Introduction
Neuronal proteins, which are usually present only
within the nervous system but may also be expressed in malignant
tumors localized outside the nervous system, are assigned to
paraneoplastic (onconeural) antigens (PNA). The immune system of
certain patients responds to PNA by producing specific
autoantibodies and/or cytotoxic T-cells that trigger the
development of a paraneoplastic neurological syndrome (PNS)
(1). One such PNA is the
photoreceptor Ca2+-binding protein recoverin (2,3), which
is normally specific to the retina and the pineal gland but may
also be aberrantly expressed in malignant tumors of the lung and
other tissues (4,5). In certain patients with lung cancer,
high-titer serum autoantibodies against recoverin (AAR) trigger the
development of a PNS, cancer-associated retinopathy (CAR) (6,7).
However, low-titer serum AAR of patients with lung cancer do not
necessarily cause CAR syndrome (8–10).
Instead, this syndrome only occurs in approximately 1% of cancer
patients, whereas the frequencies of serum AAR in patients with
small cell lung carcinoma (SCLC) and non-small cell lung carcinoma
(NSCLC) equal 15 and 20%, respectively (10).
By analogy with cancer-testis antigens, recoverin
has been classified as a cancer-retina antigen (5) for the following reasons: i) its normal
expression is restricted to an immunoprivileged tissue (retina),
ii) it is aberrantly expressed in tumor cells, iii) it subsequently
demonstrates high antigenicity (autoantibodies and/or T-cells), and
iv) aberrant demethylation of the gene promoter is involved in the
aberrant expression of recoverin (11).
In this study, we analyzed serial specimens of blood
sera of patients with a number of different malignancies to
estimate the frequency of serum AAR in oncological diseases other
than lung cancer. Since the ELISA method yields a number of
false-positive signals (12), serum
AAR were detected by immunoblotting, using recombinant recoverin as
an antigen.
Materials and methods
Collection of patient biospecimens
The analysis for the presence of AAR was performed
on the peripheral blood of 576 patients with various oncological
diseases and certain other disorders, obtained from the Institute
of Clinical Oncology at the N.N. Blokhin Russian Oncological
Scientific Centre. The oncological diseases consisted of cancers of
the breast (94 specimens), ovary (40 specimens), uterus (41
specimens), neck of the uterus (40 specimens), thyroid gland (16
specimens), stomach (41 specimens), colon (57 specimens), esophagus
(9 specimens), pancreas (25 specimens), testis (13 specimens),
bladder (21 specimens), kidney (15 specimens), and liver (12
specimens) as well as skin melanoma (33 specimens), uterine sarcoma
(2 specimens) and esophageal sarcoma (1 specimen). The benign
tumors comprised tumors of the breast (1 specimen of papilloma and
5 specimens of fibroadenoma), ovary (adenoma, 15 specimens;
fibroma, 7 specimens; Brenner's tumor, 1 specimen; and thecoma, 2
specimens), uterus (polyposis, 7 specimens; and myoma, 20
specimens), neck of the uterus (polyposis, 1 specimen), skin
(fibrous polyp, 1 specimen; keratopapilloma, 1 specimen; and
dermatofibroma, 1 specimen), thyroid gland (adenoma, 3 specimens),
and bladder (papilloma, 1 specimen). Non-neoplastic diseases were
represented by mastopathy (7 specimens), endometriosis and ovarian
cyst (11 specimens), atypical hyperplasia of the uterus (4
specimens), dysplasia of the neck of the uterus (2 specimens), a
cyst of the thyroid gland (2 specimens), nodular goiter (3
specimens), thyroiditis (1 specimen), a malignant ulcer of the
stomach (2 specimens), pancreatitis (4 specimens), hyperplasia (1
specimen) and a cyst (6 specimens) of the pancreas, hepatocirrhosis
(4 specimens), and dropsy of the testis (1 specimen).
