negative for clusterin (arrows). Residual dendritic cells or
fibroblastic reticulum cells are positive. (F) Reactive lymphoid
hyperplasia (original magnification × 100). A reactive follicle
shows intense staining of dendritic cells with weaker staining of
interfollicular reticular cells. Normal B and T lymphocytes show no
staining.
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All of the 36 systemic ALCL cases stained for clusterin. The staining
was primarily cytoplasmic and characterized by moderate to strong
dot-like staining in the Golgi areas in the majority of tumor cells
(Figure 3). Occasional cases showed membranous staining as well.
Twenty-three of the 36 ALCL cases were positive for ALK-1; 13 were negative.
Only 2 non-ALCL cases stained with the clusterin antibody. One was a
T-cell-rich B-cell lymphoma with CD30+ neoplastic B cells.
Unlike the staining in ALCL, this case showed weak cytoplasmic staining
without the characteristic Golgi staining of the ALCL cases. The pale
and diffuse quality of the cytoplasmic staining suggested that it could
be an artifact; however, we conservatively scored it as positive. The
second case was a diffuse large B-cell lymphoma that showed moderately
strong cytoplasmic staining in a subplasma membrane pattern, again
without any Golgi staining. In all cases of Hodgkin lymphoma, the
Reed-Sternberg cells were negative. It is also of interest that all 9 cases of cutaneous ALCL were negative for clusterin. This finding is
consistent with the current concept that cutaneous ALCL represents a
different clinicopathologic entity from systemic ALCL. When present in
the primary lymphoma cases, residual follicular dendritic cells or fibroblastic reticular cells (or both) were highlighted by their reactivity with the clusterin antibody. Representative cases of ALCL,
other non-Hodgkin lymphomas, Hodgkin disease, and reactive follicular
hyperplasia are shown in Figure 3.
Western blot analysis of selected ALCL cases and non-ALCL cases
confirmed the expression of clusterin in primary ALCL (Figure 4). The molecular weight of the
-clusterin reactive bands was identical to those found in the ALCL
cell lines; both the higher molecular weight band presumably
corresponding to the uncleaved precursor form of clusterin and the 40- to 42-kd chain were present. Some of the non-ALCL cases showed weak
signals for clusterin. The clusterin present in these cases was
presumably derived from trapped blood and residual dendritic or
fibroblastic reticular cells.
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| Fig 4.
Expression of clusterin in primary lymphoid neoplasms.
Western analysis of representative lymphoid neoplasms was performed
using a clusterin antibody specific to the subunit. High-level
expression of clusterin is detected in the 3 primary ALCL cases (ALCL
1-3); none or lesser amounts are seen in 3 representative cases of CLL
(CLL 1-3). The limited expression of clusterin seen in the non-ALCL
cases is presumably derived from trapped serum or from residual
dendritic cells present in the biopsy specimens. High-level expression
of clusterin is also seen in a reactive lymph node (FH1), showing
marked follicular hyperplasia with accompanying follicular dendritic
cell proliferation.
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Discussion |
The dramatic advances in human genome sequencing and the development
of gene array technologies have given investigators a new powerful tool
to assess the expression of large numbers of genes in a single
experiment. The initial uses of array technology have been to decipher
global gene expression differences resulting from physiologic changes
or changes related to disease state. For clinical oncology, this tool
promises to accelerate the identification of diagnostic and prognostic
markers. In this study, we show that analysis of gene expression using
tumor cell lines can be used to identify novel tumor-specific markers.
In the current study, we chose a series of hematopoietic cell lines,
primarily representing specific subtypes of lymphoid neoplasms, to
demonstrate the utility of array technology in identifying tumor-specific markers. The arrays from Clontech Laboratories are
relatively low-density nylon filter macroarrays containing 588 genes
representing a broad range of functional classes. These macroarrays can
be analyzed using common laboratory equipment, in contrast to the
microarrays, which require specialized detection systems not widely
available.24 Despite the modest number of genes contained
on these macroarrays (588), we were able to verify previously known,
differentially expressed genes as well as identify a previously unknown
differentially expressed gene, clusterin, that we have shown to
be specifically expressed in ALCL.
