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Keywords:

  • sperm-associated antigen 9 (SPAG9);
  • cervical carcinoma;
  • cancer testis antigen;
  • detection;
  • diagnosis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

BACKGROUND:

Cervical cancer is the second most common malignancy in women, with nearly half a million new cases diagnosed each year worldwide. The authors' recent studies have suggested an association of the cancer testis antigen sperm-associated antigen 9 (SPAG9) in ovarian carcinomas. The aim of the current study was to evaluate the clinical utility of SPAG9 expression and humoral immune response in cervical carcinomas.

METHODS:

SPAG9 mRNA expression was assessed by reverse transcriptase–polymerase chain reaction (RT-PCR) and in situ RNA hybridization. In addition, the authors investigated SPAG9 protein expression by immunohistochemistry and analyzed its association with various stages and grades of cervical cancer patients. They also tested the humoral immune response against SPAG9 in cervical cancer patients.

RESULTS:

RT-PCR, in situ RNA hybridization, and immunohistochemical analyses revealed that SPAG9 expression was significantly associated with tumor grades in 82% of early stage cervical cancer specimens. SPAG9 antibodies were detected in approximately 80% of cervical cancer patients, but not in healthy controls. Statistical analysis revealed that a significant proportion of early stage cancer patients with a high SPAG9 immunoreactivity score (IRS) exhibited significantly higher antibody response against SPAG9 compared with moderate SPAG9 IRSs, suggesting a close relation between SPAG9 protein expression and humoral immune response.

CONCLUSIONS:

The current study findings revealed that in early stage cervical cancer, a substantial number of patients exhibited SPAG9 expression and generated SPAG9 antibodies, supporting its potential role in early detection and diagnosis in cervical cancer management. Furthermore, these findings provide leads for future development of noninvasive serologic biomarkers for the early detection, diagnosis, and treatment of cervical cancer. Cancer 2009. © 2009 American Cancer Society.

Cervical cancer is the third major cause of cancer deaths in women worldwide, with an increased mortality rate in less developed countries.1 Evidence has accumulated to indicate that oncogenic types of human papillomavirus serve as an important factor in the development of the precursors of cervical cancer. However, only a small set of those infected by human papillomavirus develop cervical cancer, indicating there are other factors that contribute to the progression of cervical cancer.2 In particular, squamous cell carcinoma (SCC) of the cervix is a leading cause of death in Indian women because of limited awareness, resources, and available medical support. The prognosis is usually favorable for cervical cancer in early stages. Patients are often diagnosed with advanced stage disease, for which available chemotherapy and radiotherapy is less effective.3 Furthermore, cervical cancer patients carry a higher risk of disease recurrence in addition to poor prognosis.4 In this regard, there is a need for identification of biomarkers for early detection and diagnosis of cervical cancer.

To date several tumor antigens, such as SCC antigen (SCCA), cancer antigen 125 (CA 125), and cytokeratin fragment 19 (CYFRA 21.1), have been reported in SCC of the cervix. SCCA has been reported to be more sensitive than CYFRA 21.1 for SCC of the cervix.5 However, tumor antigens currently available for the management of patients with cervical cancer need to be validated in a large number of cervical cancer patients. A unique class of tumor-associated antigens coded by the cancer testis (CT) gene family are being investigated as cancer biomarkers because of their aberrant expression in various types of cancer.6 In this context, expression of several CT antigens, namely MAGE-A,7 GAGE,8 CAGE-1,9 NY-ESO-1,7 CRT2,10 and SSX4,11 have been reported in cervical cancer. However, humoral and cellular immune responses have been reported for only a few CT antigens.12 Especially in cervical cancer patients, autoantibody responses against SSX4 and CRT2 have been reported.10, 11 We recently reported on the expression of a new member of the CT antigen family, sperm-associated antigen 9 (SPAG9), in patients with epithelial ovarian cancer (EOC).13 We also demonstrated the presence of humoral immune response against SPAG9 in EOC patients. In addition, gene expression microarray analysis of cervix carcinoma cells14 found the SPAG9 gene expressed along with other important much-studied genes. An important feature regarding CT antigens is that these are recognized by autologous cytolytic T lymphocytes.15 Therefore, CT antigens are likely to serve as good prognostic markers for effective management of cervical cancer patients.

