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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4764-4770
Prevalence of Antibodies Against Proteins Derived From
Leukemia Cells in Patients With Chronic Myeloid Leukemia
By
Min Ling,
Yue-Jin Wen, and
Seah H. Lim
From the Department of Haematology, University of Wales College of
Medicine, Cardiff, UK.
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ABSTRACT |
Although various studies supported the notion that leukemia cells in
chronic myeloid leukemia (CML) may be recognized by the immune system,
direct evidence showing the immunogenicity in vivo of proteins derived
from the leukemia cells is lacking. In this study, we have constructed
an expression cDNA library from the leukemia cells of a patient with
CML and used the autologous serum to screen for high-titer IgG
antibodies directed at the leukemia-derived proteins. We isolated eight
distinct clones from the library, suggesting that multiple immune
responses were elicited in the autologous host. Sequence analysis
showed high degrees of homology to known gene sequences in six of the
eight clones. Neither bcr-abl nor proteinase 3 sequences were
isolated. Using Northern blot analysis, seven of the eight clones
showed ubiquitous expression in normal bone marrow, leukemia cell
lines, fresh leukemia cells, and normal tissues. However, clone no. 4 showed restricted mRNA expression, being only detected in some fresh
leukemia cells, K562 cells, and normal testicular RNA. Using bacterial
lysates in dot blot analysis, a panel of sera from normal individuals and patients with CML and other hematological malignancies were screened for high-titer antibodies against these eight clones. There
were, among the CML patients, signficantly higher prevalence of
antibodies against seven of the eight clones. They were observed even
after omitting from the analysis patients with multiple myeloma whose
associated immune paresis may impair immune responses to these
proteins. Interestingly, antibodies against these proteins were also
detected in a small number of normal individuals. Although the precise
clinical significance of our findings remains to be determined, this
study provides evidence in support of the potential immunogenicity of
leukemia-derived proteins in the autologous host. It also provides
basis for further investigations to characterize these proteins,
especially clone no. 4, and determine their potential for immune
targeting in CML.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
VARIOUS CLINICAL AND laboratory
observations suggest that the leukemia cells in chronic myeloid
leukemia (CML) are potentially immunogenic.1 Although some
of the antileukemic effect observed following allogeneic bone marrow
transplant (BMT) arises as part of a more global graft-versus-host
reaction (GVHD) due to minor histocompatibility differences between the
donor and the recipient, it is likely that additional leukemia
eradication is mediated through effector cells directed at leukemia
antigens. Such T-cell populations have been dissected from the GVH
effect in studies involving the autologous hosts. Not unlike tumor
infiltrating lymphocytes in solid tumors, the repertoire of T-cell
receptor V gene usage by T cells associated with CML was restricted
when compared with that in healthy individuals.2
Furthermore, leukemia-reactive T cells could be isolated from the
autologous host.3 More recently, leukemic cells from
patients with CML have been shown to be able to generate autologous
T-cell responses after the in vitro induction of costimulatory
molecules, CD80, and CD86 on the leukemia cells.4-6
There are potentially many protein molecules in CML from which
processed peptides may be presented by major histocompatibility complex
(MHC) molecules to elicit host immune mechanisms. They include
overexpressed normal antigens and leukemia-restricted antigens. The
latter occur due to leukemogenesis (p210bcr-abl) or
are associated with disease progression (eg, gene products of mutated
ras oncogene or p53 tumor-suppressor gene). The
bcr-abl junctional peptides7-9 and peptides from
proteinase 310 have both been shown to elicit in vitro
antileukemic T-cell responses. A recent study analyzing autologous
peptides bound by HLA class I and II molecules of leukemia cells in CML
confirmed that some of these antigens are indeed naturally processed
and presented.11 This technique has additionally isolated
other peptides that could potentially be targeted by T cells. However,
the immunogenicity in vivo of these antigens in the autologous host
remains to be determined.
