- Email: [email protected]
Detection of autoantibodies to livin and survivin in Sera from lung cancer patients
Lung Cancer (2005) 48, 217—221
Detection of autoantibodies to livin and survivin in Sera from lung cancer patients Atsuhito Yagihashia, Koichi Asanumaa, Daisuke Kobayashia, Naoki Tsujia, Yasuharu Shijubob, Shosaku Abeb, Yoshihiko Hirohashic, Toshihiko Torigoec, Noriyuki Satoc, Naoki Watanabea,∗ a
Department of Clinical Laboratory Medicine, Sapporo Medical University School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060-8543, Japan b Department of Internal Medicine, Sapporo Medical University School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060-8543, Japan c Department of Pathology, Sapporo Medical University School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060-8543, Japan Received 29 July 2004 ; received in revised form 28 October 2004; accepted 2 November 2004 KEYWORDS Livin; Survivin; Autoantibody; Lung cancer
Summary Survivin and livin are highly expressed in cancer cells and transformed cells, but show little or no expression in normal differentiated tissues. Although human antibody responses to cancer-associated antigens have been detected, the response to livin has not yet been described in lung cancer patients. We examined prevalence of anti-livin antibodies in such patients with a specific enzyme-linked immunosorbent assay (ELISA) using recombinant protein. Using a cutoff value for positivity determined as the mean absorbance +2S.D. for healthy control samples, 19 of 37 lung cancer patients (51.3%) were positive for antilivin antibodies. Of 31 samples from the same lung cancer patients, 18 (58.1%) were positive for anti-survivin antibodies. When sera from 31 lung cancer patients were assessed simultaneously by anti-survivin and anti-livin ELISAs. Twenty-one patients (71%) were positive for survivin, livin, or both. Intensity of anti-livin antibody responses did not correlate with intensity of anti-survivin responses. Like anti-survivin antibodies, anti-livin antibodies, thus, can be detected in many lung cancer patients. Testing for both antibodies together may prove useful in detecting lung cancer, but more extensive studies are needed to establish the clinical significance of anti-livin antibodies. © 2004 Elsevier Ireland Ltd. All rights reserved.
1. Introduction * Corresponding author. Tel.: +81 11 611 2111x3640;
fax: +81 11 622 7502. E-mail address: [email protected] (N. Watanabe).
Six human inhibitors of apoptosis protein (IAP) have been identified in recent years: c-IAP1, c-IAP2, XIAP, NIAP, survivin, and livin [1—3]. Each contains
0169-5002/$ — see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2004.11.002
218 one or more baculovirus IAP repeat domain, which is necessary to bind specifically to a terminal effector cell-death protease (e.g., caspase-3 and -7). This binding substantially reduces caspase activity, and reduces cell death in response to a variety of apoptotic stimuli. Among these IAP members, survivin and livin are highly expressed in cancer cells and transformed cells, but show little or no expression in normal differentiated tissues [1—11]. Antibody responses to tumor-associated antigens such as p53, p62, c-myc, and cyclin B1 are known to develop in cancer patients [12,13]. We reported recently that 25 of 63 sera from gastrointestinal cancer patients (39.7%) reacted with purified recombinant survivin an enzyme-linked immunosorbent assay (ELISA), while 17 of 35 sera from these patients (47%) were reactive in an ELISA using purified recombinant livin [14,15]. Rohayem et al. reported that 11of 51 sera from lung cancer patients (21.6%) and 4 of 49 sera from colorectal cancer patients (8.2%) reacted with purified recombinant survivin were reactive in their ELISA [16]. These reports indicate that survivin and livin can induce an antibody response in some patients with various cancers. Abundant survivin expression has been demonstrated in non-small-cell lung cancer (NSCLC) by reverse transcription-polymerase chain reaction (RT-PCR) as well as immunohistochemical detection [17—20]. These studies found high survivin expression to be related to poor survival in NSCLC patients, but not to various clinicopathologic factors [17]. In addition, overexpression of survivin in lung cancers had induced antibody responses to this protein [12,13,16]. Recently, overexpression of livin mRNA also was found in NSCLC [21]. As with survivin overexpression, livin overexpression in lung cancers may lead to anti-livin antibody responses against the cancer. However, there are no available data concerning antibody responses to livin in lung cancer patients. We, therefore, hypothesized that antibody responses to livin are induced in some lung cancer patients, and examined the prevalence such responses in this patient population.