Cell cultures and tissues
Established breast adenocarcinoma cell lines (MSF7,
MDA435, MDA231, SKBR-2, BT-20 and HS578T), colon carcinoma cell
lines (Colo320, CaCo2, CX-2, HT29 and SW948), leukemia cell lines
(ARA-10, Jurkat and K562), and cutaneous T-cell lymphoma cell lines
(CTCL) (SeAx, MyLa, HuT-78 and HH) from the Skin Cancer Unit at the
German Cancer Research Center, Heidelberg, Germany were cultivated
in RPMI-1640. The media were supplemented with 10% fetal calf
serum, L-glutamine (2 mM) and penicillin/streptomycin solution (5
U/ml), and were purchased from PAA Laboratories (Coelbe, Germany).
Cell lines were cultivated at 37°C and 5% CO2. Breast
carcinoma tissues were provided by Dr Hildenbrand, Department of
Pathology, University Hospital Mannheim, Germany. Head and neck
cancer tissues were provided by Professor Götte, Department of
Otolaryngology, Head and Neck Surgery, University Hospital
Mannheim, Germany. CTCL tissues were obtained from the Skin Cancer
Unit at the German Cancer Research Center, Heidelberg, Germany.
Obtaining of recoverin and antibodies
against recoverin
Myristoylated recoverin was heterologously expressed
in E. coli and purified as previously described (13). The purity of the preparation
obtained was estimated by SDS-polyacrylamide gel electrophoresis,
with subsequent silver staining. The recoverin preparation was then
used as an antigen in the Western blot analysis. Affinity-purified
rabbit polyclonal anti-recoverin antibodies were prepared as
previously described (2,3) and then used as a positive control in
the Western blot analysis.
Western blot analysis
Western blotting of patient sera was performed as
previously described (10,14). Briefly, following SDS
electrophoresis, recoverin (2 μg per track) was electrotransferred
to nitrocellulose membranes, the non-specific sites were saturated
by incubation with 10% delipidated dry milk for 1.5 h and then
membranes were incubated with patient sera for 12 h or with
affinity-purified rabbit polyclonal anti-recoverin antibodies (1
μg/ml) as a positive control. Blots were rinsed three times,
incubated for 1.5 h with sheep anti-human IgG peroxidase conjugate
(GE Biosciences, Amersham, UK) at a dilution of 1:1000, then rinsed
again and finally incubated with 10 mM 3,3′-diaminobenzidine (as a
substrate) in 0.01% hydrogen peroxide.
RT-PCR
Total RNA (1 μg) isolated from tumor tissues or cell
lines was reverse-transcribed with a first strand cDNA synthesis
kit at 42°C for 50 min according to the manufacturer's instructions
(Roche Diagnostics, Indianapolis, IN, USA). To perform PCR
amplification, 1 μl RT-reaction mixture was added to the solution
containing 50 pmol sense and antisense primers (total volume of 25
μl). Following the initial incubation at 94°C for 2 min, 33
amplification cycles (Tm=63) were carried out for the
recoverin gene, and 21 cycles (Tm=57) for the
housekeeping gene GAPDH. Amplified PCR products (394 bp for
recoverin and 638 bp for GAPDH) were subjected to electrophoresis
on 1.5% agarose gels followed by staining with ethidium bromide.
The samples were considered positive if a clear band was visible in
the gel in at least 2 out of 3 independent PCR experiments [for
primer sequences, see (15)].
Ophthalmological investigation
Prior to the investigation, 2% solution of ephedrine
was instilled into the conjunctival sac. The eye fundus was then
examined using direct ophthalmoscopy. Visual field limits were
determined using a hemispheric perimeter.
Results
AAR in sera of patients with malignant
tumors
We screened serial serum specimens of patients with
various oncological diseases to estimate the occurrence of AAR,
using recombinant recoverin as an antigen. Table I shows the results of the screening.
Serum AAR were detected in the patients with melanoma (at a
frequency of ~6%) and cancers of the ovary (10%), neck of the
uterus (~8%), skin (~6%), breast, uterus, stomach and bladder (each
~5%). Representative Western blots are shown in Fig. 1. Serum AAR were also found in
patients with epidermoid carcinoma of the esophagus and seminoma.