A technique has recently been developed that can be used to identify
immunogenic protein antigens in vivo.12 In this approach, expression cDNA libraries are prepared from tumor specimens and immunoscreened using absorbed and diluted autologous serum for the
detection of antigens that have elicited a high-titer IgG antibody
response. Such a humoral response may represent a B-cell response with
cognitive help from helper T cells. Therefore, even though the antigens
are intracellular and are initially identified by antibodies, this
technique identifies tumor products that can be investigated in the
context of cell-mediated immunity. Applying the serological
identification of antigens by recombinant expression cloning
(SEREX) to various solid tumors, a combination of known and
novel T-cell antigens have been isolated.12-14 In this
study, we have applied a modification of this approach to CML to
determine any B-cell responses against proteins derived from leukemia
cells in patients with CML. This study may therefore form a useful
basis for future investigation into identification of leukemia antigens that may be suitable for immune targeting.
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MATERIALS AND METHODS |
RNA extraction and construction of cDNA expression library.
Total RNA was extracted from the presentation peripheral blood
mononuclear cells of a patient (C.D.) with CML using the RNAzol method.15 Poly(A)-RNA was isolated from total RNA using the Poly-A Tract mRNA isolation systems (Promega, Southampton, UK) and a
cDNA expression library constructed from 5 µg of poly(A)-RNA. First-strand cDNA synthesis was performed using an oligo(dT) primer that contained an internal Xho I restriction site. The cDNA was ligated to EcoRI adaptors and digested with Xho I
before being size-selected, cloned directionally into  ZAP expression
vector (Stratagene, Cambridge, UK), packaged into phage particles, and transfected into XLO1-blue Escherichia coli.
Immunoscreening of cDNA library.
Immunoscreening was performed on nitrocellulose membranes containing
the phage plaques using autologous serum after protein induction with
Isopropyl -D-Thiogalactopyranoside (IPTG; 1.5 mmol/L). Before use,
the autologous serum was first preabsorbed with lysate from E
coli transfected with the blue phage. The preabsorbed serum, at a
final concentration of 1:1,000, was then incubated at room temperature
with the nitrocellulose membranes for 1 hour, washed in Tris-buffered
saline/0.01% Triton-X 100 (TBS-T) solution, and then incubated in
horseradish peroxidase-conjugated goat antihuman IgG antibody for an
additional 1 hour. Reactive phage plaques on the nitrocellulose
membrane were finally visualized, after further washing in TBS-T
solution, using the chemoluminiscence technique according to the
manufacturer's recommendation (Boehringer Mannheim, East Sussex, UK).
False-positive clones encoding normal human IgG fragments derived from
contaminating circulating B lymphocytes were eliminated by a round of
membrane screening using only horseradish peroxidase-conjugated goat
antihuman IgG antibody.
Sequence analysis of reactive clones.
Positive clones were subcloned, purified, and excised in vivo to
pBK-CMV plasmid forms (Stratagene). Plasmid DNA was prepared and the
DNA inserts evaluated by EcoRI and Xho I restriction
mapping. Nucleotide sequences of clones showing different cDNA inserts were analyzed. The sequencing reactions were performed by Oswell DNA
Sequencing Services (Southampton University, Southampton, UK) using AB1 PRISM (Perkin-Elmer, Norwalk, CT) automated
sequencers.
Northern blot analysis.
Northern blot analysis was performed to confirm the origin of the cDNA,
estimate the size and copy number of the transcripts, and to determine
the expression of the transcripts in normal and leukemia RNA. RNA was
isolated from normal bone marrow, fresh CML cells, and leukemia cell
lines (CEM, HL60, K562, Molt 4, and p39). RNA from normal brain, lungs,
small intestine, muscle, spleen, and testis was obtained from a
commercial source (Invitrogen, Leek, The Netherlands). For
Northern blot analysis, 30 µg of total RNA was resuspended in a
loading buffer containing formamide and formaldehyde, heated at
65°C for 2 minutes, and electrophoresed on a 1.2%
agarose/formaldehyde gel before being transferred by capillary method
onto nitrocellulose membranes. Subsequent hybridization to
32P-labeled probes (derived from EcoRI/Xho
I restriction digest of recombinant phagemids and labeled by random
priming) and washing were performed under high-stringency conditions.
Hybridization was performed at 60°C overnight and final washes of
the membrane at 60°C with 0.1× SSC in 0.1% (wt/vol) sodium
dodecyl sulfate solution. RNA loading in each case was
determined using a probe for the GAPDH transcript.