In the current study, we investigated the expression of SPAG9 and humoral immune response against SPAG9 in early stages and various grades of cervical cancer. Our results confirmed that a significant proportion of early stage cancer patients with high SPAG9 protein expression generated significantly higher antibody response against SPAG9 compared with moderate SPAG9 protein expression in cervical cancer patients, suggesting a close correlation between SPAG9 protein expression and humoral immune response. Our findings provide a basis for early detection and diagnosis of cervical cancer and hence, possible application in tumor biology and clinical outcome.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

Patients and Surgical Samples

A total of 66 cervical cancer patients (women diagnosed with SCC), 54 adjacent noncancerous tissues (ANCT), and the serum samples of patients undergoing surgery at Safdarjung Hospital and Vardhman Mahavir Medical College were collected for this investigation. The carcinoma samples consisted of 15 grade 1 tumors, 41 grade 2 tumors, and 10 grade 3 tumors; 31 cases were categorized as stage I (5 as stage IA and 26 as stage IB) and 35 cases as stage II (17 as stage IIA and 18 as stage IIB). Histologic diagnoses and tumor grading were based on the International Federation of Gynecology and Obstetrics (FIGO) classification system.3 After obtaining informed consent from the patients, and under the terms and conditions approved by the institutional ethics committee and the local ethical committee, specimens were obtained surgically. All ANCT and tumor tissues were immediately snap frozen and stored at −80°C for gene expression as described earlier.13

RNA Extraction and Reverse Transcriptase–Polymerase Chain Reaction

Total RNA was extracted from frozen cervical cancer tissues using TRI Reagent (Ambion, Inc., Austin, Tex) following the manufacturer's instructions. cDNA synthesis was performed using FastLane Cell cDNA kits (Qiagen GmbH, Hilden, Germany).16 Subsequently, cDNA was used as a template for reverse transcriptase–polymerase chain reaction (RT-PCR) using SPAG9 primers 5′-GACAGAGATGATTCGGGCATCACGAGAAAA-3′ (forward) and 5′-CTAAGTTGA TGACCCATTATTATACCTCGACTG-3′ (reverse). RT-PCR for β-actin mRNA expression was performed as an internal control. The PCR product was subsequently cloned into TOPO vector using the TOPO kit (Invitrogen; Life Technologies, Carlsbad, Calif), and the nucleotide sequence was confirmed.

In Situ RNA Hybridization and Immunohistochemistry

To investigate SPAG9 mRNA expression in cervical cancer specimens, in situ RNA hybridization experiments were performed using labeled antisense (experimental) and sense (control) riboprobes using the Digoxigenin RNA labeling kit (Roche Diagnostics GmbH, Mannheim, Germany) as described earlier.17 SPAG9 protein expression in 4-μm paraffin-embedded sections (serial sections) of cervical cancer tissues was analyzed with anti-SPAG9 antibody or control immunoglobulin (Ig) G by immunohistochemistry as described earlier.13 Paraffin-embedded sections of ANCT were also processed as described earlier.13 To confirm the grades of the cervical tumor, serial tumor sections were incubated with mouse monoclonal antiproliferating cell nuclear antigen (PCNA [F-2]; Santa Cruz Biotechnology, Calif) at a dilution of 1:50 at 4°C overnight in a humid chamber. After washing, antigen-bound primary antibodies were detected by horseradish peroxidase–conjugated goat antimouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa). Reactivity in the tissue specimens was observed using chromogen 0.05% 3, 3′-diaminobenzidine (Sigma-Aldrich, St. Louis, Mo), counterstained with hematoxylin solution, mounted with 1,3,-diethyl-8-phenylxanthine, and observed under a Nikon Eclipse E 400 microscope (Nikon, Fukuoka, Japan).

Determination of SPAG9 Immunoreactivity Score

SPAG9 immunostaining was examined by counting >500 cells from 5 random fields of each specimen under ×400 magnification in a stained tumor area of each section as described earlier.18 The SPAG9 immunoreactivity score (IRS) was defined as the percentage of stained cervix tissue cells. A distinct positive immunoreactivity was considered in a specimen with >10% of cancer cells demonstrating SPAG9 immunoreactivity.

