Malignant mesothelioma-associated antigens recognized by tumor-infiltrating B cells and the clinical significance of the antibody titers.
ABSTRACT Malignant pleural mesothelioma (MPM) is difficult to diagnose at an early stage. The present study attempted to obtain a tumor-specific antibody against MPM derived from tumor-infiltrating B lymphocytes in MPM by using a xenotransplanted severe combined immunodeficiency (SCID) mouse model, and to identify the antigens recognized by the antibodies. Among the antigen-antibody relationships, the clinical usefulness of antibody titers in the sera was evaluated from the viewpoint of diagnosis of MPM and monitoring of therapeutic effects. Tumor tissue specimens from two patients with MPM were engrafted subcutaneously in SCID mice and blood samples were obtained and pooled every 2 weeks after xenotransplantation until 14 weeks when the mice were killed. A cDNA library was constructed from the mRNA of a MPM cell line (K921MSO). Immunoscreening of the libraries was carried out by serological identification of antigens by a recombinant expression cloning method (SEREX) and four antigens were identified as MPM-associated antigens. Among them, antibody titers against two antigens, Gene-X and thrombospondin-2 (THBS-2), were analyzed by phage plaque assay as the first step. ELISA systems correlated with the phage plaque assay to detect antibody titers against the two antigens were constructed using 20-mer peptides of the antigen-coding genes. The cut-off value was decided by the average and standard deviation of normal healthy persons. Antibody against Gene-X was detected in 10 out of 18 (55.6%) mesothelioma patients and antibody against THBS-2 was detected in 16 out of 18 (88.9%) mesothelioma patients. No patients with lung cancer regardless of asbestos exposure exhibited positive antibody titer against the two antigens. Furthermore, the serum antibody titers decreased after surgical treatment of MPM and increased after recurrence of the disease. The titers of the antibodies against Gene-X and THBS-2 could be used as tumor markers for the diagnosis and follow up of patients with MPM.
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ABSTRACT: Combining different standard therapies with immunotherapy for the treatment of solid tumours has proven to yield a greater clinical benefit than when each is applied separately; however, the percentage of complete responses is still far from optimal, and there is an urgent need for improved treatment modalities. The latest literature data suggest that tertiary lymphoid structures (TLS), previously shown to correlate with the severity of autoimmune diseases or transplant rejection, are also formed in tumours, have a significant beneficial effect on survival and might reflect the generation of an effective immune response in close proximity to the tumour. Thus, the facilitation of TLS formation in tumour stroma could provide novel means to improve the efficiency of immunotherapy and other standard therapies. However, little is known about the mechanisms regulating the formation of tumour-associated TLS. Studies of chronic inflammatory diseases and transplant rejection have demonstrated that TLS formation and/or function requires the presence of B cells. Additionally, the infiltration of B cells into the tumour stroma has been demonstrated to be a significant prognostic factor for improved survival in different human tumours. This suggests that B cells could play a beneficial role in anti-tumour immune response not only in the context of antibody production, antigen presentation and Th1-promoting cytokine production, but also TLS formation. This review focuses on the latest discoveries in tumour-infiltrating B cell functions, their role in TLS formation and relevance in human tumour control, revealing novel opportunities to improve cancer therapies.Cancer Immunology and Immunotherapy 04/2014; · 3.64 Impact Factor
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ABSTRACT: Hyaluronic acid (HA) has been proposed as a biochemical marker of malignant pleural mesothelioma (MPM). The present study focused on the implications of HA and CD44 interaction in the proliferation and invasiveness of MPM. The proliferation and invasive activity was evaluated in two human mesothelioma cell lines, ACC-MESO-1 and K921MSO, by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the transwell chamber model. The knockdown of CD44 gene expression was accomplished by transfection of the cells with small interfering RNA. Flow cytometry revealed that both the ACC-MESO-1 and K921MSO cell lines highly expressed CD44. Treatment with HA enhanced the proliferation in both mesothelioma cell lines in comparison to cells without HA treatment. The treatment with HA (25 μg/ml) also significantly upregulated the invasion of both types of cells. The silencing of CD44 significantly abrogated the effect of HA treatment on the proliferation of ACC-MESO-1 cells and significantly suppressed the proliferation of K921MSO cells. HA-CD44 binding is important for the migration and proliferation of mesothelioma cells. Therefore, the HA-CD44 interaction is a potentially useful therapeutic target in MPM.Tumor Biology 08/2012; · 2.84 Impact Factor
Chapter: B Lymphocytes in Cancer Immunology[Show abstract] [Hide abstract]