Reverse transcription-polymerase chain reaction (RT-PCR).
To evaluate the mRNA expression, total RNA was first reverse
transcribed using oligo-dT primers and used with gene-specific oligonucleotide primers for PCR amplification of cDNA segments of 300 to 400 bp in length. All PCR reactions were performed using the
following cycling conditions: 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute for 30 cycles. RT-PCR was performed in a thermal cycler (Perkin-Elmer), and the PCR products were visualized on an ethidium bromide agarose gel for DNA of expected size.
The positive control amplified the plasmid DNA containing the DNA
insert, and the negative control was composed of the PCR reaction
mixture lacking cDNA.
Preparation of crude fusion proteins.
Freshly prepared E coli cultures containing the appropriate
phagemid constructs were grown at 37°C to an absobance at 600 nm of
0.8 to 1.0 in 200 mL of NZY bacterial broth containing 50 µg/mL
kanamycin. IPTG was added to induce the expression of the fusion
proteins. The E coli were harvested by centrifugation at 3,600 rpm for 15 minutes at 4°C. After removal of the supernatant, cells
were weighed and 3 mL of lysis buffer (50 mmol/L Tris, pH 8.0, 1 mmol/L
EDTA, 100 mmol/L NaCl) was added to each gram (wet weight) of E
coli pellet. The cells were then lysed using the lysozyme-deoxycholate method.16 The cell debris was removed by centrifugation at 10,000 rpm for 15 minutes at 4°C.
Detection of antigen-reactive antibodies in allogeneic sera.
This procedure was performed using dot blot analysis.
Briefly, bacterial supernatant containing the crude fusion proteins was
dotted onto nitrocellulose membranes and dried at 37°C for 15 minutes. After blocking, the membranes were incubated for 1 hour with
1:800 diluted sera, which had been preabsorbed with lysate from E
coli transfected with the blue phage, followed by an additional 1 hour of incubation with horseradish peroxidase-conjugated rabbit
antihuman IgG antibody (Sigma, Poole, UK). The membranes were extensively washed with TBS-T solution between each incubation step. Binding of the antibodies to the fusion proteins was visualized by incubating with DAB peroxidase substrate (Sigma FastTM
3,3 -diaminobenzidine set; Sigma) for 2 minutes. Each set of blots contained a control consisting of the bacterial lysate from E
coli transfected with the blue phage. Sera were analyzed in a
random order and with the observer blinded to their origin.
Statistics.
 2 was used to determine the power of significance
between the analyzed groups.
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RESULTS |
SEREX analysis of CML cDNA library.
To isolate clones expressing antigens reactive with the autologous
serum, an expression cDNA library containing 1.8 × 106 primary phage clones, constructed from the leukemic
cells of a patient with CML, was used for immunoscreening. After
excluding false-positive clones encoding the human Ig fragments, eight
positive plaques were identified. They were subcloned, purified, and
excised into pBK-CMV phagemid. The inserts were analyzed by restriction mapping, which suggested that they represented eight different clones.
The size of the DNA inserts ranged from approximately 800 bp to greater
than 3 kb.
Molecular analysis of the identified antigens.
Nucleotide sequence analysis of the cDNA inserts was performed to
determine the identity and sequence homology of the clones using the
BLAST software on US National Molecular Biology Laboratory and GenBank
data bases (released January 1998; Table
1). Clones no. 2, 3, 5, and 8 showed strong homology to human nuclear
chloride ion channel protein (NCC27), heterogenous nuclear RNA binding protein M4, human modulator recognition factor I, and human MAF oncogene, respectively. Interestingly, although high degrees of homology (>95%) were observed, there appeared to be an internal nucleotide deletion of more than 100 bp, respectively, in clones no. 2 and 3 when compared with their homologue genes. No significant sequence
homology was observed for clones no. 4 and 7. Clones no. 1 and 6 appeared to be fusion genes previously not reported. Further work is
ongoing to confirm that they are not cloning artefacts.
Expression of the identified genes in the autologous host.