2. Material and methods 2.1. Samples Blood samples were collected from 28 patients with non-small-cell lung cancers (NSCLS) and 9 patients
A. Yagihashi et al.
Table 1 Characteristics of patients and absorbances of anti-livin and survivin antibodies Case
Histology
Clinical Anti-livin stage antibody
Anti-survivin antibodya
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCLC SCLC SCLC SCLC SCLC SCLC SCLC SCLC SCLC
I I III III IV IV IV IV IV IV IV IV IV IV IV IV IV I I II III III III III III IV IV IV II III III III III IV IV IV IV
0.9238 0.7042 1.1619 1.2731 0.7894 1.1544 0.7182 1.0291 1.176 1.1604 1.2095
0.356 0.681 0.269 0.934 0.284 0.331 0.431 0.624 0.789 0.994 1.021 1.103 1.106 1.3 1.426 1.844 2.406 0.25 0.577 0.27 0.267 1.494 1.109 1.738 2.12 0.089 0.725 1.733 0.629 0.263 1.078 1.303 1.547 0.234 0.673 1.228 1.344
1.1501 1.5471 1.0611 1.3683 1.0681 1.4573 1.152 1.0934 1.3367 1.4852 1.4148 1.4813 1.1126 1.4496 1.347 1.6757 1.152 1.2167 1.46 1.4204
Clinical stages were evaluated according to the criteria of the Japanese Society of Lung Cancer. Ad: adenocarcinoma; SCC: squamous cell carcinoma; SCLC: small-cell carcinoma. Anti-livin and anti-survivin antibodies were measured using the anti-livin or anti-survivin ELISAs. a Absorbance was measured at 492 nm.
with small-cell lung cancer (SCLC) after histologic diagnosis. As a control, blood samples were collected from 7 nonhospitalized adults who were over 40 and had no malignancy. Informed consent was obtained from all blood donors. After centrifugation, sera were divided into aliquots and stored at −80 ◦ C. These lung cancer patients were diagnosed as being in stages I—IV based on TNM classification (UICC) (Table 1).
Anti-livin antibody against lung cancer
219
2.2. Immunoassays for anti-livin antibody and anti-survivin antibody Preparation of purified recombinant livin, survivin and green fluorescence protein (GFP) was performed according to the method described previously [14,15]. As an antigen for coating wells in an ELISA, purified recombinant livin or survivin was diluted in 50 mM bicarbonate buffer (pH 9.5) to a final protein concentration of 5 g/ml. Purified recombinant GFP was used as a control antigen at the same concentration in the same buffer. Livin, survivin or control antigen solution was placed in wells of 96well plates (Corning, NY) and incubated overnight at 4 ◦ C. After removing antigen solutions and washing five times with phosphate-buffered saline (PBS) including 0.05% Tween 20 (T-PBS), plates were blocked with 1% bovine serum albumin (BSA) in PBS for 2 h at room temperature (RT). After emptying the wells and washing five times with T-PBS, 100 l of serum sample diluted (1:100) in PBS was added to each well and incubated for 1 h at RT. Then samples were removed and wells were washed five times with T-PBS, after which each well was incubated for 30 min with a 1:2000 dilution of rabbit antihuman IgG F(ab )2 conjugated with horseradish peroxidase (DAKO, Carpinteria, CA). After removal of this antibody solution and washing five times with T-PBS, each well was developed by o-phenyldiamine. After a 10-min incubation in darkness, the reaction was stopped with 0.5N H2 SO4 and absorbance was measured at 492 nm. Data were obtained in triplicate for each sample.
Fig. 1 Enzyme-linked immunosorbent assay results for anti-livin antibodies in sera from patients with lung cancers (n = 37) were compared with findings in healthy controls (n =7). The cutoff value for positivity was 0.816. A492 , absorbance at 492 nm.
stage was as follows: 0 of 6 stage I and II patients, 8 of 11 stage III patients, and 12 of 20 stage IV patients (Table 1). The cutoff value for positivity in the anti-survivin ELISA, determined from healthy donor samples as the mean absorbance +2S.D., was 1.1594. Sera from 18 of 31 lung cancer patients (58.1%) were positive by the ELISA using recombinant survivin protein (Fig. 2). Subgroup positivity according to clinical stage was as follows: 2 of 6 stage I and II patients, 5 of 9 stage III patients, and 12 of 16 stage IV patients (Table 1).