ABSTRACT: The role of B lymphocytes in the pathogenesis and treatment of cancer has not received as much attention as the role of T cells. However, most patients with solid tumors harbor circulating antitumor antibodies and most tumors contain a population of infiltrating B cells implying an association between oncogenic events and B-cell activation. B-cell immunity can be beneficial by providing antibody-mediated protection from oncogenic viruses or a source of recombinant tumor-specific antibodies that can be used in combination with chemotherapeutic regimens. However, activation of B cells may also be detrimental to an effective antitumor response. Tumor-reactive antibodies and B cells often recognize antigens that are generated during the unscheduled apoptotic and necrotic death processes, which accompany tumor progression and may be involved in wound-healing processes that promote tumor growth and impair protective T-cell responses. Therefore, methods to eliminate autoreactive B cells, or switch them to a B effector-1 (Be-1) phenotype that amplifies Th1/Tc1-type T-cell responses, which are typically associated with effective antitumor responses, may improve the clinical outcomes of T-cell-mediated immunotherapies. Possible strategies include the administration of B-cell-depleting monoclonal antibodies, use of targeted B-cell stimulatory agents such as Toll-like Receptor agonists, and adoptive transfer of large numbers of ex vivo generated tumor-reactive Be-1 cells. KeywordsB lymphocytes-Cancer vaccines-Chronic lymphocytic leukemia-Regulatory B cells-Tumor immunology12/2010: pages 37-57;
Cancer Sci|July 2009|vol. 100|no. 7|1326–1334doi: 10.1111/j.1349-7006.2009.01181.x
© 2009 Japanese Cancer Association
Blackwell Publishing Asia
Malignant mesothelioma-associated antigens
recognized by tumor-infiltrating B cells and
the clinical significance of the antibody titers
Yoshiki Shigematsu, Takeshi Hanagiri, Koji Kuroda, Tetsuro Baba, Makiko Mizukami, Yoshinobu Ichiki,
Manabu Yasuda, Mitsuhiro Takenoyama, Kenji Sugio and Kosei Yasumoto1
Second Department of Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
(Received December 3, 2008/Revised March 24, 2009/Accepted March 27, 2009/Online publication May 3, 2009)
Malignant pleural mesothelioma (MPM) is difficult to diagnose at an
early stage. The present study attempted to obtain a tumor-specific
antibody against MPM derived from tumor-infiltrating B lymphocytes in
MPM by using a xenotransplanted severe combined immunodeficiency
(SCID) mouse model, and to identify the antigens recognized by the
antibodies. Among the antigen–antibody relationships, the clinical
usefulness of antibody titers in the sera was evaluated from the
viewpoint of diagnosis of MPM and monitoring of therapeutic effects.
Tumor tissue specimens from two patients with MPM were engrafted
subcutaneously in SCID mice and blood samples were obtained and
pooled every 2 weeks after xenotransplantation until 14 weeks when
the mice were killed. A cDNA library was constructed from the mRNA
of a MPM cell line (K921MSO). Immunoscreening of the libraries was
carried out by serological identification of antigens by a recombinant
expression cloning method (SEREX) and four antigens were identified
as MPM-associated antigens. Among them, antibody titers against
two antigens, Gene-X and thrombospondin-2 (THBS-2), were analyzed
by phage plaque assay as the first step. ELISA systems correlated
with the phage plaque assay to detect antibody titers against the
two antigens were constructed using 20-mer peptides of the antigen-
coding genes. The cut-off value was decided by the average and
standard deviation of normal healthy persons. Antibody against
Gene-X was detected in 10 out of 18 (55.6%) mesothelioma patients
and antibody against THBS-2 was detected in 16 out of 18 (88.9%)
mesothelioma patients. No patients with lung cancer regardless of
asbestos exposure exhibited positive antibody titer against the two
antigens. Furthermore, the serum antibody titers decreased after
surgical treatment of MPM and increased after recurrence of the
disease. The titers of the antibodies against Gene-X and THBS-2
could be used as tumor markers for the diagnosis and follow up of
patients with MPM. (Cancer Sci 2009; 100: 1326–1334)
fatal. MPM is thought to be associated with previous exposure
to asbestos fibers. Due to the widespread use of asbestos fibers
in the latter half of the 20th century, the incidence of MPM is
predicted to rise sharply in industrialized countries in the next
few decades after 30–40 years of a latent period.(1) The number
of new cases in Europe is expected to double between 1998 and
2018.(2) Based on the prevalence in USA and Europe, Japan is
also facing an epidemic of MPM belatedly. In Japan, 500
patients with MPM died in 1995 and the number of deaths
increased to approximately 900 patients in 2003.(3) Not only
occupational exposure, but also environmental exposure becomes
a public health problem, because asbestos was used widely in
buildings as a construction material and asbestos cement pipes.(4,5)
Malignant pleural mesothelioma is difficult to diagnose
radiographically at an early stage and often progresses to an
advanced stage without symptoms. Although a new chemotherapy
alignant pleural mesothelioma (MPM) is an aggressive
neoplasm arising from mesothelial cells and it is usually
regimen using cis-diamminedichloroplatinum (CDDP) plus peme-
trexed has shown some improvement, the median survival is
generally only 9–12 months.(6–9) The potentially curative approach
to MPM seems to be extrapleural pheumonectomy (EPP) at an
early stage, followed by chemotherapy and radiotherapy (trimodality
treatment). Sugarbaker et al. reported that the median survival
after EPP is 19 months and the 2- and 5-year survival rates are
38 and 15% respectively.(9) However, the perioperative mortality of
EPP has been reported to be as high as 11%.(9) No randomized
controlled study between EPP and an alternative treatment has
been carried out because of the limited number of MPM patients
at stages I and II.
Tumor-infiltrating B lymphocytes (TIB) in lung cancer produce
antibodies against tumor-associated antigens and the antibody
titer against certain antigens is useful for evaluation of the
outcome of anticancer treatments.(10–12) Therefore, the present
study focused on the roles of TIB in MPM. The present study
was designed to obtain tumor-specific antibodies derived from
TIB of malignant mesothelioma by using a xenotransplanted
SCID mouse model.(12,13) TIB-derived IgG was applied to the
serological identification of antigens using the recombinant
expression cloning method (SEREX) in order to identify tumor
antigens associated with MPM and to discover “diagnostic
biomarkers” useful for diagnosis and/or evaluation of the effect
of treatment for MPM.
