Humoral immune responses of lung cancer patients against tumor antigen NY-ESO-1

Cancer Letters 236 (2006) 64–71 www.elsevier.com/locate/canlet

Humoral immune responses of lung cancer patients against tumor antigen NY-ESO-1 ¨ zlem Tu¨recia, Ulrich Mackb, Ulrich Luxemburgera, Helma Heinenb, O Frank Krummenauerc, Martina Sesterd, Urban Sesterd, Gerhard W. Sybrechtb, Ugur Sahina,* a

Department of Internal Medicine, Johannes Gutenberg University, Obere Zahlbacherstr.63, D-55131 Mainz, Germany b Department of Pulmonary Medicine, Saarland University, Homburg/Saar, Germany c Department for Medical Biometry, Epidemiology and Informatics, Johannes Gutenberg University, Mainz, Germany d Department of Nephrology, Saarland University, Homburg/Saar, Germany Received 14 February 2005; received in revised form 1 May 2005; accepted 2 May 2005

Abstract The cancer-associated antigen NY-ESO-1 is expressed in a number of malignancies of different histological type. Patients with NY-ESO-1 expressing tumors have been shown to bear circulating autoantibodies against this antigen. In this study, we have assessed the NY-ESO-I autoantibody response in patients with lung cancer by a serum ELISA. Using a serum dilution of 1:400 we detected seroreactivity in 35 of 175 (20%) of patients. Incidence of autoantibodies was significantly higher in patients suffering from non small cell lung cancer (NSCLC, 23%) as compared to those with small cell lung cancer (SCLC, 9%). In the NSCLC group, NY-ESO-I antibody was significantly more frequent in patients with undifferentiated tumors (40%) as compared to patients with either adenocarcinoma or squamous cell carcinoma (15 and 29%). Our observations indicate that induction of NY-ESO-I autoantibodies depends on the histological subtype within a given tumor entity. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Tumor serology; Lung cancer; NY-ESO-I

1. Introduction The molecular characterization of human tumor antigens (TAA) has allowed an improved analysis and a better understanding of human immune responses to cancer. Based on the specific recognition of cancer * Corresponding author. Tel.: C49 66131 3933488; fax: C49 66131 3933364. E-mail address: [email protected] (U. Sahin).

cells by autologous cytotoxic T lymphocytes [1] or autologous sera of cancer patients [2], a large number of TAA has been identified [3,4]. Among these, the cancer/testis (CT) class of antigens, e.g. MAGE [5], SSX2/HOM-MEL-40 [6] and NY-ESO-1 [7] are of particular interest, since they are expressed in a wide range of human tumors, but not in normal tissues except for testis [8]. The CT antigen NY-ESO-1, initially cloned by an autoantibody from a patient with esophageal cancer

0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.05.008

¨ . Tu¨reci et al. / Cancer Letters 236 (2006) 64–71 O

[7] is expressed in a significant proportion of tumors of different histological type. It elicits both cellular and humoral immune responses in patients with NY-ESO-1 expressing tumors. MHC class II [9–11] as well as MHC class I [12,13] restricted epitopes have been identified, the latter being currently tested in clinical trials as peptide vaccines against malignant melanoma [14]. Antibody responses indicative of strong immunogenicity have been detected exclusively in cancer patients and reported in more than half of late stage melanoma patients bearing NY-ESO-1 positive tumors, in patients with breast, ovarian cancer [15] as well as with transitional cell carcinoma [16]. There is evidence, that antibody responses correlate with tumor grade [16], with presence of spontaneous T-cell responses [13] and with the evolution of the disease [17]. This is in contrast with other TAA described so far, which often induce cytotoxic T-lymphocyte (CTL) responses, but rarely detectable serum antibodies (i.e. melanocyte differentiation antigens), or TAA of low immunogenicity, that are associated with cellular and humoral immune responses in !5% of patients with antigen expressing tumors (i.e. MAGE-1, MAGE-3) [15,18,19]. Lung cancer is a tumor type, in which NY-ESO-1 is found frequently and proposed as potential target [20]. However, immune responses in patients suffering from this tumor type have not been systematically explored. In the present study, we have assessed the prevalence of autoantibodies in sera of lung cancer patients and correlated it with clinical and histological criteria.