Enzyme-linked Immunoadsorbent Assay

The recombinant SPAG9 protein was expressed and purified as described earlier.13 The recombinant protein was purified to >95% purity when analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and was used as an antigen for the detection of SPAG9 antibodies to monitor the immune response against SPAG9 in patients with cervical cancer. Humoral immune response against SPAG9 in the serum of cervical cancer patients was analyzed in serial dilutions of cancer patients' serum with enzyme-linked immunoadsorbent assay (ELISA) as described earlier.19 Results for patients' serum dilution (1:100) were accepted with estimated ELISA titers above the mean + 2 standard deviations (SD) of the healthy sera. All cervical cancer samples and normal healthy donor samples were tested in duplicates, and the mean was used for data analysis. A subset of samples was reassayed 5 times in every ELISA plate for quality control. The intra-assay and interassay coefficients of variation were 4.5% and 6.3%, respectively.

Statistical Analysis

The Pearson chi-square test, Student t test for unpaired data, Mann-Whitney U test, and Kruskal-Wallis test were performed using the SPSS 16.0 statistical software package (SPSS Inc., Chicago, Ill). Pearson correlation coefficients were used to examine the association between SPAG9 IRSs and ELISA scores. Results were expressed as the mean ± standard error (SE). All P values were 2-sided, and a P value of <.05 was considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

SPAG9 Expression in Cervical Tumors

SPAG9 mRNA expression was investigated in 66 cervical cancer specimens and 54 ANCT by RT-PCR (Fig. 1). As shown in Figure 1, SPAG9 expression was observed in tumors, but not in ANCT. Table 1 summarizes the RT-PCR results. SPAG9 mRNA expression was found in 54 of 66 (82%) tissue specimens irrespective of tumor stages (Table 1). The size of the PCR product in tumors was the same as in the SCC tumor specimens and in testes (Fig. 1). The PCR product was confirmed as SPAG9 by nucleotide sequencing. The relation between SPAG9 mRNA expression and pathologic and clinical features is detailed in Table 1. SPAG9 mRNA expression was observed in 5 of 5 (100%) stage IA, in 21 of 26 (81%) stage IB, in 15 of 17 (88%) stage IIA, and in 13 of 18 (72%) stage IIB (Table 1) cervical cancer specimens. In addition, SPAG9 expression was detected in 87% (13 of 15) grade 1, 90% grade 2 (37 of 41), and 40% (4 of 10) grade 3 cervical cancer tissues. The statistical analysis revealed that SPAG9 expression was independent of tumor stages, indicating no correlation between tumor stages and SPAG9 expression. However, SPAG9 expression was found to be significantly associated among the grades using Pearson chi-square test (P = .001).

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Figure 1. Reverse transcriptase–polymerase chain reaction results of sperm-associated antigen 9 (SPAG9) expression are shown. SPAG9 was found to be expressed in various stages of tumor tissues compared with the paired adjacent noncancerous tissues in cervical cancer patients. β-Actin gene expression was used as the internal control. ANCT indicates associated noncancerous tissues.

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Table 1. SPAG9 Expression (RT-PCR/IHC-IRS), Humoral Immune Response (ELISA), and Clinicopathologic Characteristics of Cervical Cancer
 SPAG9 Expression
Pathologic and Clinical FeaturesRT-PCR, No. Positive/No. TestedELISA, No. Positive/No. TestedIHC-IRS, No. Positive/No. Tested
  1. SPAG9 indicates sperm-associated antigen 9; RT-PCR, reverse transcriptase–polymerase chain reaction; IHC-IRS, immunohistochemistry-immunoreactivity score; ELISA, enzyme-linked immunoadsorbent assay.