We chose to study tumor cell lines derived from representative subtypes
of lymphomas rather than primary lymphoma specimens for the following
reasons. Primary tumors are, in reality, complex tissues that are
comprised not only of tumor cells but also of varying percentages of
infiltrating lymphocytes, blood vessels, and other stromal components.
The use of cell lines avoids the problem of having mixed populations of
cells contributing to the extracted mRNA and complicating the analysis
of the hybridization signals. Secondly, it is difficult, if not
impossible, to control the condition of the primary tissues with regard
to both in vivo factors and the results of degradation occurring
between the time of the biopsy and the extraction of the RNA. Cell
culture conditions are easily standardized, eliminating many of the
differences that might exist between primary tumors as a result of
specimen handling and local conditions affecting the tumor in vivo.
Thirdly, material from primary tumors is often limited, and frequently
the RNA extracted is of poor quality, whereas cell line RNA is
essentially unlimited and of good quality. Although cell lines may not
be perfect replicates of primary tumors in some respects, most cell
lines retain a majority of known phenotypic and differentiation-related
markers possessed by the parent primary tumor. We reasoned that there
would likely be many unstudied proteins shared between the derived cell
lines and primary tumors that could serve as tumor-specific markers.
To assess whether we could identify known differentially expressed
markers in the cell lines, we first looked at a series of markers
contained on the blot and known to be differentially expressed. This
exercise revealed that, in general, the results from the arrays were
capable of identifying previously reported differentially expressed
genes. As examples, CD19, a B-cell lineage-specific marker, was
specifically expressed only in B-cell lines. CD4, a T-cell-restricted
marker within lymphoid neoplasms, was expressed only in T-cell lines.
Cyclin D1, a specific marker for mantle cell lymphomas and
t(11;14)-carrying myelomas, was expressed only in the 2 mantle-cell-lymphoma cell lines and in 1 myeloma cell line having a
t(11;14) translocation.
Having satisfied ourselves that the Clontech macroarrays were reliable
in assessing differential expression of known genes, we next wished to
assess their ability to identify previously unknown, differentially
expressed genes, because these would be potential new diagnostic
markers. For this initial demonstration, we used a fairly strict
definition of differential gene expression. We required that the
candidate gene be expressed in all, or all but one, of the
representative members of the lymphoma subtype and that it be expressed
in no more than one example of another lymphoma subtype. Among the
genes that showed differential expression limited to a particular group
of cell lines, we identified a single gene, clusterin, which
was strongly expressed in all 4 ALCL cell lines and in none of the
remaining cell lines. We obtained an antibody to human clusterin and
confirmed expression at the protein level by Western blotting and
immunohistochemistry. We next studied by immunohistochemistry and in
some cases by Western blotting, a series of primary lymphomas including
36 ALCL cases. This study confirmed the differential expression of
clusterin in primary cases of ALCL and illustrated its potential use as
a diagnostic marker.
Anaplastic large-cell lymphoma is a form of T-cell lymphoma that is
defined by CD30 positivity and anaplastic morphology.25 Recently it has become apparent that there are at least 2 forms of ALCL
based on the presence or absence of a characteristic cytogenetic abnormality, the t(2;5), that leads to the overexpression of an unusual
protein kinase designated ALK-1.26-31 ALK-1-positive cases occur more frequently in children and young adults and have a relatively good prognosis. ALK-1-negative ALCL occurs in older individuals and has a poorer prognosis. It is interesting that clusterin marks both forms of primary nodal ALCL and is not correlated with the presence or absence of ALK protein. On the other hand, clusterin did not mark 9 cases of cutaneous ALCL, a lymphoma resembling nodal ALCL, but now thought to be distinct because of its specific presentation, indolent clinical course, and lack of ALK-1 staining in
virtually all cases.27
It is also of interest that clusterin expression does not appear to be
correlated with CD30 expression, because none of the strong
CD30+ Reed-Sternberg cells in cases of Hodgkin lymphoma
marked for clusterin. Furthermore, although 1 of the 2 possible
clusterin-expressing tumors among the B-cell non-Hodgkin lymphomas
weakly expressed CD30, none of 11 other diffuse large B-cell lymphomas
(Table 3), showing weak or focal CD30 positivity, expressed clusterin.