2.3. Preabsorption of samples with recombinant survivin, livin or GFP Serum samples (100 l of a 1:100 dilution) were incubated with 30 g/ml of recombinant livin, survivin or GFP control antigen for 1 h at 37 ◦ C and then subjected to the anti-livin or anti-survivin ELISA described above.
3. Results 3.1. Detection of anti-livin and anti-survivin antibodies The cutoff value for positivity in the anti-livin ELISA, determined from healthy donor samples as the mean absorbance +2S.D., was 0.816. Sera from 19 of 37 lung cancer patients (51.3%) were positive by the ELISA using recombinant livin protein (Fig. 1). Subgroup positivity according to clinical
Fig. 2 Enzyme-linked immunosorbent assay results for anti-survivin antibodies in sera from patients with lung cancers (n = 31) and from controls (n = 7). The cutoff value for positivity was 1.1594. A492 , absorbance at 492 nm.
220
Fig. 3 Lack of a relationship between intensities of antilivin antibodies and anti-survivin antibodies in sera from lung cancer patients. Spectrophotometric absorbance for the anti-survivin antibody enzyme-linked immunosorbent assay was plotted against that for anti-livin antibody (y = 0.444x + 0.234, r = 0.225). A492 , absorbance at 492 nm.
Sera from 31 lung cancer patients were assessed simultaneously with the anti-survivin and antilivin ELISAs. Intensity of anti-survivin antibody responses did not correlate with intensity of anti-livin antibody responses (y = 0.444x + 0.234, r = 0.225) (Fig. 3). Among the 31 sera, 22 (71%) were positive for one or both ELISAs using the respective proteins. There are no significant differences in the intensities of antibody responses to livin or survivin between NSCLC and SCLC. In addition, the intensities of antibody responses to livin or survivin did not correlate with tumor size, lymph node status or distant metastasis.
3.2. Preabsorption of anti-livin and antisurvivin antibodies To determine the specificity of the livin and survivin ELISAs, all sera were preabsorbed with recombinant livin, survivin, or control GFP antigen. Data were obtained in triplicate for each sample. Reactivity of sera decreased significantly (p < 0.05) after absorption with recombinant livin or survivin protein, but not after absorption with recombinant control GFP (Mann—Whitney U-test).
4. Discussion Semiquantitative RT-PCR methods have detected survivin and livin mRNA in NSCLC, while indicating little or no expression in noncancerous lung tissues [17—21]. In addition, the finding of anti-survivin antibodies has been reported in sera from lung cancer
A. Yagihashi et al. patients, suggesting their tumors may overexpress survivin inducing a specific antibody response. However, similar data had not been reported concerning anti-livin antibody. In this study, anti-livin antibodies were detected for the first time in sera from lung cancer patients by an anti-livin ELISA using full-length recombinant livin protein. Anti-livin antibodies were detected in excess of cutoff values in 19 of 37 lung cancer patients (51.3%). When most of the same sera were analyzed for anti-survivin antibodies, 18 of 31 were positive (58.1%). The results suggest that similarly to finding for survivin, overexpression of livin in lung cancers had induced antibody responses to this protein. Specificity of livin or survivin recognition was confirmed by lack of reactivity with GFP protein using the same procedure described for livin or survivin and by competition for anti-livin or antisurvivin reactivity by preabsorption of the sera with livin or survivin protein; preabsorption with GFP did not affect these reactivities. In this study of lung cancer, anti-survivin antibodies were found in all clinical stages, but anti-livin only in advanced stages. We, previously, reported for gastric cancer that anti-livin antibodies were detected only in sera from patients with advanced disease [15]. These results may imply that larger expression of livin in lung cancer is needed for inducing antibody responses to livin compared with survivin. Thus, positivity for antilivin antibodies may have a prognostic significance in lung cancer. In addition, in this study, 71% of lung cancer patients were seropositive in ELISAs using recombinant survivin protein, or recombinant livin protein, or in both. This result suggests that in combination, ELISA for anti-livin antibodies