All tumors54/66 (82%)53/66 (80%)54/66 (82%)
Tumor stage   
 Stage I (IA+IB)26/31 (84%)26/31 (84%)26/31 (84%)
 Stage IA5/5 (100%)5/5 (100%)5/5 (100%)
 Stage IB21/26 (81%)21/26 (81%)21/26 (81%)
 Stage II (IIA+IIB)28/35 (80%)27/35 (77%)28/35 (80%)
 Stage IIA15/17 (88%)14/17 (82%)15/17 (88%)
 Stage IIB13/18 (72%)13/18 (72%)13/18 (72%)
Lymph node involvement   
 Positive11/13 (85%)11/13 (85%)11/13 (85%)
 Negative43/53 (81%)42/53 (79%)43/53 (81%)
Histologic grade   
 Grade 113/15 (87%)13/15 (87%)13/15 (87%)
 Grade 237/41 (90%)36/41 (88%)37/41 (90%)
 Grade 34/10 (40%)4/10 (40%)4/10 (40%)

SPAG9 mRNA and Protein Are Expressed in the Cervical Cancer Specimens

To determine whether the SPAG9 mRNA is expressed in cervical cancer tissues and ANCT specimens, we used in situ RNA hybridization with digoxigenin-labeled riboprobes. SPAG9 mRNA expression was observed in 82% of investigated cases. Using antisense riboprobe, positive tumor cells presented chocolate brown reaction product, indicating the presence of SPAG9 mRNA in cervical cancer tissues in various stages (I and II), as shown in Figure 2 (Panels b, e, h, and k). However, the sense riboprobe failed to demonstrate any localization (Fig. 2c, 2f, 2i, and 2l).

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Figure 2. In situ RNA hybridization demonstrates SPAG9 expression in cervical squamous cell carcinoma. Representative hematoxylin and eosin (H and E)-stained sections of (a) stage IA, (d) stage IB, (g) stage IIA, and (j) stage IIB tissue are shown. Serial tissue sections from the same specimens demonstrating a chocolate brown color with digoxigenin-labeled SPAG9 antisense riboprobe (b, e, h, and k) whereas sense riboprobe (c, f, i, and l) failed to show any signal. The signal was developed using alkaline phosphatase nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. SPAG9 indicates sperm-associated antigen 9 (original magnification ×200; objective, ×20).

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To examine the SPAG9 protein expression in cervical cancer specimens, immunohistochemistry was performed. On the basis of IRS, a distinct SPAG9 protein expression positive immunoreactivity (>10% of cancer cells were stained) was noted in 82% of patient tissues. Representative photomicrographs of SPAG9 reactivity in various stages of cervical cancer tissues are shown in Figure 3. The immunohistochemical staining results indicated that SPAG9 expression was localized to the cytoplasm of squamous cells in cervical cancer tissues in various stages (Fig. 3a, 3d, 3g, and 3j). However, tumor tissue probed with control IgG (Fig. 2b, 2e, 2h, and 2k) or ANCT probed with anti-SPAG9 antibodies (Fig. 3c, 3f, 3i, and 3l) failed to demonstrate any SPAG9 reactivity. In addition, tumor grades were assessed using PCNA.20 Our results confirmed high expression of PCNA, with increasing grades as shown in representative photomicrographs (Fig. 4b, 4d, and 4f).

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Figure 3. Immunohistochemical analysis of sperm-associated antigen 9 (SPAG9) expression in various cervical cancer stage tissues probed with anti-SPAG9 antibodies ([a] stage IA, [d] stage IB, [g] stage IIA, and [j] stage IIB) demonstrated SPAG9 cytoplasmic localization. Serial tissue sections from the same specimens probed with control immunoglobulin G (b, e, h, and k) and associated noncancerous tissues (ANCT) probed with anti-SPAG9 antibodies (c, f, i, and l) failed to demonstrate any reactivity (original magnification ×200; objective, ×20).

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Figure 4. Immunohistochemical analysis of sperm-associated antigen 9 (SPAG9) and proliferating cell nuclear antigen (PCNA) protein expression in different grades of cervical cancer is shown. The cytoplasmic localization of SPAG9 protein in (a) grade 1, (c) grade 2, and (e) grade 3 cervical cancer is shown. Representative photomicrographs of (b) grade 1, (d) grade 2, and (f) grade 3 cervical cancer demonstrate positive nuclear-stained cells with antibodies against PCNA (original magnification ×400; objective, ×40).