However, these frequencies could not be calculated as the number of
available specimens was <20. It is significant that none of the
serum specimens from healthy individuals exhibited the presence of
AAR.
 | Table IAAR in sera of patients with various
oncological diseases of different localization and etiology. |
Table I
AAR in sera of patients with various
oncological diseases of different localization and etiology.
| Tumor
localization | Total number of
specimens | Number (percentage)
of AAR-positive sera |
|---|
| Breast cancer,
including: | 94 | 5 (~5%) |
| Infiltrative ductal
carcinoma | 50 | 2 |
| Infiltrative lobular
carcinoma | 12 | 0 |
| Mixed
(lobular-ductal) carcinoma | 7 | 1 |
| Papillary
carcinoma | 3 | 0 |
| Medullary
carcinoma | 2 | 0 |
| Undefined
histotypea | 20 | 2 |
| Ovary,
including: | 40 | 4 (10%) |
| Adenocarcinoma | 29 | 3 |
| Low-grade
differentiated adenocarcinoma | 3 | 1 |
| Teratoma | 3 | 0 |
| Undefined
histotypea | 5 | 0 |
| Uterus,
including: | 43 | 2 (~5%) |
| Adenocarcinoma | 36 | 0 |
| Low-grade
differentiated carcinoma | 2 | 1 |
| Epidermoid
carcinoma | 3 | 0 |
| Sarcoma | 2 | 1 |
| Neck of uterus,
including: | 40 | 3 (~8%) |
| Adenocarcinoma | 3 | 0 |
| Epidermoid
carcinoma | 28 | 1 |
| Undefined
histotypea | 9 | 2 |
| Skin, including: | 36 | 2 (~6%) |
| Melanoma | 33 | 2 |
| Squamous cell
carcinoma of skin | 3 | 0 |
| Thyroid gland,
including: | 16 | 0 |
| Papillary
carcinoma | 12 | 0 |
| Medullary
carcinoma | 4 | 0 |
| Stomach,
including: | 42 | 2 (~5%) |
| Adenocarcinoma | 27 | 2 |
| Signet ring cell
carcinoma | 10 | 0 |
| Angiosarcoma | 4 | 0 |
| Undefined
histotypea | 1 | 0 |
| Colon,
adenocarcinoma | 40 | 0 |
| Esophagus,
including: | 10 | 1 |
| Epidermoid
carcinoma | 7 | 1 |
|
Adenocarcinoma | 1 | 0 |
| Sarcoma | 1 | 0 |
| Undefined
histotypea | 1 | 0 |
| Pancreas,
including: | 15 | 0 |
|
Adenocarcinoma | 8 | 0 |
| Carcinoid | 1 | 0 |
| Undefined
histotypea | 6 | 0 |
| Male genital
system, seminoma | 13 | 1 |
| Bladder,
including: | 21 | 1 (~5%) |
| Transitional cell
carcinoma | 18 | 0 |
| Signet ring cell
carcinoma | 1 | 1 |
| Undefined
histotypea | 2 | 0 |
| Kidney,
including: | 10 | 0 |
| Renal cell
carcinoma | 8 | 0 |
| Undefined
histotypea | 2 | 0 |
| Liver,
hepatocellular carcinoma | 7 | 0 |
| Melanoma | 125 | 8 (~6%) |
| Lung,
including: | 143 | 24 |
| Small cell lung
carcinoma (10) | 99 | 15 (~15%) |
| Non-small cell
lung carcinoma (10) | 44 | 9 (~20%) |
It is of note that the presence of serum AAR in
patients with cancers of the neck of the uterus, stomach and
bladder, seminoma and epidermoid carcinoma of the esophagus was
demonstrated for the first time. According to our previous studies
(bottom of Table I), the frequency
of serum AAR in patients with SCLC and NSCLC (10) equals ~15 and ~20%, respectively.