and anti-survivin antibodies may be useful in detection of lung cancer [12,13]. However, the number of patients presently examined was very small, so further studies are needed to establish the clinical significance of anti-livin antibodies. Little is known about the mechanism by which livin and survivin are presented to the immune system. Recently, livin-derived HLA-A2 and -A3 restricted epitopes were found to be presented on surfaces of various cancer cells as were, survivinderived HLA-A1, -A2, A3, -A11, -A24 and -B35 restricted epitopes [22—27]. Spontaneous immune responses against these antigen-derived peptides were detected in patients with a variety of cancers. These reports suggest that livin and survivin act as major cancer antigens and can induce both T- and B-cell responses in cancer patients. Schmollinger et al. recently reported that a melanoma patient vaccinated with irradiated autologous melanoma cells incorporating a transgene
Anti-livin antibody against lung cancer causing overexpression of granulocyte-macrophage colony-stimulating factor developed cytotoxic T lymphocyte reactivity against livin-derived peptides and antibody responses to recombinant full-length livin protein [28]. This suggests that anti-livin antibodies might serve to identify patients likely to benefit from new modalities of cancer immunotherapy that target livin. In conclusion, we demonstrated that like antisurvivin antibodies, anti-livin antibodies can be detected in lung cancer patients. Overexpression of livin in lung cancers apparently incites antibody responses to livin. In addition, our results suggest that sensitive assay for anti-livin antibodies in combination with anti-survivin antibodies could be of use in detecting lung cancer, but larger studies are needed.
References [1] Li F. Survivin study: what is next wave? J Cell Physiol 2003;197:8—29. [2] Kasof GM, Gomes BC. Livin, a novel inhibitor of apoptosis protein family member. J Biol Chem 2001;276:3238—46. [3] Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 1997;3:917—21. [4] Vucic D, Stennicke HR, Pisabarro MT, Salvesen GS, Dixit VM. ML-IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas. Curr Biol 2000;10:1359—66. [5] Lin JH, Deng G, Huang Q, Morser J. KIAP, a novel member of the inhibitor of apoptosis protein family. Biochem Biophys Res Commun 2000;279:820—31. [6] Ashhab Y, Alian A, Plliack A, Panet A, Yehuda D. Two splicing variants of apoptosis gene with different biological properties and tissue distribution pattern. FEBS Lett 2001;495:56—60. [7] Sarela AI, Macadam RCA, Farmery SM, Markham AF, Guillou PJ. Expression of the antiapoptosis gene, survivin, predicts death from recurrent colorectal carcinoma. Gut 2000;46:645—50. [8] Nakamura M, Tsuji N, Asanuma K, Kobayashi D, Yagihashi A, Hirata K, et al. Survivin as a predictor of cisdiamminedichloroplatinum sensitivity in gastric cancer patients. Cancer Sci 2004;95:44—51. [9] Lu CD, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res 1998;58:1808—12. [10] Nasu S, Yagihashi A, Izawa A, Saito K, Asanuma K, Nakamura M, et al. Survivin mRNA expression in patients with breast cancer. Anticancer Res 2002;22:1839—43. [11] Gazzaniga P, Gradilone A, Giuliani O, Silvestri I, Nofroni I, Frati L, et al. Expression and prognostic significance of livin, survivin and other apoptosis-related genes in
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the progression of superficial bladder cancer. Ann Oncol 2003;14:85—90. Zhang JY, Casiano CA, Peng XX, Koziol JA, Chan EKL, Tan EM. Enhancement of antibody detection in cancer using panel of recombinant tumor-associated antigens. Cancer Epidemiol Biomarkers Prev 2003;12:136—43. James A, Zhang JY, Casiano CA, Peng XX, Shi FD, Feng AC, et al. Recursive partitioning as an approach to selection of immune markers for tumor diagnosis. Clin Cancer Res 2003;9:5120—6. Yagihashi A, Asanuma K, Nakamura M, Araya J, Mano Y, Torigoe T, et al. Detection of anti-survivin antibody in gastrointestinal cancer patients. Clin Chem 2001;47:1729—31. Yagihashi A, Asanuma K, Tsuji N, Torigoe T, Sato N, Hirata K, et al. Detection of anti-livin antibody in gastrointestinal cancer patients. Clin Chem 2003;49:1206—8. Rohayem J, Diestelkoetter P, Weigle B, Oehmichen A, Schmitz M, Mehhorn J. Cancer Res 2000;60:1815—7. Monz´ o M, Rosell R, Felip E, Astudillo J, S´ anchez JJ, Maestre