Materials and Methods
The study protocol was approved by the Human and Animal
Ethics Review Committee of the University of Occupational and
Environmental Health, Japan, and written informed consent was
obtained from each patient regarding the usage of surgical
specimens and sera.
Patients. Eighteen patients with MPM listed in Table 1 were
treated in our hospital. Among the 18 patients with MPM, 17
patients were male and one was female. The average age was
65.9 years (range 54–82 years). According to the histological
classification, eight patients were epithelioid type, six were
sarcomatoid type, and four were biphasic type. According to the
international MPM staging system,(14) three patients were at
stage I, four were at stage II, eight were at stage III, and
three were at stage IV. The sera of the 18 patients with MPM
were obtained before any treatment. Among them, nine patients
1To whom correspondence should be addressed.
Abbreviations: CT, threshold cycle number; EPP, extrapleural pneumonectomy; IFT-
88, intraflagellar transport 88 homolog; MPM, malignant pleural mesothelioma;
SEREX, serological identification of antigens by a recombinant expression closing
method; STUB, stress-induced-phosphoprotein 1 homology and U-box containing
protein; TIB, tumor-infiltrating B lymphocytes; THBS, thrombospondin; TOPIIβ,
Shigematsu et al.
Cancer Sci|July 2009|vol. 100| no. 7| 1327
© 2009 Japanese Cancer Association
were subjected to EPP. The sera of 47 patients with lung cancer
were also collected and stored at –20°C until use. Among them,
27 had a history of asbestos exposure in the work place and 20
did not. The sera of 16 patients with breast cancer were also
collected. Healthy donor sera were collected from 25 volunteers
aged from 26 to 44 years as a control.
Engraftment of human mesothelioma tissue into SCID mice.
Female SCID mice (6 weeks old) were purchased from Charles
River Japan (Tokyo, Japan) and were maintained in specific
pathogen-free conditions throughout the study. Approximately
5 × 5 × 5 mm of the fresh mesothelioma tissue specimens of
patient no. 1 and 2 were engrafted subcutaneously into the
lateral flank of the SCID mice. Blood from the SCID mice was
obtained by retro-orbital venipuncture and the tumor volume
was measured every 2 weeks. The serum was collected and
pooled at –20°C after being centrifuged at 1500g for 10 min at
20°C. The SCID mice were killed at 14 weeks after engraftment
and whole blood was then collected to obtain the serum.
Tumor tissue specimens and cell lines. The two mesothelioma
tissue specimens (M1 and M2) engrafted into SCID mice were
grown in SCID mice. One mesothelioma cell line designated as
K921MSO was established from patient M4. The method for
establishing the tumor cell line has been described previously.(15)
This cell line was cultured in complete culture medium consisting
of RPMI-1640 (Gibco-BRL, Grand Island, NY, USA) supplemented
with 10% heat-inactivated fetal calf serum (Equitech-Bio,
Ingram, TX, USA), 10 mmol/L HEPES, 100 units/mL penicillin
G, and 100 mg/mL streptomycin sulfate.
Immunohistochemical staining for B lymphocytes. Immunohisto-
chemical staining of B lymphocytes infiltrating into the tumor
tissue was carried out using peroxidase-conjugated mouse
monoclonal antihuman CD20 antibody (L26; Dako Japan,
Tokyo, Japan), as described previously.(10) Briefly, the formalin-
fixed paraffin-embedded tissue sections were deparaffinized,
dehydrated, and incubated with 3% hydrogen peroxidase in
distilled water for 5 min. They were rinsed with distilled water
and placed in TBS for 5 min, incubated with L26 diluted with
TBS 1 : 50 for 60 min at room temperature, and then the labeled
Streptavidin Biotin Kit (Dako Japan) was used. Thereafter,
the slides were washed with water, dehydrated, mounted, and
RNA extraction and construction of the cDNA library. mRNA from
the mesothelioma tissue from patient M1 and the mesothelioma
cell line K921MSO were prepared using the Fast Track mRNA
Extraction Kit (Invitrogen, Carlsbad, CA, USA). A cDNA
library was constructed in a λ-ZAP Express vector, using a
cDNA library kit (Stratagene, La Jolla, CA, USA). Wild-type λ-
ZAP Express bacteriophage were amplified in Escherichia coli
XL1 Blue MRF (Stratagene) by overnight propagation of
5000pfu on NZY agar plates (ForMedium, Norfolk, UK). A 12-mL
aliquot of sphingomyelin buffer (5.8 g NaCl, 2 g MgSO4–7H2O,
20 mM Tris-HCl [pH 7.5], 0.01% gelatin) was then added to the
plates, which were gently agitated at 4°C overnight in a Bio-Shaker
(Taitec, Saitama, Japan). The resultant suspension was collected
and centrifuged at 500g for 10 min at 20°C, and the supernatants
were collected and stored at 4°C. The established libraries
contained 5 × 105 recombinant cDNA each, and were screened
for the existence of antibody by SEREX.
SEREX method. In order to identify the antigens recognized by
the IgG from TIB, the amplified cDNA expression library was
screened using the sera from the SCID mice with engrafted
tumors from M1 and M2 patients. The serum was incubated
overnight at 4°C with nitrocellulose membranes containing
phage plaques at a density of 10 000 pfu per plate. After
washing, the membranes were incubated with a 1 : 1000 dilution
of peroxidase-conjugated, Fc fragment-specific goat antihuman
IgG (Jackson Immunoresearch, West Grove, PA, USA) for 1 h at
room temperature and the reactive phage plaques were then
visualized using 3-3′-diaminobenzidine.