The origin of the isolated sequences was confirmed by Northern blot
analysis on total RNA isolated from the primary leukemia cells
(Fig 1). Positive signals were obtained
using probes derived from seven of the eight clones (no. 1, 2, 4, 5, 6, 7, and 8). However, no signal was obtained in the Northern blot
analysis using a probe derived from clone no. 3, for which one
potential explanation is a very low level of mRNA expression. The
expression of the mRNA from which clone no. 3 was derived was
subsequently confirmed by RT-PCR using a pair of sequence-specific
oligonucleotide primers (Fig 2).
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| Fig 1.
Northern blot analysis of the expression of the eight
isolated sequences in the primary leukemia cells. Signals were obtained
using probes derived from clones no. 1, 2, 4, 5, 6, 7, and 8. The level
of gene expression of these seven clones was variable compared with
signals obtained from a control probe, GAPDH.
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| Fig 2.
RT-PCR analysis for the expression of clone no. 3 (lane
1, molecular marker; lane 2, positive control consisted of PCR
amplification of a recombinant plasmid containing sequence 3; lane 3, RT-PCR amplification of total RNA isolated from the primary leukemia
cells; lane 4, negative control). Clone no. 3 PCR primer sequences were
as follows: clone no. 3-1, 5-CAA AGA AGA TCC TGA TGG-3;
clone no. 3-2, 5-GCA GTG CAT TGG TCT ATC-3.
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Expression of the identified genes in leukemia cells.
The expression of the isolated gene sequences was evaluated in the
other CML patients by Northern blot analysis using total RNA derived
from the leukemia cells of four other patients. We found that all of
the eight clones were detected in most, but not all, of the four other
CML patients (Table 2 and
Fig 3). In addition, we also determined the gene sequence
expression of six (clones no. 1, 2, 3, 4, 5, and 7) of these eight
clones in five leukemia cell lines and found that clones no. 1, 2, 3, 5, and 7 were detected in RNA from all cell lines, except for P39 cells, in which gene sequence 7 was not detected (Table 2).
Interestingly, in contrast to clones no. 1, 2, 3, 5, and 7, clone no. 4 was only detected in K562 cells and not CEM, HEL, Molt 4, and P39 cells (Fig 4). The results provide supporting
evidence for the preferential expression of gene sequence from clone
no. 4 in CML.
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| Fig 3.
Northern blot analysis for the expression of clone no. 4 and GAPDH, showing clone no. 4 mRNA in leukemia cells derived from two
of the four other CML patients (lane 1, RNA from leukemia cells derived
from patient C.D.; lanes 2, 3, 4, and 5, RNA from leukemia cells
derived from four other CML patients).
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| Fig 4.
Northern blot analysis for the expression of clone no. 4 and GAPDH in leukemia cell lines, normal bone marrow, and patient C.D.,
showing clone no. 4 mRNA in K562 cells (lane 1, P39; lane 2, Molt 4;
lane 3, K562; lane 4, HEL; lane 5, CEM; lane 6, normal bone marrow;
lane 7, patient C.D.).
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Expression of the identified genes in normal tissues.
The mRNA expression of these eight gene sequences was also evaluated by
Northern blot analysis using total RNA derived from a small panel of
normal tissues, namely, bone marrow, brain, lungs, small intestine,
muscle, spleen, and testis. Table 3 shows
that clones no. 1, 2, 3, 5, 6, 7, and 8 are widely expressed amongst the normal tissues. However, expression of gene sequence from clone no.
4 appears to be restricted to only normal testis
(Fig 5). In particular, we did not detect
expression of the gene sequence in normal bone marrow.
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| Fig 5.
Northern blot analysis for the expression of clone no. 4 and GAPDH showing clone no. 4 mRNA in normal testis (lane 1, lungs;
lane 2, spleen; lane 3, muscle; lane 4, testis; lane 5, small
intestine; lane 6, brain; lane 7, normal bone marrow).
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Prevalence of high-titer antibodies against the identified antigens
amongst sera from patients and healthy donors.
Screening was performed using diluted sera from a group of patients
with CML and other hematological diseases and normal healthy donors.
The clinical characteristics of these patients are shown in
Table 4. Because the prevalence of
autoantibody is influenced by the sex and age of the patients, we first
determined any differences in the distribution of these two parameters
in each of the sample groups. There was no difference in the sex
distribution of patients with CML compared with the groups of normal
healthy donors or patients with other hematological malignancies.
