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Technical Briefs |
1 Department of Radiation Oncology,3 Department of Otorhinolaryngology, Head and Neck Surgery, and4 Division of Hematology/Oncology, Department of Internal Oncology, Chang Gung Memorial Hospital, Taoyuan, Taiwan;2 Institute of Public Health, National Yang-Ming University, Taipei, Taiwan;5 School of Medical Technology, Chang Gung University, Taipei Chang Gung Head and Neck Oncology Group, Taoyuan, Taiwan
aaddress correspondence to this author at: School of Medical Technology, Chang Gung University, 259 Wen-Hwa 1st Road, Taoyuan 333, Taiwan; fax 886-3-2118247, e-mail ajchen{at}mail.cgu.edu.tw
Several proteins associated with malignant transformation of cells can induce autoantibodies (1)(2)(3)(4). These autoantibodies, such as those to p53, are detectable in serum and may serve to monitor tumor progression (4)(5). Survivin, a recently cloned 16.5-kDa apoptosis inhibitor belonging to the IAP3 family (6), is expressed in the G2-M phase of the cell cycle (7). Its overexpression in cancer is thought to overcome an apoptotic checkpoint and favor aberrant progression of transformed cells through mitosis. Survivin is not produced in adult tissues except for the thymus and placenta, but it is abundantly produced in fetal tissues and in various human tumors, including lung, colon, gastric, breast, and bladder cancers as well as high-grade lymphomas (6)(7)(8)(9)(10). We developed an enzyme-labeled assay to measure anti-survivin autoantibody in sera and evaluated the relationship between anti-survivin and clinicopathologic variables.
We obtained blood from 294 consecutive patients, before cancer treatment, at the Otorhinolaryngology or Head and Neck Surgery Clinics at Chang Gung Memorial Hospital with written informed consent. The standard treatment was radical surgery for early-stage patients, plus adjuvant radiotherapy for intermediate-risk patients, such as those with close surgical margins or lymph node metastases. Concomitant chemoradiotherapy was given to patients with lymph node metastases and extracapsular spread. All cancers were histologically graded as well-differentiated, moderately differentiated, or poorly differentiated, according to the WHO classification (11). Tumor pathology staging was classified according to the system of the American Joint Committee on Cancer (12). Blood samples from 40 gender- and age-matched healthy individuals were obtained as controls. After centrifugation, the sera were stored at –20 °C until use.
Survivin cDNA was obtained by reverse transcription-PCR using total RNA from Jurkat cells with survivin-specific primers. The PCR product was ligated into the pET29a expression plasmid (pET-survivin; Novagen), which contains a histidine-tag fusion protein. The correct sequence of the cloned PCR product was verified by sequencing. Escherichia coli BL21 were transformed with pET-survivin. Expression of recombinant survivin (r-survivin) was induced with isopropyl ß-p-thiogalactopyranoside. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to assess induction of recombinant protein. The cells were harvested by centrifugation and sonicated on ice in short bursts in a binding buffer solution containing 20 mmol/L imidazole. The r-survivin was purified from the supernatant by His-Bind (Novagen) metal chelate chromatography. The purity of the protein was examined by Western blot analysis using polyclonal anti-survivin antibody.
Purified r-survivin was diluted in 20 mmol/L phosphate buffer (pH 7.4) to a final concentration of 5 mg/L (Coomassie assay; Pierce), dispensed into 96-well plates (150 µL/well), and incubated overnight at 4 °C. After being washed with washing buffer [phosphate-buffered saline (PBS) containing 1 mL of Tween 20 and 1 mL of Nonidet P-40 per liter], the plates were blocked with 10 g/L bovine serum albumin in washing buffer and incubated 2 h at room temperature. The plates were washed five times and dried under reduced pressure. Sera were diluted 1:100 in a washing buffer containing 2 g/L bovine serum albumin and 30 mL/L fetal calf serum, and 100 µL was placed in r-survivin-coated wells and incubated 1 h at 37 °C with gentle shaking. A calibrator was prepared with rabbit antiserum raised against r-survivin protein. After being washed, each well was incubated with 100 µL of a 1:5000 dilution (in sample dilution solution) of goat anti-human IgG F(ab')2 labeled with horseradish peroxidase (Zymed) and incubated for 1 h at 37 °C with gentle shaking, followed by washing. We added 100 µL of tetramethylbenzidine substrate solution (Sigma) and, after 15 min at room temperature, 100 µL of 2 mol/L HCl. The absorbance was then measured at 450 nm. For each patient, the data were calculated as the ratio of absorbance for the unknown patient sample to the absorbance of the calibrator.