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Although significant proportions of cervical cancer tissues of various stages were found positive by RT-PCR, in situ RNA hybridization, and immunohistochemistry analysis, no significant association was found between SPAG9 expression and various stages using the Pearson chi-square test, suggesting that SPAG9 expression was independent of FIGO stages. However, a significant association was found between SPAG9 expression and grade 1, grade 2, and grade 3 (P = .001) using the Pearson chi-square test. On the basis of SPAG9 expression, while comparing grades, a significant difference was found between grades 2 and 3 (P = 0.049) using the Student t test, but no significant difference was observed between grades 1 and 2 or between grades 1 and 3 with the Student t test.

SPAG9 Association With Cervical Carcinoma

SPAG9 IRS was defined as the percentage of carcinoma cells in cervical cancer tissue demonstrating SPAG9 immunolocalization. SPAG9 IRSs of each stage in cervical cancer patients were 68.8 (mean) ± 5.58 (SE) in stage IA, 65.29 ± 3.24 in stage IB, 67.73 ± 4.9 in stage IIA, and 64.77 ± 3.04 in stage IIB. The overall SPAG9 IRSs were 65.96 ± 2.8 in stage I (IA & IB) and 66.35 ± 2.93 in stage II (IIA & IIB) (Fig. 5a). SPAG9 IRS apparently exhibited immunoreactivity in all the grades of cervical carcinoma. Within various tumor-differentiated specimens, different stage specimens demonstrated SPAG9 immunoreactivity. Grade 1, which included specimens of stage IA (n = 3), stage IB (n = 7), and stage IIA (n = 5), demonstrated 63.33 ± 8.24, 66.33 ± 4.18, and 60.25 ± 7.03 SPAG9 IRSs, respectively (Table 2). Grade 2, which included specimens of stage IA (n = 2), stage IB (n = 14), stage IIA (n = 12), and stage IIB (n = 13), exhibited 77 ± 1.98, 68.92 ± 3.68, 70.45 ± 6.11, and 64.54 ± 3.55 SPAG9 IRSs, respectively (Table 2). Grade 3, which included specimens of stage IB (n = 5) and stage IIB (n = 5), demonstrated 38.5 ± 8.52 and 66 ± 5.01 SPAG9 IRSs, respectively (Table 2). The overall SPAG9 IRSs among the grades were 63.77 ± 3.20, 68.51 ± 2.45, and 52.25 ± 38.9 in grade 1, 2, and 3, respectively (Fig. 5b). Furthermore, based on SPAG9 IRS in cervical tumors, 2 groups (moderate: <50% tumor cells expressing SPAG9; and high: >50% tumor cells expressing SPAG9) were analyzed. The results indicated that 83% of cervical cancer patients (30 of 36 patients with stages IB and IIA) revealed higher SPAG9 IRSs of 71.67 ± 2.1, compared with 17% of cervical cancer patients (6 of 36 patients with stages IB and IIA) having moderate SPAG9 IRSs 39.50 ± 4.5, as shown in Figure 5c. In addition, when we examined overall stages, a higher SPAG9 IRSs of 70.95 ± 1.5 was observed in 83% of cervical cancer patients (45 of 54), compared with moderate SPAG9 IRSs of 42.20 ± 3.20 in 17% of cervical cancer patients (9 of 54), as shown in Figure 5d.

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Figure 5. Sperm-associated antigen 9 (SPAG9) immunoreactivity score (IRS) in various (a) stages and (b) grades of cervical cancer tissues and its association with pathologic stages and grading are shown. SPAG9 IRS was significantly associated among various grades of cervical cancer (P = .001). (c) In the combined group of stage IB and stage IIA tissues, high SPAG9 IRSs (>50% SPAG9-expressing cells) were found to be significantly more common (P < .0001) compared with moderate SPAG9 IRSs (<50% SPAG9-expressing cells). (d) In the combined group of stage I (IA and IB) and stage II (IIA and IIB) tissues, high SPAG9 IRSs (>50% SPAG9-expressing cells) were found to be significantly more common (P < .0001) compared with moderate SPAG9 IRSs (<50% SPAG9-expressing cells). Point indicates mean; bars, standard error; *, P = .049; **, P = .001; ***, P < .0001.