Whereas there was no difference in the age distribution of CML patients
and normal healthy donors, CML patients were significantly younger than
those with other hematological malignancies (P < .0003).
To perform screening for the presence of high-titer antibodies against
the identified antigens by dot blot analysis, crude fusion proteins
were prepared from lysates of the bacterial transformants. Because the
cDNAs were cloned into the -galactosidase gene in the plasmid,
successful preparation of fusion proteins was demonstrated by the
ability of the proteins to bind polyclonal anti--galactosidase antibodies, ie, the production of fusion proteins between
-galactosidase and leukemia proteins
(Fig 6). All sera were then used for
screening at a dilution of 1:800 and a negative control consisting of
bacterial lysate from a blue phage was included in all cases. Those
sera that did not show any reactivity at this serum dilution were
confirmed at a serum dilution of 1:200.
Table 5 shows the distribution of serum
reactivity to these antigens in the various groups of individuals.
There was a significantly higher prevalence of antibodies directed
against the leukemia-derived antigens in patients with CML compared
with normal healthy donors. This was observed for seven of the eight
antigens (clones no. 1, 2, 3, 4, 6, 7, and 8). Such differences were
observed even when four CML patients who were on interferon-
(IFN-) therapy were excluded from the analysis (.001 < P < .01). Interestingly, one normal donor exhibited antibodies to all
eight and another to seven of the eight antigens.
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| Fig 6.
Dot blot analysis using crude fusion protein extracts.
(A) Bacterial extracts from all eight clones contained fusion proteins
capable of binding to anti--galactosidase antibodies. (B) Dot blot
screening of sera for antibodies to leukemia derived proteins. (Top)
Negative reactivity to all eight proteins. (Middle) Positive reactivity
to all eight proteins. (Bottom) Positive reactivity to proteins derived
from clone no. 5 only. B1, bacterial extract from a clone of blue
phage; B2, bacterial extract from a nontransformed E coli.
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Table 5.
Prevalence of IgG Antibodies Directed at 8 Leukemia-Derived Antigens (P Value Compared With CML
Patients)
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The high prevalence of antibodies appears to be disease-related. Only 7 of the 38 patients with other hematological malignancies (1 myeloma, 1 myeloproliferative disease, 1 non-Hodgkin's lymphoma, 2 monoclonal
gammopathy of unknown significance, 1 myelodysplastic syndrome, and 1 acute myeloid leukemia) also exhibited antibodies to most of the eight
antigens. Despite being of a younger patient group, more CML patients
produced antibodies to these antigens when compared with the group of
older patients with other hematological malignancies (.001 < P < .02). This was observed even after excluding patients
with multiple myeloma (.01 < P < .05) whose associated immune-paresis may have limited any antibody responses.
To determine the durability of the B-cell responses, sera from a number
of individuals were tested on two or three separate occasions. Sera
were collected between 3 and 6 months apart. In all cases, the results
were identical.
In 20 randomly selected samples, we also compared our method of
antibody screening with screening using phage plaques as described in
previous studies.12,13 We found concordance between results obtained by these two techniques. However, in general, results obtained
with phage plaques tended to be less clear, probably reflecting lower
concentrations of the antigens present in the plaques compared with
bacterial lysate.
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DISCUSSION |
Recent advances in tumor immunology suggest the potential
immunogenicity of various tumors in the autologous setting.
Furthermore, with the successful generation of autologous cytotoxic
T-lymphocytes (CTLs) against tumor cells, an array of antigenic targets
on the tumor cells has been defined, either through the screening of target cells transfected with recombinant tumor DNA
libraries17 or biochemical purification of peptides eluted
from the MHC molecules.18 Unfortunately such approaches are
limited by the ability to generate in vitro tumor-reactive CTLs.
Therefore, these techniques cannot be easily adapted to define antigens
in most tumors. Based on the knowledge of overexpressed normal proteins
or novel fusion proteins in tumor cells, workers in CML have identified
immunogenic epitopes within the bcr-abl7-9 and
proteinase 310 proteins. Peptides within these proteins are
able to stimulate the generation of specific CTLs that also recognize
HLA-matched fresh leukemia cells. However, such an approach does not
provide any information regarding the immunogenicity of these peptides
in vivo. It remains to be determined if the CTL reactivity with fresh
leukemia cells represents true immune responses against peptides from
these proteins that have been endogenously processed and presented on
the leukemia cells or cross-reactivity with other endogenously
processed peptides expressed on leukemia cells. In this study, we have
used the SEREX approach to investigate any B-cell responses to
antigens derived from the leukemia cells in CML. This work therefore
provides foundation for future identification of leukemia antigens in
CML.