J, et al. A novel anti-apoptosis gene: re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers. J Clin Oncol 1999;17:2100—4. Ikehara M, Oshita F, Kameda Y, Ito H, Ohgane N, Suzuki R, et al. Expression of survivin correlated with vessel invasion is a marker of poor prognosis in small adenocarcinoma of the lung. Oncol Rep 2002;9:835—8. Falleni M, Pellegrini C, Marchetti A, Oprandi B, Buttitta F, Barassi F, et al. Survivin gene expression in early-stage nonsmall cell lung cancer. J Pathol 2003;200:620—6. Hofmann HS, Simm A, Hammer A, Silber RE, Bartling B. Expression of inhibitors of apoptosis (IAP) proteins in nonsmall cell human lung cancer. J Cancer Res Clin Oncol 2002;128:554—60. Tanabe H, Yagihashi A, Tsuji N, Shijubo Y, Abe S, Watanabe N. Expression of survivin mRNA and livin mRNA in non-smallcell lung cancer. Lung Cancer 2004;46:299—304. Shmitz M, Diestelkoetter P, Weigle B, Schmachtenberg F, Stevanovic S, Ockert D, et al. Generation of survivinspecific CD8+ T effector cells by dendritic cells pulsed with protein or selected peptides. Cancer Res 2000;60:4845—9. Reker S, Meier A, Holten-Anderson L, Svane IM, Becker JC, thor Straten P, et al. Identification of novel survivn-derived CTL epitopes. Cancer Biol Ther 2004;3:173—9. Hirohashi Y, Torigoe T, Maeda A, Nabeta Y, Kamiguchi K, Sato T, et al. An HLA-A24-restricted cytotoxic T lymphocyte epitope of a tumor-associated protein, survivin. Clin Cancer Res 2002;8:1731—9. Reker S, Becker JC, Svane IM, Ralfkaier, thor Straten P, Anderson MH. HLA-B35-restricted immune responses against survivin in cancer patients. Int J Cancer 2004;108:937—41. Anderson MH, Reker S, Becker JC, thor Straten P. The melanoma inhibitor of apoptosis protein: a target for spontaneous cytotoxic T cell responses. J Invest Dermatol 2004;122:392—9. Anderson MH, Becker JC, thor Straten P. Identification of an HLA-A3-restricted cytotoxic T lymphocyte (CTL) epitope from ML-IAP. J Invest Dermatol 2004;122:1336—7. Schmollinger JC, Vonderheide RH, Hoar KM, Maecker B, Schultze J, Hodi FS, et al. Melanoma inhibitor of apoptosis protein (ML-IAP) is a target for immune-mediated tumor destruction. Proc Natl Acad Sci USA 2003;100:3398—403.
Detection of autoantibodies to livin and survivin in Sera from lung cancer patients Atsuhito Yagihashia, Koichi Asanumaa, Daisuke Kobayashia, Naoki Tsujia, Yasuharu Shijubob, Shosaku Abeb, Yoshihiko Hirohashic, Toshihiko Torigoec, Noriyuki Satoc, Naoki Watanabea,∗ a
Department of Clinical Laboratory Medicine, Sapporo Medical University School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060-8543, Japan b Department of Internal Medicine, Sapporo Medical University School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060-8543, Japan c Department of Pathology, Sapporo Medical University School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060-8543, Japan Received 29 July 2004 ; received in revised form 28 October 2004; accepted 2 November 2004 KEYWORDS Livin; Survivin; Autoantibody; Lung cancer
Summary Survivin and livin are highly expressed in cancer cells and transformed cells, but show little or no expression in normal differentiated tissues. Although human antibody responses to cancer-associated antigens have been detected, the response to livin has not yet been described in lung cancer patients. We examined prevalence of anti-livin antibodies in such patients with a specific enzyme-linked immunosorbent assay (ELISA) using recombinant protein. Using a cutoff value for positivity determined as the mean absorbance +2S.D. for healthy control samples, 19 of 37 lung cancer patients (51.3%) were positive for antilivin antibodies. Of 31 samples from the same lung cancer patients, 18 (58.1%) were positive for anti-survivin antibodies. When sera from 31 lung cancer patients were assessed simultaneously by anti-survivin and anti-livin ELISAs. Twenty-one patients (71%) were positive for survivin, livin, or both. Intensity of anti-livin antibody responses did not correlate with intensity of anti-survivin responses. Like anti-survivin antibodies, anti-livin antibodies, thus, can be detected in many lung cancer patients. Testing for both antibodies together may prove useful in detecting lung cancer, but more extensive studies are needed to establish the clinical significance of anti-livin antibodies. © 2004 Elsevier Ireland Ltd. All rights reserved.