The functional significance of clusterin expression in ALCL is unclear,
although in other tissues it has been implicated in a variety of
functions.23,32 Clusterin was originally isolated from ram
rete testis fluid33 and so named because of its ability to
induce clustering of Sertoli cells.34 It has since been
shown to be widely expressed in the epithelial cells of many other
organs including liver, stomach, and brain, and is secreted in numerous body fluids such as semen, urine, breast milk, cerebrospinal fluid, and
plasma.23 Clusterin has been implicated in lipid transport, reproduction, complement regulation, tissue remodeling, cell-cell interaction, programmed cell death (apoptosis), and cell
survival.23,32 Most intriguing is its proposed role in
apoptosis. Initially it was believed to be proapoptotic because of its
accumulation in tissues undergoing apoptosis.35 However,
recent data suggest that clusterin is accumulated in the surviving
cells adjacent to areas undergoing apoptosis, leading investigators to
reassess the role of clusterin in apoptosis.36 In accord
with these more recent observations, clusterin has been shown to have
potent chaparone-like activities and protects a wide variety of
proteins from heat or mercaptoethanol-induced
denaturation.37,38 These data are consistent with a
generalized protective antiapoptotic function rather than a
proapoptotic one.
Few studies have investigated clusterin expression in lymphoid cells
because early studies of its tissue distribution failed to identify
clusterin in peripheral blood T cells.39 Because of its
hypothesized role in apoptosis, clusterin had been proposed to play a
role in the generation of T-cell tolerance in the thymus. However, its
expression was limited to the epithelial cells of the medulla, whereas
thymocytes lacked clusterin expression.40,41 These findings
suggested that clusterin was not an important regulator of programmed
cell death in thymocytes and confirmed the lack of clusterin expression
in a second source of T-lineage lymphocytes. Finally, in their attempts
to develop inducible models of clusterin expression, French et
al36 were not able to show clusterin expression or
induction in a T-cell lymphoblastic leukemia cell line. Although most
ALCL cases have a T-cell phenotype, we also were not able to identify a
normal T-cell population expressing clusterin, and at the present time
we have no clues regarding its differential expression in ALCL. We have
previously shown that ALCL displays some unusual phenotypic features in
that the majority of cases express both CD4 and cytotoxic granular
proteins.42 Thus, it is possible that the normal cellular
counterpart is a rare cell or is present only at particular stages of
differentiation or development. Alternatively, clusterin may be a true
tumor antigen in ALCL and may not be expressed in the normal cellular counterpart.
In summary, we have described an approach for the identification and
confirmation of novel tumor-specific markers, using a combination of
gene array technology on selected representative cell lines and
immunochemical screening of primary case material. Using relatively
small format gene expression macroarrays with 588 genes and stringent
criteria for differential gene expression, we were able to identify
differentially expressed gene products, including clusterin, a new
specific marker for ALCL. Undoubtedly, the use of larger format
microarrays with many thousands of genes will increase the yield of new
markers as will more sophisticated computer-assisted analysis of
quantitative differences in expression levels. In the future,
improvements in microdissection and mRNA/cDNA amplification technology
will allow the direct use of amplified cDNA derived from primary
tumors, without the confounding effect of contaminating mRNA from
nonneoplastic cells. The use of primary tumor material will be
particularly important for the identification of prognostic markers.
| |
Acknowledgments |
We wish to thank Dr G. Jokhadze (Clontech Laboratories) for helpful
discussions regarding the Clontech Atlas arrays, Ms T. Davies-Hill and
Dr L. Quintanella-Martinez for assistance with the Western blotting,
and Drs A. Karpas and M. Shimakage for kindly providing the Karpas 299 and Ki-JK cell lines, respectively.
| |
Footnotes |
Submitted December 13, 1999; accepted March 8, 2000.
A.W. and C.T. contributed equally to this work.
Reprints: Mark Raffeld, Laboratory of Pathology,
National Cancer Institute, Bldg 10, Rm 2N110, 9000 Rockville Pike, Bethesda, MD 20892; e-mail: mraff{at}box-m.nih.gov.
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Abstract
Introduction