We added 4 µg of purified r-survivin protein to a loading buffer and boiled the solution for 5 min. Subsequently, we subjected the r-survivin sample to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to a polyvinylidene difluoride membrane. The membrane was then blocked overnight at 4 °C with 50 g/L nonfat dry milk in PBS containing 1 mL/L Tween. After decanting the blocking buffer, we cut the membrane into pieces and incubated the pieces separately with 1:100 diluted serum (in PBS containing 50 g/L bovine serum albumin) for 1 h at 37 °C with agitation. After washing the membrane six times with PBS-Tween, we incubated the membrane with anti-human IgG antibody conjugated with horseradish peroxidase (Santa Cruz) for 1 h at 37 °C. After being washed, the membrane was submerged in ECL developing solution (Amersham) and autoradiographed.
The Pearson 
2 test was used to look for an association between anti-survivin and clinicopathologic variables, including tumor extent (tumor, node, and overall stage) and pathology findings (degree of differentiation). All P values presented were two-sided, and significance was set at <0.05.
The r-survivin protein appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1A in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue7/) and Western blot analysis (Fig. 1B in the online Data Supplement). The assay was linear on dilution (r = 0.988). For three samples with absorbance ratios of 1.6–2.2, the within-run imprecision (CV) was 2.4–2.7% (n = 10) and the between-run CV was 2.7–3.1% (n = 10). Sera with rheumatoid factor (250 kIU/L), anti-streptolysin O (420 kIU/L), and increased C-reactive protein (50 mg/L) were negative for anti-survivin. No bands were detected by Western blot analysis in samples with ELISA signals below the cutoff, but clear bands were found in samples with signals above the cutoff (Fig. 2 in the online Data Supplement).
The median age of the patients was 49.5 years (range, 26–79 years), 94% were male, 66% consumed alcohol, 85% smoked tobacco, and 84% chewed betel quid. The diagnosed cancers were of the hypopharynx (9%), oropharynx (6%), larynx (3.7%), and oral cavity (81%); of the latter, 96 (33%) of the cancers were in the buccal mucosa, 66 (22%) were in the tongue, and 76 (25.8%) were in other sites. All cancers were squamous cell carcinomas, with 106 (36%) well differentiated, 148 (50%) moderately differentiated, and 40 (13.6%) poorly differentiated. The mean age of the controls was 46 years (range, 18–72 years), and 66% were male.
The mean (SD) absorbance ratio was 0.27 (0.14) in sera from controls and 0.60 (0.40) in sera from cancer patients (Fig. 1
). At a cutoff value equal to the mean + 2 SD in controls (13)(14), anti-survivin was increased in 1 (2.5%) noncancer and 134 (46%) cancer samples (P <0.001). Anti-survivin signals did not differ significantly among cancers at different anatomic sites (data not shown). The relationship between anti-survivin autoantibody and clinicopathologic variables is shown in Table 1
. Anti-survivin was not statistically associated with patient sex or age or lymph node status (N stage). Correlations were significant between anti-survivin and tumor stage (T stage; P = 0.040), overall stage (P = 0.003), and histologic grade (P = 0.012).
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Autoantibodies can be induced by several cellular molecules when cells are in a high-proliferation state, such as in malignancy (1)(2)(3)(4)(15)(16)(17). Although the clinical value of these antibodies remains subject to debate, consistent results have been observed in breast, colon, and gastric cancers, in which the antibodies have been associated with high-grade tumors or poor prognosis. Although there is no previous report regarding the association of anti-survivin with tumor status, the correlation of survivin expression and more aggressive disease has been found in many cancers (18)(19)(20). In this study, anti-survivin was correlated with tumor aggressiveness, indicating that the serum autoantibody may represent cellular status. These results suggest that the detection of circulating anti-survivin autoantibody could potentially serve as a useful noninvasive marker for determining head-and-neck cancer status.
Detection of serum survivin autoantibody by immunoblotting may be of limited value for routine use in most pathology or diagnostic laboratories. The simple, rapid enzyme-linked assay we have developed, which uses r-survivin protein as the antigen, may be advantageous for screening of healthy individuals or patients with head-and-neck cancer to monitor the development of survivin autoantibody during oncogenesis.
In conclusion, we present the first report of a convenient method to detect survivin antibodies in patients with head-and-neck cancer. This high prevalence of survivin antibody in patients with various cancers makes this molecule an attractive biomarker for assessing such tumors, particularly in patients lacking anti-p53 or other clinically useful serum tumor markers.
Acknowledgments
This work was supported by National Science Research Grant NSC 90-2320-B-182-059 of Taiwan (to A.J.C.) and National Science Research Grant NSC 90-2314-B-182A-105 of Taiwan (to J.T.C.). We thank Mary Jeanne Buttrey, MD, for critical reading and correction of the manuscript.
References
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