Sequence analysis of the reactive cDNA clones. Immunoreactive
cDNA was subcloned, purified, and excised to the pBK-CMV
Phagemid Vector (Stratagene). Plasmid DNA was prepared
using the Wizard Miniprep DNA purification system (Promega,
Madison, WI, USA). The inserted DNA was evaluated by
EcoRI–XhoI restriction mapping and each cDNA insert was
sequenced. The sequencing reactions were carried out using
ABI PRISM 3100 (PE Biosystems, Tokyo, Japan). DNA
sequence alignments were carried out using BLAST (http://
www.ncbi.nlm.nih.gov/BLAST/) on the EMBL and GenBank
Reverse transcription of the antigen-coding gene in mesothelioma
and normal tissues. Individual total RNA from the frozen
Table 1.Characteristics of patients with malignant pleural mesothelioma
PatientAge (years) SexStage TreatmentHistology Autoimmune
M1 and M2 patients with malignant mesothelioma engrafted in SCID mice were studied.
M1–M18 patients had blood samples taken in order to analyze their antibody titers against the identified antigens.
EPP, extrapleural pneumonectomy; ITP, idiopathic thrombocytopenic purpura; SC, supportive care.
© 2009 Japanese Cancer Association
malignant mesothelioma tissue specimens from patients (M1–M5,
M7, M10, M14, M15) and K921MSO and L324MSO cell lines
was obtained using the RNeasy plus mini kit (Qiagen Science,
German Town, MD, USA). The commercially available panel of
total RNA obtained from the 20 normal tissues including adrenal
gland, bone marrow, brain, fetal brain, fetal liver, heart, kidney,
liver, lung, placenta, prostate, salivary gland, skeletal muscle,
testis, thymus, thyroid gland, trachea, uterus, colon, and spinal
cord (Clontech Laboratories, Palo Alto, CA, USA) was used as
normal controls. RNA was converted to cDNA using a First
Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech,
Tokyo, Japan). The cDNA were used as templates for PCR
amplification. Gene-specific oligonucleotide primers were
designed for each DNA segment at the estimated proper melting
temperature. Gene-specific primers were synthesized commercially
by Hokkaido System Science (Sapporo, Japan). Four antigens
were identified by the SEREX method as shown in Table 2. All
primers for the four antigens detected were designed with
Primer Express (Applied Biosystems, Foster, CA, USA) as
shown in Table 3. The expected sizes of the PCR products were
251, 851, 851, and 500 bp for BAC clone CIT987SK-A-328A3
(AC002301) (designated as Gene-X by us; clone 1), THBS-2
(clone 2), STUB-1 (clone 3), and IFT-88 (clone 4) respectively.
Each PCR amplification contained 2.5 μL cDNA supplemented
with 2.5 μL of 10× PCR buffer (Takara Bio, Shiga, Japan), 1 μL
of 10 mmol/L dNTP, 0.5 μL each of a 20-mmol/L solution of
primers, 1 unit of Taq DNA polymerase, and water added to a
total volume of 25 μL. The mixture was heated to 94°C for
5 min. Amplification was then carried out in a thermal cycler
(Biometra, Göttingen, Germany) with 30 cycles for all antigens.
The cycles of amplification for clones 1, 2, and 4 were: 1 min at
94°C, 1 min at 65°C, and 1 min at 72°C; and for clone 3, 1 min
at 94°C, 1 min at 63°C, and 1 min at 72°C. The PCR products
were extended in a final step of 10 min at 72°C. The integrity
and quantity of the cDNA were determined by comparison with
β-actin levels. The PCR products were analyzed by 1.5% gel
electrophoresis and ethidium bromide visualization.
Quantitative RT-PCR of the antigen-coding gene. Quantitative RT-
PCR was carried out using the ABI PRISM 7000 (Applied
Biosystems). The relative amounts of Gene-X, THBS-2, STUB-
1, and IFT-88 mRNA were measured by means of detection of
intercalated SYBR green. PCR was done with 10 μL of SYBR
Green PCR Master Mix (Applied Biosystems), 1 μL of cDNA,
and each primer set described below in a total volume of 20 μL.
The PCR cycles were 95°C for 10 min, followed by 45 cycles of
95°C for 15 s and 60°C for 1 min. The primer sequences of
the four genes for quantitative RT-PCR are shown in Table 3.
The quantitative PCR primer sequences of β-actin were
GGCATCGTGATGGACTCCG and GCTGGAAGGTGGACA-
GCGA. The concentration of each primer set was 200 nmol/L
for the identified gene and β-actin. The CT was defined as the
fractional cycle number at which the amount of amplified target
product reached a fixed threshold. ΔCT was calculated by
comparing the proportion of the CT between the identified
antigen and β-actin in the same amount of each templete.
Relative quantification was achieved by comparisons with the
ΔCT of normal lung. The relative expression was calculated
using the following formula: relative expression = 2 – (ΔCT
sample – ΔCT normal lung).