We demonstrated that multiple B-cell immune responses occur in the
autologous host to proteins derived from the leukemia cells in CML. A
total of eight cDNA clones encoding the proteins were identified. Six
of these eight sequences showed strong homology to known genes, whereas
the other two were novel sequences. Interestingly, although the six
sequences showed strong homology to known genes, there was an internal
nucleotide deletion in two of these sequences. Two fusion sequences
were also identified that are currently under further investigations to
exclude that they are cloning artefacts. It remains to be determined if
the antibody responses arose as a result of these genetic anomalies.
Using sequence comparison, we have so far not identified any mutation
in these gene fragments. Surprisingly, potential CML antigens such as
the bcr-abl fusion protein and proteinase 3 were not
represented.
We then determined the tissue distribution of these gene sequences in
CML cells, leukemia cell lines, and a limited panel of normal tissue.
Although seven of the eight clones showed ubiquitous expresssion, clone
no. 4 appeared to exhibit restricted tissue expression, being detected
only in normal testis and in CML cells. Interestingly, it was also
detected in CML cell line, K562. Although the restricted expression of
clone no. 4 requires to be confirmed using a bigger panel of normal
tissues, clone no. 4 with a novel gene sequence may be a new member of
the group of cancer-testicular antigens in which normal testicular
antigens are aberantly expressed in tumor cells. Work is ongoing to
confirm this and to express the cDNA in mammalian cells for further
characterization of the protein.
The observation that antibodies against seven of the eight antigens
were found predominantly in disease-bearing patients suggests that the
immune responses may be disease-related. This was observed even after
excluding CML patients on IFN- therapy, because IFN- therapy may
potentially induce autoimmunity. However, we also detected antibodies
in two healthy donors. Although it is possible that the antibodies
detected in healthy donors may not be directed at the same B-cell
epitopes on these proteins, one possible explanation for the presence
of antibodies in these individuals is that the antigen may also be
expressed under nonmalignant conditions such as viral infections or
other inflammatory processes. Another explanation may be the presence
of malignant CML cells in these apparently normal individuals, because
a previous study19 indicated the ability to detect the
bcr-abl transcripts in apparently normal individuals.
The antibodies against the leukemia-derived proteins detected by our
screening system were of the IgG subclass, implying the possible
involvement of CD4 cognitive help in the B-cell responses. The
coexistence of cellular and humoral responses have indeed been reported
in tumor antigens such as NY-ESO-1 in esophageal carcinoma14 and HER-2/neu in breast cancer.20
In CML, this is particularly relevant, because, based on works
involving selective T-cell depletion for allogeneic BMT,21
CD4 T cells appear to have an important role in mediating antileukemic
immune responses. Obviously such antileukemic immune responses would
only occur if the antigens are also processed and presented by the
leukemia cells in the context of MHC class II molecules.
The clinical significance of B-cell responses to leukemia-derived
proteins is unclear. In breast cancer, the presence of antibodies directed to p53 protein has been associated with a poor
clinical outcome.22 It is therefore important to determine
if the presence of antibodies to these proteins influences the
responses to IFN- therapy and outcome after allogeneic BMT in CML.
However, this could only be achieved through analysis of many more CML
patients. Further work would also permit the determination of any
correlation between antibody production and mRNA expression by the
leukemia cells.
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FOOTNOTES |
Submitted May 4, 1998;
accepted August 5, 1998.
Supported by the Leukaemia Research Fund, UK, the Leukaemia Research
Appeal for Wales, and the Welsh Bone Marrow Transplant Research Fund.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Seah H. Lim, MD, PhD, Myeloma and
Transplantation Research Center, University of Arkansas for Medical
Sciences, 4301 W Markham, Slot 776, Little Rock, AR 72205.
| |
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Abstract
Introduction