65

the others extensive local or lymph node involvement (9 patients in stage IIIA, 57 patients in stage IIIB). Only few patients with more limited stages of disease could be recruited (1 patient in stage IB, 5 patients in stage IIB). 2.2. Lambda phage plaque lift assay This method was described in detail elsewhere. Briefly, a lambda phage clone containing a NY-ESO-1 construct was mixed with wild-type phage as internal negative control at a ratio of 1:10. Nitrocellulose membranes were immunoscreened after plaque-lift with 1:200 diluted sera from patients for the presence of high-titered specific IgG [21]. 2.3. Preparation of recombinant His-tagged NY-ESO-1 NY-ESO-1 cDNA was cloned from testis tissue derived total RNA. The PCR product representing the entire coding sequence of this gene was ligated into the prokaryotic expression vector pQE30 (Qiagen, Hilden, Germany), which allows the overexpression of recombinant protein with a 6!His tag and the subsequent purification using nickel-nitrilotriacetic acid resin. Induction of recombinant protein synthesis and purification by NiC2 column were performed according to the manufacturer’s instructions. Purity of the recombinant protein was determined by SDS-PAGE and Coomassie Blue staining. The tumor-associated antigen CT8/HOM-Tes-85 was recombinantly expressed and purified under the same conditions and used as a negative control. 2.4. Enzyme linked immuno sorbent assay

2. Materials and methods 2.1. Sera and patients Human sera were obtained from 175 patients with lung cancers of various subtypes and from 51 normal blood donors and were stored at K80 8C. Most of the patients had large primary tumors (3 patients with T1, 21 patients with T2, 21 patients with T3 and 127 patients with T4 tumors). The majority of the patients had distant metastasis (103 patients), most of

The ELISA was performed as described by Stockert [15] with minor modifications. 50 ml/well of a 1 mg/ml NY-ESO-I protein solution in coating buffer (15 mM Na2CO3, 30 mM NaHCO3, pH 9.6, with 0.02% NaN3) was adsorbed to Maxisorp microwell plates 12!8 (Nunc, Roskilde, Denmark) overnight at 4 8C. Plates were washed with PBS and blocked for 2 h at 37 8C with 100 ml/well of 2% BSA/ PBS. After washing, 50 ml/well of serum dilutions in PBS/2% BSA was added and incubated for 2 h at room temperature. Plates were washed and 50 ml/well

¨ . Tu¨reci et al. / Cancer Letters 236 (2006) 64–71 O

secondary antibody diluted in PBS/2% BSA was added (Goat anti-human IgG-AP; Jackson Immuno research, West Grove, PA, USA) and incubated for 1 h at room temperature. Plates were washed, incubated with 50 ml/well of substrate solution (4-Nitrophenyl phosphate disodiumsalt!6 H2O, Merck, Darmstadt, Germany) for 30 min at room temperature, and immediately read (Wallac Victor2; ¨ berlingen, Germany). Perkin Elmer Instruments, U Sera were tested in a dilution of 1:400 in triplicates. In parallel, sera were tested on the unrelated recombinant protein CT8/HOM-Tes-85 expressed and purified in the same way to disclose reactivity due to unspecific stickiness. OD values obtained as NY-ESO-1 reactivity were corrected for OD values of CT8/HOM-Tes-85 reactivity. To deduce levels of serum-titers from absorbance values, positive sera were tested over a range of 4-fold dilutions from 1:400 to 1:102,400.

statistics as applied by others [15]. There, in analogy to the published gold standard to which we compared, the cut-off for positive antibody reactivity against NY-ESO-1 was set as an OD value of a 1:400 diluted serum that exceeded the mean absorbance of sera from normal donors by 3 standard deviations thus measuring events beyond the 99% percentile. The current investigation, however, was aimed at clinical hypothesis-building rather than comparison of assay techniques. The primary objective was to assess healthy donors

A 2.5

A 405nm

66

2.0 1.5 1.0

cut-off

0.5

2.5. Statistics

0

3. Results

NSCLC

B 2.5

A 405nm

2.0

1.0

0 Adenocarcinoma

In a similar study, which compared a novel autoantibody ELISA approach with an established one [22] we had used the same parametrical

undifferentiated

squamous cell carcinoma

SCLC

C 2.5 2.0 1.5 1.0 0.5

3.1. Establishment of an ELISA for detection of NY-ESO-1 autoantibodies

cut-off

1.5

0.5

A 405nm

Data description was based on medians and quartiles for continuous endpoints (graphical representation on box whisker plots, accordingly) and on absolute and appropriate relative frequencies for binary endpoints. Significance comparisons between patient subsamples were based on two sample Wilcoxon tests for continuous and on exact Fisher tests for binary endpoints. A logistic regression model was used to compare patient subsamples along several competing explanatory factors, where a forward selection was performed due to rather restricted subsample sizes. The results of this regression modeling were summarized via Likelihood ratio tests. For any significance estimation, a p-value !0.05 was regarded as an indication of local statistical significance.

cut-off

0

Fig. 1. Anti-NY-ESO-1 antibodies detected by ELISA in (A) sera from healthy blood donors (control population, nZ51), (B) sera from patients with NSCLC (nZ132) and (C) from patients with SCLC (nZ43). The results shown are mean values of triplicates. The horizontal line indicates the cut-off value for positivity.