Analysis of the antibody titer against antigens detected in the sera
of patients. To determine the antibody titer against immunoreactive
clones, a phage plaque assay was carried out as described
previously.(10,13) Briefly, the phage from a positive clone were
mixed with irrelevant phage as an internal negative control at a
ratio 1:1 and then transfected into E. coli. Plaques were blotted
onto nitrocellulose membranes and washed. Next, they were
Table 2.Antigen-coding genes identified by IgG derived from tumor-infiltrating B lymphocytes from xenotransplanted SCID mice
cDNA clone MPM patient Homology search Function of the gene
STIP1 homology and U-box containing protein 1 (STUB1)
Intraflagellar transport 88 homolog (IFT-88)
Ubiquitin-protein ligase activity
Table 3.Specific primers of identified antigens for RT-PCR
Gene PrimersSize (bp)Annealing (°C)
Gene-X5′-GAG GCA AGA GAA TCA CAT GAA TCC-3′
5′-GCT GTA CTG ATC CTT TTC TCC TGA A-3′
5′-GGC GCA TCT AAC GCG TAT CT-3′
5′-CAG CTC CAC ACG CAA AAA AG-3′
5′-TAC ACC AAC CGG GCC TTG T-3′
5′-CCA GAG TCC AAC AGC AGA ACT TG-3′
5′-CGG CTA GAT GAG GCT TTG GA-3′
5′-TGT GCA GAG ACG AAC TAA GAA ACG-3′
Gene-X5′-AGT CCA GGG CTC CTG CTG AA-3′
5′-TGG AAG CTC CGA CCG ACA T-3′
5′-CTT TGA GGT TGA TCG TTG TGT TGT-3′
5′-TTC GTT GGT CTC GGG AAT ACA-3′
5′-GGC CAA GCA CGA CAA GTA CAT-3′
5′-GCT GAT CTT GCC ACA CAG GTA GT-3′
5′-GAA ACT TCA CGC AAT CCT ACG A-3′
IFT88 111 60
Shigematsu et al.
Cancer Sci| July 2009| vol. 100| no. 7| 1329
© 2009 Japanese Cancer Association
incubated with sera at 1:200, 1:1000, 1:5000, and 1:25 000
dilutions with TBS containing 0.02% NaN3, which had been
incubated at 4°C over 16 h. Before these assays, patients’ sera
were incubated with membranes blotted with irrelevant phage
plaques at 4°C over 16 h in order to remove IgG against E. coli.
The spots were evaluated as positive only if the tested clones
were clearly distinguishable from the negative phage.
Establishment of ELISA for antibodies against Gene-X and THBS-2.
Three 20-mer peptides were predicted with a computed algorithm
system on the basis of their hydrophilicity, probability of surface
exposure, flexibility, and antigenic index from the base sequence
of the open reading frame of Gene-X and THBS-2, each peptide
synthesized with over 95% purity was purchased from BEX Co.,
Ltd. (Tokyo, Japan) as shown in Figure 1. Each synthesized
peptide of Gene-X and THBS-2 was diluted in PBS and
coated onto a flat-bottomed 96-well ELISA plate (Iwaki, Asahi
Techno Glass, Chiba, Japan) at 4°C overnight. After serial
dilution of the peptides, the optimal peptide concentrations of
Gene-X and THBS-2 were decided to be 1 μg/mL and 100 ng/mL
respectively. The plates were washed with PBS in Tween and
blocked with 1% BSA in PBS at room temperature for 1 h. After
washing three times, the patient sera (diluted 1/100, 1/500, and
1/2500 in 1% BSA in PBS) were added and incubated at 4°C
overnight to assure the correlation of the antibody titers between
the phage plaque assay and ELISA. The plates were washed and
incubated with the secondary antibody, horseradish peroxidase-
conjugated goat antihuman IgG (H + L-chain) (Medical and
Biological Laboratories, Nagoya, Japan) at 1/4000 dilution for
5 h at 4°C. The plates were washed and incubated with
tetramethylbenzidine substrate solution (1,2-phenylenediamine
dihydrochloride) (Thermo Fisher Scientific, Waltham, MA, USA)
for 20 min at room temperature. After the addition of 0.18 M
H2SO4 (100 μL), the absorbance was determined with an iMark
Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules,
CA, USA) at 450 nm.
Immunohistochemical detection of B cells in the tumor specimens.
The tumor specimens from two patients with malignant
mesothelioma were engrafted into SCID mice. The mesothelioma
from the M1 patient was diagnosed pathologically as sarcomatoid
type and M2 as epithelioid type. To confirm the infiltration of B
lymphocytes, the mesothelioma specimens were stained with
anti-CD20 antibody. Photomicrographs of the infiltration of
CD20-positive B lymphocytes are shown in Figure 2.
Human IgG production in the SCID mice transplanted with tumor
tissue. The human IgG level in the sera of SCID mice and
volume of the engrafted tumors were measured every 2 weeks.
The human IgG levels increased gradually and reached a peak
level at 4 and 8 weeks after transplantation of M2 and M1,
respectively, but declined gradually thereafter. The peak level
of human IgG of M1 and M2 were 570 and 660 μg/mL
respectively. The transplanted tumors grew up on the lateral
flanks of the mice.
Identification of immunoreactive cDNA clones by SEREX. The cDNA
libraries of the mesothelioma tissue (M1) were screened by
the sera from the SCID mice xenotransplanted with M1 and the
with a computed algorithm system on the basis
of hydrophilicity, probability of surface exposure,
flexibility, and antigenic index from the base
sequence of the ORF
thrombospondin (THBS)-2, and synthesized and
purified commercially to over 95%. The identified
antigen in this study was located downstream of
the registered ORF of THBS-2. The identified
second ORF of THBS-2 was located from 4506 to
4719 bp, and the prediction of antigenic peptide
of THBS-2 was carried out in the second ORF.