Determination of the titers of AAR in the sera of 23
patients with various malignant tumors yielded varying values
ranging between 1:20 and 1:320. These values are far lower than
those determined previously (7) for
patients with CAR syndrome, in which the titer varied from 1:3000
to 1:6000. In the present study, five AAR-positive patients with
cancers of the breast, ovary, stomach and bladder were available
for ophthalmological investigation. Of these, only one patient with
ovarian adenocarcinoma had narrowing of the visual field and loci
of degeneration in the retina, indicating the presence of CAR
syndrome in the patient.
AAR in sera of patients with benign
tumors and non-neoplastic diseases
Patients with a number of benign tumors were
examined, including tumors of the breast (papilloma and
fibroadenoma), ovary (adenoma, fibroma, Brenner's tumor and
thecoma), uterus (polyposis and myoma), neck of the uterus
(polyposis), skin (fibrous polyp, keratopapilloma and
dermatofibroma), thyroid gland (adenoma), and bladder (papilloma).
In all of these cases, AAR were found only in the patients with
benign tumors of the uterus with a frequency of ~7%. None of the
serum specimens of patients with the following non-neoplastic
diseases demonstrated the presence of AAR: mastopathy,
endometriosis, ovarian cyst, atypical hyperplasia of the uterus,
dysplasia of the neck of the uterus, cyst of the thyroid gland,
nodular goiter, thyroiditis, malignant ulcer of the stomach,
pancreatitis, hyperplasia and cyst of the pancreas, hepatocirrhosis
and dropsy of the testis.
mRNA for recoverin is aberrantly
expressed in different tumor types
We additionally analyzed the expression of mRNA for
recoverin (Rc mRNA) in tissues and/or cell lines of carcinomas of
the breast, colon and head and neck. In addition, we aimed to
establish whether the recoverin gene was capable of being expressed
in non-solid tumors, such as leukemia and cutaneous T-cell
lymphoma. Representative electropherograms of RT-PCR tumor samples
are shown in Fig. 2. Table II shows that the Rc mRNA expression
was detected in each tumor and cell line tested, with the exception
of carcinoma of the head and neck and leukemia.
| Table IIExpression of mRNA for recoverin (Rc
mRNA) expression in tumor tissues and cell lines. |
Table II
Expression of mRNA for recoverin (Rc
mRNA) expression in tumor tissues and cell lines.
| Type of tumor | Type of tumor
specimens | Number of specimens
(Total/Rc mRNA-positive) |
|---|
| Breast
adenocarcinoma | Cell lines | 12/9 |
| Tissue | 14/8 |
| Colon (without
typing) | Cell lines | 5/1 |
| Head and neck
carcinoma | Tissue | 5/0 |
| Leukemia | Cell lines | 3/0 |
| Cutaneous T-cell
lymphoma | Cell lines | 4/3 |
| Tissues | 5/2 |
Discussion
In our previous studies (8–10), we
concluded that AAR was detected in the sera of patients with lung
cancer who had no manifestation of CAR syndrome. The present study
confirms that this conclusion is also true for other malignancies,
including cancers of the breast, stomach and bladder. A similar
situation occurs in the case of Hu antigens, one of the most
extensively studied groups of PNA (16). Autoantibodies against Hu antigens
underlie a multifocal neurological disease, paraneoplastic sensory
neuropathy. The frequency of this PNS in SCLC patients with high
titers of anti-Hu and corresponding PNS is only 1% (17). However, SCLC patients with low
titers of anti-Hu and without PNS have also been found. The
frequency for such cases was in the range of 15–20% (18), which is much higher than that of the
high-titer anti-Hu cases. The presence of anti-PNA antibodies in
patients without PNS has also been demonstrated for Yo and Ri
antigens, but in that case autoantibodies were detected in 2–4% of
the patients only (19).