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Table 2. Distribution of FIGO Stage and Grade in Patients With Cervical Cancer
GradesStage IAStage IBStage IIAStage IIBTotal
  1. FIGO indicates International Federation of Gynecology and Obstetrics.

137515
2214121341
35510
Total526171866

Furthermore, to analyze our results, we assessed the significance of SPAG9 IRSs in various stages and grades using various statistical tests. There was no significant difference in SPAG9 IRSs among stages IA, IB, IIA, and IIB (P = .634) and grades 1, 2, and 3 (P = .076) by multiple comparisons with the Kruskal-Wallis test. In addition, there was no significant difference noted in overall stages I and II using Student t test (P = .833). Our analysis further revealed that there was no significant difference between SPAG9 IRSs and stages within each grade using multiple comparisons with the Kruskal-Wallis test and Mann-Whitney U test, respectively (grade 1: stage IA/IB/IIA, P = .669; grade 2: stage IA/IB/IIA/IIB, P = .309; and grade 3: stage IB/IIB, P = .333). On the basis of SPAG9 IRSs, while comparing grades, a significant difference was found between grades 2 and 3 (P = .049), but no significant difference was observed between grades 1 and 2 or between grades 1 and 3 by the Student t test. However, a significant association was found between SPAG9 IRS and grades 1, 2, and 3 (P = .001) by the Pearson chi-square test. On the basis of SPAG9 IRS in cervical tumors, 2 groups (those with moderate and high SPAG9-expressing tumor cells) were analyzed. When moderate and high SPAG9 IRSs in stage IB and IIA were examined, a significant difference (P = .000) was found, with a higher number of cervical patients (n = 30 of 66) demonstrating SPAG9 expression, as examined using the Mann-Whitney U test (shown in Fig. 5c). In addition, overall stage I (IA and IB) and II (IIA and IIB) also demonstrated a similar trend of significantly (P <.0001) higher SPAG9 IRSs (>50% cells) in a higher number of cervical cancer patients (n = 45 of 66) compared with patients having moderate SPAG9 IRSs (<50% cells) (n = 9 of 66), as shown in Figure 5d using the Student t test.

Antibody Response to SPAG9 in Cervical Cancer Patients

Our immunohistochemistry analyses confirmed the SPAG9 protein expression; therefore, we further investigated whether SPAG9 was immunogenic in cervical cancer patients using ELISA. Initially, we calculated the cutoff signal intensities at A492nm for positive antibody reactivity against SPAG9, which was 0.30 (absorbance greater than 2 SDs above the mean value of the controls). Fifty-four healthy blood donors matched for age and sex with the cancer population were selected as a control group. As shown in Figure 6a, SPAG9 antibodies were detected in approximately 80% of cervical cancer patients (SPAG9-expressing tumors) (Table 1). Although patients with early stage disease demonstrated a humoral response against SPAG9, no significant association was found between SPAG9 antibodies and stages, indicating that the humoral immune response was independent of stages of cervical cancer (Table 1). In contrast, a significant association was found between SPAG9 antibodies and various differentiated tumors (P = .002) using the Pearson chi-square test. Positive SPAG9 ELISA results were confirmed by Western blot analysis, which demonstrated binding to the SPAG9 recombinant protein band (data not shown). Furthermore, we also compared SPAG9 antibodies based on SPAG9 IRS in cervical tumors in 2 groups (moderate and high SPAG9-expressing tumor cells). When high SPAG9 IRSs (>50% cells) and moderate SPAG9 IRSs (<50% cells) in overall stages I and II were compared, a significant difference (P < .0001) was observed in a higher number of patients with high SPAG9 expression, as shown in Figure 6b. In addition, when high SPAG9 IRSs (>50% cells) and moderate SPAG9 IRSs (<50% cells) within stages IB/IIA were compared, a significant difference (P = .004) was observed (Fig. 6c) by the Mann-Whitney U test, suggesting that higher SPAG9 antibodies ELISA scores were detected in cervical tumors with high SPAG9 IRSs, as shown in Figures 6b and c.