Three 20-mer peptides were predicted
of Gene-X and
thelioma specimens obtained from patients M1
and M2. B lymphocytes were infiltrated in tumor
tissues. Left, hematoxylin–eosin staining; right,
immunohistochemical staining with anti-CD20
Photomicrograph of malignant meso-
© 2009 Japanese Cancer Association
cDNA libraries of the mesothelioma cell line K921MSO were
screened by the sera from the SCID mice xenotransplanted with
M2 tissue (dilution 1:200). Four positive cDNA clones were
identified. On the basis of the DNA sequence analysis, DNA
homology search of these genes was carried out using BLAST
on the EMBL and GenBank databases and the four genes were
identical to Gene-X, THBS-2, STUB-1, and IFT-88. Gene-X
and THBS-2 were identified from the serum of SCID mice
xenotransplanted with the mesothelioma tissue from patient M1.
STUB-1 and IFT-88 were identified from the serum of SCID
mice xenotransplanted with the mesothelioma tissue from patient
M2 (Table 2). THBS-2 is known as a potent inhibitor of tumor
growth and angiogenesis and STUB-1 is as a ubiquitin-protein
ligase. The function of the other two genes (Gene-X and IFT-88)
is unknown. Expression of the four genes was examined by
RT-PCR in the five mesothelioma tissues and two mesothelioma
cell lines. Gene-X was expressed in the autologous M1 and the
allogeneic M2 mesothelioma, and STUB-1 was expressed in the
autologous M2 and the allogeneic M1, M4, and M5 mesotheliomas
and the two cell lines. THBS-2 and IFT-88 were also expressed
in all of five allogeneic mesothelioma and the two cell lines.
(Fig. 3a). The expression of these antigen-coding genes in
normal tissues is shown in Figure 3(b). Gene-X was expressed
in the liver, testis, thyroid gland, and colon. THBS-2 was
expressed in the brain, heart, and spinal cord. STUB-1 was
expressed in the adrenal gland, brain, fetal brain, heart, kidney,
and skeletal muscle. IFT-88 was expressed in the heart, kidney,
testis, and thyroid gland. Quantitative RT-PCR data of the four
genes are shown in Figure 3(c), when the expression level of the
normal lung was estimated as 1.0. Gene-X was expressed in
three of nine MPM tissues over 15.7-fold of normal testis,
which was highest expressed in normal tissues. THBS-2 was
expressed in seven of nine MPM tissues over 10.1-fold of
normal heart, which was highest expressed in normal tissues.
However, STUB-1 and IFT-88 were not expressed in any MPM
over 7.8-fold of normal heart or 8.8-fold of normal testis,
respectively, which were highest expressed in normal tissues.
Antibody titers of Gene-X and THBS-2 among patients with
mesothelioma, lung cancer, and breast cancer. The titer of antibody
against the four antigens was evaluated by a phage plaque assay.
Detection rates of antibodies against the four antigens evaluated
by phage plaque assay are shown in Table 4. Among the four
antigens, antibodies were frequently detected against Gene-X
and THBS-2. Therefore, ELISA systems to detect antibodies
against these two genes were constructed. As shown in Figure 4,
the antibody titers the antibody titers were examined by the
ELISA system against 6 synthesized peptides of Gene-X and
THBS-2. As shown in Figure 4, the antibody titers of Gene-X40–59
and THBS-226–45 were well correlated with the results by phage
plaque assay. Therefore, antibody titers against Gene-X40–59 and
THBS-226–45 were evaluated quantitatively in 25 healthy persons.
The cut-off values of antibody titers were decided on the basis
of the average plus two times the standard deviation of the
healthy persons. The estimated cut-off value of Gene-X40–59 was
0.414 and the value of THBS-226–45 was 0.079. On the basis of
these cut-off values, the antibody against Gene-X was decided as
positive in 10 of 18 (55.6%) MPM patients and 0 of 25 (0%)
healthy donors. The antibody against THBS-226–45 was decided
as positive in 16 of 18 (88.9%) MPM patients and 2 of 25
(8.0%) healthy donors (Table 5). The titers of antibodies against
Gene-X40–59 and THBS-226–45 were analyzed in lung cancer patients
with or without asbestos exposure and breast cancer patients
(Fig. 5a,b). Among the 47 patients with lung cancer and 16 patients
with breast cancer, all patients showed negative levels of the
antibodies against Gene-X40–59 and THBS-226–45. In order to evaluate
the effect of surgical resection of MPM on the antibody titers,
postoperative sera of patients M15 and M18 were subjected to
the analyses. The titers of the M15 and M18 patients’ antibodies
against Gene-X40–59 and THBS-226–45 decreased 2–4 months after
surgical resection. The M15 and M18 patients encountered
recurrence of the disease 10 and 9 months after the operation
respectively. The titers of antibody against THBS-2 were increased
at the time of recurrence (Fig. 5c).
The humoral immune response against tumor antigens has been
investigated since the 1970s.(16–19) The present study focused
on the role of TIB from the viewpoint of the tumor-specific
immune response. Previous studies demonstrated that TIB produce
IgG in SCID mice xenotransplanted with human lung cancer
tissue.(10–13,20) TIB-derived IgG was analyzed by the SEREX
method and 22 antigens were identified in a patient with lung
cancer. A mutated p53 was one of the identified antigens and the
change in the antibody titer against mutated p53 was correlated
with the clinical course of the patient.(13) In another patient with
pleomorphic carcinoma of the lung, antibodies against SEREX
defined antigens (MAGE-B2 and two function unknown antigens)
that could be used as tumor markers reflecting the clinical course.(10)
These findings indicated that TIB plays an important role in the
humoral responses against tumor-associated antigens.