1. Introduction * Corresponding author. Tel.: +81 11 611 2111x3640;
fax: +81 11 622 7502. E-mail address: [email protected] (N. Watanabe).
Six human inhibitors of apoptosis protein (IAP) have been identified in recent years: c-IAP1, c-IAP2, XIAP, NIAP, survivin, and livin [1—3]. Each contains
0169-5002/$ — see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2004.11.002
218 one or more baculovirus IAP repeat domain, which is necessary to bind specifically to a terminal effector cell-death protease (e.g., caspase-3 and -7). This binding substantially reduces caspase activity, and reduces cell death in response to a variety of apoptotic stimuli. Among these IAP members, survivin and livin are highly expressed in cancer cells and transformed cells, but show little or no expression in normal differentiated tissues [1—11]. Antibody responses to tumor-associated antigens such as p53, p62, c-myc, and cyclin B1 are known to develop in cancer patients [12,13]. We reported recently that 25 of 63 sera from gastrointestinal cancer patients (39.7%) reacted with purified recombinant survivin an enzyme-linked immunosorbent assay (ELISA), while 17 of 35 sera from these patients (47%) were reactive in an ELISA using purified recombinant livin [14,15]. Rohayem et al. reported that 11of 51 sera from lung cancer patients (21.6%) and 4 of 49 sera from colorectal cancer patients (8.2%) reacted with purified recombinant survivin were reactive in their ELISA [16]. These reports indicate that survivin and livin can induce an antibody response in some patients with various cancers. Abundant survivin expression has been demonstrated in non-small-cell lung cancer (NSCLC) by reverse transcription-polymerase chain reaction (RT-PCR) as well as immunohistochemical detection [17—20]. These studies found high survivin expression to be related to poor survival in NSCLC patients, but not to various clinicopathologic factors [17]. In addition, overexpression of survivin in lung cancers had induced antibody responses to this protein [12,13,16]. Recently, overexpression of livin mRNA also was found in NSCLC [21]. As with survivin overexpression, livin overexpression in lung cancers may lead to anti-livin antibody responses against the cancer. However, there are no available data concerning antibody responses to livin in lung cancer patients. We, therefore, hypothesized that antibody responses to livin are induced in some lung cancer patients, and examined the prevalence such responses in this patient population.
2. Material and methods 2.1. Samples Blood samples were collected from 28 patients with non-small-cell lung cancers (NSCLS) and 9 patients
A. Yagihashi et al.
Table 1 Characteristics of patients and absorbances of anti-livin and survivin antibodies Case
Histology
Clinical Anti-livin stage antibody
Anti-survivin antibodya
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad Ad SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCLC SCLC SCLC SCLC SCLC SCLC SCLC SCLC SCLC
I I III III IV IV IV IV IV IV IV IV IV IV IV IV IV I I II III III III III III IV IV IV II III III III III IV IV IV IV
0.9238 0.7042 1.1619 1.2731 0.7894 1.1544 0.7182 1.0291 1.176 1.1604 1.2095
0.356 0.681 0.269 0.934 0.284 0.331 0.431 0.624 0.789 0.994 1.021 1.103 1.106 1.3 1.426 1.844 2.406 0.25 0.577 0.27 0.267 1.494 1.109 1.738 2.12 0.089 0.725 1.733 0.629 0.263 1.078 1.303 1.547 0.234 0.673 1.228 1.344
1.1501 1.5471 1.0611 1.3683 1.0681 1.4573 1.152 1.0934 1.3367 1.4852 1.4148 1.4813 1.1126 1.4496 1.347 1.6757 1.152 1.2167 1.46 1.4204
Clinical stages were evaluated according to the criteria of the Japanese Society of Lung Cancer. Ad: adenocarcinoma; SCC: squamous cell carcinoma; SCLC: small-cell carcinoma. Anti-livin and anti-survivin antibodies were measured using the anti-livin or anti-survivin ELISAs. a Absorbance was measured at 492 nm.