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Figure 6. Analysis of sperm-associated antigen 9 (SPAG9) antibodies in cervical cancer patients is shown. (a) Enzyme-linked immunoadsorbent (ELISA) analysis of sera from cervical cancer patients of different stages (stages IA, IB, IIA, and IIB) and grades (grade 1, grade 2, and grade 3) and from healthy women donors is shown. X indicates the cutoff A492 nm at mean + 2 standard deviations from healthy donors for positivity above or negativity below the line. There was a significant difference in the seroreactivity between normal healthy women donors and patients with cervical cancer (P < .0001). (b) In the combined stage I (IA and IB) and stage II (IIA and IIB) group, SPAG9 antibody ELISA scores were significantly higher (P < .0001) in the high SPAG9 IRSs group compared with the moderate SPAG9 IRSs group. (c) In the combined stage IB and stage IIA group, the SPAG9 antibody ELISA scores were significantly higher (P = .004) in the high SPAG9 IRSs group compared with the moderate SPAG9 IRSs group. OD indicates optical density; nm, nanometers; point, mean; bars, standard error; ***, P < .0001.

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SPAG9 Expression, Immune Responses, and Various Cervical Cancer Stages

Furthermore, based on SPAG9 IRS in cervical tumors, 2 groups (moderate and high SPAG9 IRSs) were analyzed to investigate the correlation with SPAG9 antibodies ELISA scores. When high SPAG9 expression (SPAG9 IRSs: high indicates >50% cells expressing SPAG9) and ELISA scores were examined, SPAG9 expression was found to be correlated significantly with ELISA scores in cervical cancer stages I and II (Pearson correlation, .333; P < .025), but not in cervical cancer stages IB and IIA (Pearson correlation, 0.293; P = .116). In the combined (moderate and high SPAG9 IRSs) group, SPAG9 expression was found to correlate the strongest with the SPAG9 antibodies ELISA scores in cervical cancer stages I and II (Pearson correlation, 0.512; P = .000) and in cervical cancer stages IB and IIA (Pearson correlation, 0.478; P < .003), suggesting that SPAG9 expression and SPAG9 antibodies may be useful as an ideal noninvasive serum biomarker for early stage cervical cancer. The analysis of SPAG9 expression and its humoral response in cervical cancer patients of different stages is presented in Table 1.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

Numerous cervical cancer biomarkers have been identified to date, such as SCCA, CYFRA 21.1, and 2 mucins (CA 15-3 and CA 125).21 However, the clinical relevance of these tumor-associated antigens is still debated.22 Because to our knowledge there is no validated tumor marker,5 there is an urgent need to identify new biomarkers for the early detection and management of patients with cervical cancer. CT antigens are predominantly expressed in normal gametogenic tissues as well as in different histologic types of tumors, and therefore they represent ideal diagnostic and therapeutic candidates.6 In the current study, we analyzed the expression of SPAG9, a CT antigen, and humoral immune response in patients in the early stages of cervical SCC and evaluated its association with the early detection of cervical cancer patients.

To our knowledge, the current study is the first to describe SPAG9 expression and in vivo immunogenicity in cervical cancer patients. The SPAG9 expression was observed in a significant proportion of cervical cancer patients when compared with other known CT antigens. When we examined cervical tumors based on SPAG9 IRSs in 2 groups (moderate [<50% tumor cells expressing SPAG9] and high [>50% tumor cells expressing SPAG9]), we found a significant difference between high SPAG9 expression in a higher number of cervical patients in stage IB and IIA, compared with moderate SPAG9-expressing tumors. In addition, in overall stages I (IA and IB) and II (IIA and IIB), a similar trend toward significantly higher SPAG9 IRSs (>50% cells) was observed in a higher number of cervical cancer patients compared with cancer patients having moderate SPAG9 IRSs. Furthermore, a significant association was also found between SPAG9 IRSs and grades 1, 2, and 3. This association suggests a possible, yet unknown, biologic mechanism for the spread of cervical cancer and recurrence. A recent report on EphA2 and EphrinA-1 expression in early cervical SCCs suggested that these 2 proteins may play a role in the development of a subset of early cervical cancers.23 A recent study demonstrated a very strong expression of membrane type 1 matrix metalloproteinase (MT1-MMP) in nearly all invasive carcinomas; however, to our knowledge little is known regarding the role of MT1-MMP in mediating the invasiveness of cervical cancer cells.24 Our similar results may suggest that high SPAG9 expression may contribute to tumor invasion and aggressive phenotype. It is quite possible that the high SPAG9 expression in early stages and various grade tumors may initiate parallel signaling pathways, which may support cellular proliferation and growth, resulting in tumor spread and progression of disease. However, this needs to be investigated further.