By using these IgG antibodies derived from TIB, four anti-
gens (Gene-X, THBS-2, STUB-1, and IFT88) were identified.
Gene-X has 100% homology to the registered gene in GenBank
(accession no. AC002301) that is located on chromosome 16p11
and encodes 251 amino acids. The function of this gene is not yet
known. It was reported that a 26.5-kb gene rich duplication is shared
by human Xq28 and 16p11.(21) The overall nucleotide similarity
within the interchromosomal duplication was found to be 94–97%,
on the basis of a genomic DNA sequence analysis.(22)
Thrombospondin-2 is located on the long arm of chromosome
6 and belongs to the thrombospondin family. It is a disulfide-linked
homotrimeric glycoprotein located at the cell membrane(23,24)
that mediates cell-to-cell and cell-to-matrix interactions. This
protein is known to be a potent inhibitor of tumor growth and
angiogenesis. However, the thrombospondin family, in particular
THBS-2, is reported to be pleiotrophic in function.(25) The
pericellular levels of MMP2 are controlled to a large extent by
THBS-2 (and potentially also by THBS-1) and the elevated
levels of MMP2 are likely to play a role in reduction of cellular
adhesion with abnormal collagen fibril structure and augmentation
of the proliferation of vascular endothelial cells.(25) Therefore,
THBS-2 is intimately involved in the promotion of invasion and
sera of mesothelioma patients and healthy donors by phage plaque
Detection of antibodies against the identified antigens in
Antigen MPM patient Healthy donors
Judged at †1/1500 dilution, ‡1/1000 dilution, §1/200 dilution.
sera of mesothelioma patients and healthy donors by ELISA
Detection of antibodies against the identified antigens in
Antigen MPM patientHealthy donors
Shigematsu et al.
Cancer Sci| July 2009| vol. 100| no. 7| 1331
© 2009 Japanese Cancer Association
mesothelioma tissue. Gene-X was expressed in allogeneic M2 mesothelioma tissue, and stress-induced-phosphoprotein 1 homology and U-box
containing protein (STUB)-1 was expressed in allogeneic M1, M4, and M5 mesothelioma tissues. Thrombospondin (THBS)-2 and intraflagellar
transport 88 homolog (IFT-88) were also expressed in all allogeneic mesothelioma tissues. Underlines are positive bands. (b) RT-PCR expression of
the four antigens in the panel of normal tissues. Lane 1, adrenal gland; lane 2, bone marrow; lane 3, brain; lane 4, fetal brain; lane 5, fetal liver;
lane 6, heart; lane 7, kidney; lane 8, liver; lane 9, lung; lane 10, placenta; lane 11, prostate; lane 12, salivary gland; lane 13, skeletal muscle; lane
14, testis; lane 15, thymus; lane 16, thyroid gland; lane 17, trachea; lane 18, uterus; lane 19, colon; lane 20, spinal cord; P, positive control.
Underlines are positive bands. (c) Quantitative RT-PCR expression of the four antigens in mesothelioma tissue and normal tissues. The bars are fold
expression compared to normal lung tissues. Gene-X was expressed in three of nine malignant pleural mesothelioma (MPM) tissues over 15.7-fold
of normal testis, which was highest expressed in normal tissues. THBS-2 was expressed in seven of nine MPM tissues over 10.1-fold of normal heart,
which was highest expressed in normal tissues. However, STUB-1 and IFT-88 were not expressed in any MPM over 7.8-fold of normal heart and 8.8-
fold of normal testis, respectively, which were highest expressed in normal tissues. M1–M15, MPM tissues. K921MSO and L324MSO are MPM cell
lines. Line 1–20 are normal tissues corresponding to the panel used in Figure 3(b).
RT-PCR expression of the four antigens in mesothelioma and normal tissues. (a) All four antigens were expressed in autologous
1332 doi: 10.1111/j.1349-7006.2009.01181.x
© 2009 Japanese Cancer Association
metastasis of cancer cells. The open reading frame (3510 bp)
encodes a protein of 1170 amino acids with known features of
the THBS-2 subunit.(26) The THBS mRNA 3′-untranslated region
has a role in the regulation of THBS mRNA and the mRNA
stability was observed in cultured cells in response to platelet-
derived growth factor.(27,28) The identified antigen in the present
study was located downstream of the registered ORF of THBS-2.
The ORF of THBS-2 was located from 249 to 3767 bp. The
identified second ORF of THBS-2 was located from 4506
to 4719 bp. It is not known why this small protein is translated
in MPM patients. However, Oyama et al. reported that the
small protein was unexpectedly translated from upstream or
downstream of the registered ORF in a proteomic analysis of an
erythroleukemia cell line K562.(29) Mechanisms including leaky
scanning or sequence such as an internal ribosome entry site
may be associated with the transcription of the variants.(29,30)
There are some reports showing overexpression of THBS-1 in
bladder, breast, and lung cancers.(31–34) THBS-2 protein has a
homologous structure to THBS-1; therefore, the antibody against
THBS-2 may show immunological cross reaction against
THBS-1. However, in the present study, we detected antibody
against the second ORF of THBS-2; therefore, the antibody against
the second ORF of THBS-2 may not cross react with THBS-1.