To generate AAR, tumor cells should express Rc mRNA
and recoverin protein. The expression of AAR at the mRNA and/or
protein levels has been shown for tumor tissues and/or cell lines
in the case of lung tumors (7–10),
malignant melanoma (15,20) and breast, endometrial and cervical
carcinomas (14). Recently, the
recoverin-positive immune reaction was demonstrated for brain
glioma and glioblastoma, squamous cell carcinoma, and carcinomas of
the esophagus, stomach, colon, rectum, pancreas, kidney, prostate,
ovary, neck of the uterus and breast; in addition, the ability of
lung tumors to express recoverin was confirmed (21). In this study, we found for the first
time the expression of Rc mRNA in cutaneous T-cell lymphoma and
confirmed that Rc mRNA is expressed in carcinomas of the breast and
colon. Rc mRNA expression was, however, not detected in carcinoma
of the head and neck or in leukemia. Therefore, the aberrant
expression of recoverin is not only a feature of tumors of
neuroendocrinal and epithelial origin, but can also be an attribute
of tumors of mesothelial origin.
We concluded in our previous study (10) that the frequency of recoverin
expression in lung tumors is higher than that of the AAR-positive
cases in lung cancer patients and much higher than that of CAR
syndrome patients. It is possible that this conclusion is also true
for a number of other cancer types. If the maximal AAR frequency in
patients with cancers determined thus far does not exceed 20%
(10), the frequency of the
recoverin expression in various recoverin-positive malignant tumors
generally varies between 30% (21)
and 85% (10). It is of note that,
to the best of our knowledge, normal tissues do not express
recoverin at the protein level. Brain glioblastoma appears to be
exempt from this rule (21).
However, in this case normal tissues were obtained from the brain
area adjacent to the malignant tumors, the cells of which may
contaminate the specimens obtained for the analysis. It should be
added that the recoverin expression at the mRNA level has been
detected in normal human lung, brain, thymus, uterus and
melanocytes (15).
The frequency of AAR in the patients with various
malignancies determined in this study was below 10% (Table I); thus, these autoantibodies alone
cannot be considered as a reliable marker of cancer. This is also
true even for lung cancer, in which the AAR frequency did not
exceed 20% (10). Nevertheless, the
usefulness of AAR in clinical practice cannot be ruled out,
particularly when used in combination with other tumor markers, as
the symptoms of a PNS (and thus the presence of a PNA) may be
manifested long before the clinical diagnosis of the underlying
tumor, thus enabling the clinician to predict the future
development of a particular cancer (1,22).
Subsequently, it is significant that AAR were absent in the sera of
healthy individuals and patients with non-neoplastic diseases as
well as in the vast majority of patients with benign tumors. It is
of note that in one of the patients with breast cancer, serum AAR
were detected at a titer of 1:40 prior to treatment. However, AAR
disappeared from the serum following surgical removal of the tumor,
which had been a source of the aberrantly expressed recoverin prior
to surgery. A similar effect has previously been described for a
patient with SCLC (8); low-titer
AAR were present in the patient's serum prior to treatment but
disappeared from the blood stream following a course of
chemotherapy.
According to the data obtained in the present study,
the frequencies of serum AAR in patients with various malignancies
other than lung cancer do not exceed 10%. Of the five AAR-positive
patients with various cancers only one had CAR syndrome; thus, the
presence of AAR in patient serum does not necessarily trigger the
development of CAR syndrome. The analysis of Rc mRNA expression in
tumors of different etiology allows us to conclude that the
aberrant expression of recoverin is not a feature of tumors of
neuroendocrinal and epithelial origin only, but can also be an
attribute of tumors of mesothelial origin.
Acknowledgements
We are indebted to E.V. Bragina for the assistance
in the preparation of this article. This study was supported in
part by grant no. 09-04-00395 to P.P.P. from the Russian Foundation
for Basic Research.
Abbreviations:
|
AAR
|
autoantibodies against recoverin
|
|
CAR
|
cancer-associated retinopathy
|
|
NSCLC
|
non-small cell lung carcinoma
|
|
SCLC
|
small cell lung carcinoma
|
|
Rc mRNA
|
mRNA for recoverin
|
|
PNA
|
paraneoplastic antigens
|
|
PNS
|
paraneoplastic syndrome
|
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