with small-cell lung cancer (SCLC) after histologic diagnosis. As a control, blood samples were collected from 7 nonhospitalized adults who were over 40 and had no malignancy. Informed consent was obtained from all blood donors. After centrifugation, sera were divided into aliquots and stored at −80 ◦ C. These lung cancer patients were diagnosed as being in stages I—IV based on TNM classification (UICC) (Table 1).
Anti-livin antibody against lung cancer
219
2.2. Immunoassays for anti-livin antibody and anti-survivin antibody Preparation of purified recombinant livin, survivin and green fluorescence protein (GFP) was performed according to the method described previously [14,15]. As an antigen for coating wells in an ELISA, purified recombinant livin or survivin was diluted in 50 mM bicarbonate buffer (pH 9.5) to a final protein concentration of 5 g/ml. Purified recombinant GFP was used as a control antigen at the same concentration in the same buffer. Livin, survivin or control antigen solution was placed in wells of 96well plates (Corning, NY) and incubated overnight at 4 ◦ C. After removing antigen solutions and washing five times with phosphate-buffered saline (PBS) including 0.05% Tween 20 (T-PBS), plates were blocked with 1% bovine serum albumin (BSA) in PBS for 2 h at room temperature (RT). After emptying the wells and washing five times with T-PBS, 100 l of serum sample diluted (1:100) in PBS was added to each well and incubated for 1 h at RT. Then samples were removed and wells were washed five times with T-PBS, after which each well was incubated for 30 min with a 1:2000 dilution of rabbit antihuman IgG F(ab )2 conjugated with horseradish peroxidase (DAKO, Carpinteria, CA). After removal of this antibody solution and washing five times with T-PBS, each well was developed by o-phenyldiamine. After a 10-min incubation in darkness, the reaction was stopped with 0.5N H2 SO4 and absorbance was measured at 492 nm. Data were obtained in triplicate for each sample.
Fig. 1 Enzyme-linked immunosorbent assay results for anti-livin antibodies in sera from patients with lung cancers (n = 37) were compared with findings in healthy controls (n =7). The cutoff value for positivity was 0.816. A492 , absorbance at 492 nm.
stage was as follows: 0 of 6 stage I and II patients, 8 of 11 stage III patients, and 12 of 20 stage IV patients (Table 1). The cutoff value for positivity in the anti-survivin ELISA, determined from healthy donor samples as the mean absorbance +2S.D., was 1.1594. Sera from 18 of 31 lung cancer patients (58.1%) were positive by the ELISA using recombinant survivin protein (Fig. 2). Subgroup positivity according to clinical stage was as follows: 2 of 6 stage I and II patients, 5 of 9 stage III patients, and 12 of 16 stage IV patients (Table 1).
2.3. Preabsorption of samples with recombinant survivin, livin or GFP Serum samples (100 l of a 1:100 dilution) were incubated with 30 g/ml of recombinant livin, survivin or GFP control antigen for 1 h at 37 ◦ C and then subjected to the anti-livin or anti-survivin ELISA described above.
3. Results 3.1. Detection of anti-livin and anti-survivin antibodies The cutoff value for positivity in the anti-livin ELISA, determined from healthy donor samples as the mean absorbance +2S.D., was 0.816. Sera from 19 of 37 lung cancer patients (51.3%) were positive by the ELISA using recombinant livin protein (Fig. 1). Subgroup positivity according to clinical
Fig. 2 Enzyme-linked immunosorbent assay results for anti-survivin antibodies in sera from patients with lung cancers (n = 31) and from controls (n = 7). The cutoff value for positivity was 1.1594. A492 , absorbance at 492 nm.
220
Fig. 3 Lack of a relationship between intensities of antilivin antibodies and anti-survivin antibodies in sera from lung cancer patients. Spectrophotometric absorbance for the anti-survivin antibody enzyme-linked immunosorbent assay was plotted against that for anti-livin antibody (y = 0.444x + 0.234, r = 0.225). A492 , absorbance at 492 nm.