The expression of several CT antigens, such as MAGE (29.7%),8 GAGE (35%),8 SSX1 (4%), SSX2 (4%), SSX4 (20%),11 and CRT2 (50%),10 have been reported in cervical cancer. However, protein expression has been demonstrated by immunohistochemistry in tissues of cervical cancer patients only for MAGE-A (34%)7 and NY-ESO-1 (49%).7 In contrast, SPAG9 mRNA and protein expression was detected in 82% of cervical cancer tissues irrespective of disease stages. This is an important feature that could lead to the identification and characterization of tumor-specific targets for immunotherapy and the development of cancer biomarkers. The predominant SPAG9 expression in patients with early stage cervical cancer thereby ensures its significant role as a potential biomarker for the early diagnosis of cervical cancer.

The generation of humoral immune response against CT antigens can be regarded as a signal that indicates the presence of the tumor in the host.25 Our earlier studies demonstrated the presence of anti-SPAG9 antibodies in approximately 67% of patients with epithelial ovarian cancer with SPAG9-expressing tumors.13 It is interesting to note that in this study, we found SPAG9 antibodies in the sera of approximately 80% of cervical cancer patients with SPAG9-expressing tumors. However, when compared with other CT antigens, antibodies to SSX4 were detected in only 1 of 38 cervical cancer patients.11 Another study recently reported an antibody response to the CT antigen CRT2 in 58% of cervical cancer patients.10 It is noteworthy that all of the sera tested for SPAG9 antibodies were taken from resected specimens from cervical cancer patients in the early stages of disease. In contrast, our finding demonstrated a significant proportion of early stage cervical cancer patients exhibiting an antibody response against SPAG9, supporting its potential role as a serum biomarker.

The aberrant expression of CT antigens in tumors has been proposed to recapitulate the germline expression program, which is characteristic of neoplastic phenotypes such as immortality, invasiveness, immune evasion, and metastatic capacity.26 Furthermore, our earlier MAPK interaction studies of SPAG9 with the JNK signaling pathway demonstrated that SPAG9 functions as a scaffolding protein.16 The JNK signaling interaction is well known to play an important regulatory role in cell proliferation, differentiation, apoptosis, cellular transformation, and tumor cell growth, and may be equally important for the outcome of SPAG9 expression in the cervix.16, 27, 28 To our knowledge, the mechanism underlying the association between SPAG9 and carcinogenesis is still unclear. Perhaps the overexpression of SPAG9 changes the stability or kinetics of MAPK signaling pathways, leading to facilitation of cell invasion and migration; this warrants further studies.

In conclusion, the results of the current study demonstrated that a substantial number of cervical cancer patients exhibit SPAG9 expression and generate SPAG9 antibodies. Our important findings indicate that a significant proportion of patients with early stage cancer with high SPAG9 protein expression exhibit a significantly higher antibody response against SPAG9, compared with patients with moderate SPAG9 protein expression, supporting its potential role as a noninvasive serum biomarker for the early detection and diagnosis of this disease entity. Our preliminary observations need to be validated in a larger number of cervical cancer patients. Furthermore, the potential clinical utility of SPAG9 should be explored as a biomarker in tissue biopsies and in monitoring for recurrence of cervical cancer after surgery. SPAG9 should be examined in a larger study to validate and expand the findings of the current study.

Conflict of Interest Disclosures

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

Supported by the Cancer Research Program, Associated Cancer Center for Immunotherapy, Department of Biotechnology, Government of India.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References
  • 1
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