Futhermore, the antibody titer against THBS-2 was significantly
higher in MPM patients than in lung cancer patients. Because
MPM is sometimes difficult to distinguish from lung cancer, the
antibody titer against THBS-2 might become a useful tumor
marker for making a differential diagnosis.
(a) Gene-X and (b) thrombospondin (THBS)-2
between phage plaque assay and ELISA. The
antibody titers against Gene-X40–59 and THBS-226–45
were well correlated with the results by phage
plaque assay. R = coefficient of correlation. A
significant correlation was considered between
phage plaque assay and ELISA when the P-value
was less than 0.05.
The correlation of antibody titer against
(b) thrombospondin (THBS)-2 according to
malignant pleural mesothelioma (MPM) patients,
normal healthy persons, lung cancer patients
with or without asbestos exposure, and breast
cancer patients. To determine the clinical
significance of these antigens, a comparison of
antibody titers was carried out by ELISA. These
data were analyzed with 1/100 dilutions of the
sera, and the optical density at 450 nm is shown.
The cut-off values of Gene-X and THBS-2 were
determined to be 0.414 and 0.079 respectively.
(c) Postoperative time course of antibody titers
against Gene-X and THBS-2 in patients M15 and
M18. To determine the therapeutic effect before
and after the operation, a comparison of
antibody titers was carried out in patients M15
and M18 by ELISA. The optical densities at
450 nm were analyzed in the two patients. In
both patients, the titers of antibodies against
Gene-X and THBS-2 decreased from 2 to
4 months after surgical resection. The titers of
antibody against THBS-2 were increased at the
time of recurrence.
The antibody titer against (a) Gene-X and
Shigematsu et al.
Cancer Sci| July 2009|vol. 100|no. 7| 1333
© 2009 Japanese Cancer Association
Stress-induced-phosphoprotein 1 homology and U-box con-
taining protein-1 is located on the short arm of chromosome 16.
This protein had been identified using the SEREX method in
patients with colon cancer.(35) STUB-1 protein is also designated
carboxyl terminus of Hsp70-interacting protein (CHIP). Forced
expression of STUB-1 in fibroblasts by transduction of the
STUB-1 gene increased the refolding of proteins under stress
conditions such as thermal denaturation, and inhibition of Hsp70
chaperone activity by ATP depletion abrogated the effects of
STUB-1 on protein folding, indicating that the STUB-1-mediated
events were Hsp70 dependent.(36) STUB-1 is also reported to be
a direct chaperone of wild-type p53 and to maintain p53 in
wild-type conformation under physiological conditions.(37)
Intraflagellar transport 88 homolog is located on the long arm
of chromosome 13 and the function of this gene remains
unknown. This gene belongs to the tetratricopeptide repeat
family. It was reported that mutations of a similar gene in mouse
could cause polycystic kidney disease.(38) Polycystic disease of
the kidney is known to be associated with nephrotic syndrome
with membranous syndrome.(39) IFT-88 was identified by using
the sera of SCID mice engrafted with tumor tissue derived
from patient M2. The M2 patient suffered from membranous
nephropathy and the membranous nephropathy was improved
after an extrapleural pneumonectomy for MPM. Moreover,
membranous nephropathy had been reported to be associated
with MPM.(40,41) The causal relationship between IFT-88 and
membranous nephropathy should therefore be elucidated in a
Among the four genes, Gene-X and THBS-2 were overexpressed
frequently in MPM. Furthermore, antibodies against Gene-X
and THBS-2 were detected in 55.6 and 88.9%, respectively,
of sera of patients with MPM. The antibody titer against these
antigens decreased after surgical resection of MPM. Therefore,
these antibody titers could be useful not only for diagnosis of
MPM but also evaluation of the outcome of therapy.
Mesothelin has been reported to be a new mesothelioma-
associated antigen.(42) It is highly expressed in several cancers
including pancreatic, ovarian, pulmonary adenocarcinoma, and
mesothelioma. Several investigators have reported that a high
level of serum soluble mesothelin is associated with MPM and
it is a promising marker for the diagnosis and clinical monitoring
of MPM.(43) The antibody against mesothelin detected by ELISA
is the IgG isotype in MPM patients, implying that cognate
helper T-cell immunity might be present and activated in
the patients with a respective B-cell response.(44) Mesothelin is
a glycosylphosphatidylinositol-linked membrane protein over-
expressed on the cell surface. Therefore, mesothelin could be a
potential target for antibody-based immunotherapy. A clinical
trial using monoclonal antibodies against mesothelin is underway
for MPM patients.(45)
Robinson et al. have reported eight antigens identified by the
SEREX method using sera of patients with MPM. Among them
TOPIIβ was detected in 13 out of 14 patients with MPM.(46)
Interestingly, the antibody titer against TOPIIβ in patients
with MPM was correlated with poor survival of MPM
In conclusion, the B cells infiltrated in MPM are sensitized
and produce antibody against tumor-associated antigens. Among
the SEREX-defined antigens, the titers of antibodies against
Gene-X and THBS-2 could be used as tumor markers for the
diagnosis of patients with malignant mesothelioma.
This study was supported in part by a University of Occupational and
Environmental Health Research Grant for the Promotion of Occupational
Health and Grant-in-Aid for scientific research from the Ministry of
Education, Culture, Sports, Science, and Technology, Japan. We thank
Yukari Oshibuchi, Misako Fukumoto, Yuki Goto, and Aya Katayama for
their expert technical help.
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