Sera from 31 lung cancer patients were assessed simultaneously with the anti-survivin and antilivin ELISAs. Intensity of anti-survivin antibody responses did not correlate with intensity of anti-livin antibody responses (y = 0.444x + 0.234, r = 0.225) (Fig. 3). Among the 31 sera, 22 (71%) were positive for one or both ELISAs using the respective proteins. There are no significant differences in the intensities of antibody responses to livin or survivin between NSCLC and SCLC. In addition, the intensities of antibody responses to livin or survivin did not correlate with tumor size, lymph node status or distant metastasis.
3.2. Preabsorption of anti-livin and antisurvivin antibodies To determine the specificity of the livin and survivin ELISAs, all sera were preabsorbed with recombinant livin, survivin, or control GFP antigen. Data were obtained in triplicate for each sample. Reactivity of sera decreased significantly (p < 0.05) after absorption with recombinant livin or survivin protein, but not after absorption with recombinant control GFP (Mann—Whitney U-test).
4. Discussion Semiquantitative RT-PCR methods have detected survivin and livin mRNA in NSCLC, while indicating little or no expression in noncancerous lung tissues [17—21]. In addition, the finding of anti-survivin antibodies has been reported in sera from lung cancer
A. Yagihashi et al. patients, suggesting their tumors may overexpress survivin inducing a specific antibody response. However, similar data had not been reported concerning anti-livin antibody. In this study, anti-livin antibodies were detected for the first time in sera from lung cancer patients by an anti-livin ELISA using full-length recombinant livin protein. Anti-livin antibodies were detected in excess of cutoff values in 19 of 37 lung cancer patients (51.3%). When most of the same sera were analyzed for anti-survivin antibodies, 18 of 31 were positive (58.1%). The results suggest that similarly to finding for survivin, overexpression of livin in lung cancers had induced antibody responses to this protein. Specificity of livin or survivin recognition was confirmed by lack of reactivity with GFP protein using the same procedure described for livin or survivin and by competition for anti-livin or antisurvivin reactivity by preabsorption of the sera with livin or survivin protein; preabsorption with GFP did not affect these reactivities. In this study of lung cancer, anti-survivin antibodies were found in all clinical stages, but anti-livin only in advanced stages. We, previously, reported for gastric cancer that anti-livin antibodies were detected only in sera from patients with advanced disease [15]. These results may imply that larger expression of livin in lung cancer is needed for inducing antibody responses to livin compared with survivin. Thus, positivity for antilivin antibodies may have a prognostic significance in lung cancer. In addition, in this study, 71% of lung cancer patients were seropositive in ELISAs using recombinant survivin protein, or recombinant livin protein, or in both. This result suggests that in combination, ELISA for anti-livin antibodies and anti-survivin antibodies may be useful in detection of lung cancer [12,13]. However, the number of patients presently examined was very small, so further studies are needed to establish the clinical significance of anti-livin antibodies. Little is known about the mechanism by which livin and survivin are presented to the immune system. Recently, livin-derived HLA-A2 and -A3 restricted epitopes were found to be presented on surfaces of various cancer cells as were, survivinderived HLA-A1, -A2, A3, -A11, -A24 and -B35 restricted epitopes [22—27]. Spontaneous immune responses against these antigen-derived peptides were detected in patients with a variety of cancers. These reports suggest that livin and survivin act as major cancer antigens and can induce both T- and B-cell responses in cancer patients. Schmollinger et al. recently reported that a melanoma patient vaccinated with irradiated autologous melanoma cells incorporating a transgene
Anti-livin antibody against lung cancer causing overexpression of granulocyte-macrophage colony-stimulating factor developed cytotoxic T lymphocyte reactivity against livin-derived peptides and antibody responses to recombinant full-length livin protein [28]. This suggests that anti-livin antibodies might serve to identify patients likely to benefit from new modalities of cancer immunotherapy that target livin. In conclusion, we demonstrated that like antisurvivin antibodies, anti-livin antibodies can be detected in lung cancer patients. Overexpression of livin in lung cancers apparently incites antibody responses to livin. In addition, our results suggest that sensitive assay for anti-livin antibodies in combination with anti-survivin antibodies could be of use in detecting lung cancer, but larger studies are needed.
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