¨ . Tu¨reci et al. / Cancer Letters 236 (2006) 64–71 O

the number of sera of patients with significantly different absorbance as compared to the healthy population. Continuous data were assumed to be of non-parametrical distribution. The cut-off for positive seroreactivity was defined as an absorbance of 0.053 (Fig. 1(a)) and determined as OD value of events beyond the 95% percentile of a cohort of 51 normal donors. The median absorbance seen with the sera of healthy blood donors was 0.016 (interquartile range 0.003–0.027). Representatives of sera found as reactive or non-reactive in the ELISA were confirmed by the lambda phage assay as independent method.

67

3.2. Prevalence of NY-ESO-1 autoantibody in lung cancer sera A total of 175 patients with lung cancer were recruited after histopathological confirmation of the tumor. Their sera were subjected to the ELISA and tested for reactivity not only for NY-ESO-I but in parallel also on CT8/HOM-Tes-85, an irrelevant recombinant control protein expressed and purified under the same conditions. Concomitant reactivity to this protein was neither detected in sera from healthy controls nor from patients. Sera with NY-ESO-I

Table 1 Characteristics of patients with NY-ESO-I antibody, including gender, age of patients and histology of their tumor as well as stage of their disease Code

Gender

Age

Absorbance

P577 P004 P592 P477 P305 P097 P026 P638 P571 P125 P676 P366 P111 P154 P176 P160 P269 P417 P219 P169 P593 P585 P358 P016 P165 P178 P112 P006 P424 P488 P213 P414 P002 P107 P053

f f m f m m m m m m m f f m m m m f m m m m m m m m f m m m m m m m f

48 58 64 67 50 66 69 74 60 73 75 69 74 65 61 62 63 77 64 71 58 63 66 73 48 60 60 61 51 72 56 70 69 66 58

1.878 2.594 2.799 0.061 0.055 0.058 2.166 0.260 0.066 0.869 0.067 0.938 0.540 0.287 0.068 0.055 0.114 0.104 0.061 0.079 1.214 0.133 0.531 0.064 0.060 0.075 0.083 0.816 0.256 2.191 0.060 0.102 0.093 0.441 0.060

Tumor-stage T4 T4 T4 T4 T4 T4 T3 T4 T4 T2 T4 T4 T2 T4 T4 T4 Tx T4 T4 T1 T4 T1 T3 T4 T4 T4 T4 T4 T4 T4 T2 T4 Tx T4 T4

N3 Nx N3 N3 N3 N2 N1 N3 N3 N2 N2 N0 N2 N1 N2 N2 Nx N2 N2 N2 N2 N2 N1 N2 N0 N3 N2 Nx N0 N3 N0 N2 Nx N3 N2

M0 M0 M1 M1 M1 M0 M1 M1 M0 M1 M0 M1 M0 M1 M1 M0 M1 M0 M1 M0 M0 M0 M0 M0 M1 M1 M0 M0 M0 M0 M0 M1 M1 M0 M1

Histology IIIB IIIB IV IV IV IIIB IV IV IIIB IV IIIB IV IIIA IV IV IIIB IV IIIB IV IIIA IIIB IIIA IIIA IIIB IV IV IIIB IIIB IIIB IIIB IB IV IV IIIB IV

NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC NSCLC SCLC SCLC SCLC SCLC

Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma undifferentiated undifferentiated undifferentiated undifferentiated undifferentiated undifferentiated squamous squamous squamous squamous squamous squamous squamous squamous squamous squamous squamous squamous squamous squamous squamous squamous

¨ . Tu¨reci et al. / Cancer Letters 236 (2006) 64–71 O

68

A 3.0

A 405nm

2.5 2.0 1.5 1.0 0.5

n=

176

61

0

healthy donors

B

patients with lung cancer

lung cancer 3.0

A 405nm

2.5 2.0 1.5 1.0 0.5

n=

43

132

0

SCLC

C

NSCLC

NSCLC 3.0

A 405nm

2.5 2.0 1.5 1.0 0.5

n= 0

62

adeno

55 squamous

15 undifferentiated

Fig. 2. Box Whisker plots of anti-NY-ESO-1 antibody prevalence comparing (A) healthy and diseased populations, (B) patients with NSCLC versus with SCLC, (C) patients with three different histological subtypes of NSCLC.

reactivity were tested at least three times, and the majority of negative sera were tested twice. Using the cut-off level defined above we detected autoantibodies against NY-ESO-I in 35 patients (Table 1), representing 20% of the cohort (Fig. 1 (b) and (c), Fig. 2). The absorbance of the positive lung cancer sera were between 0.055 and 2.799, with average absorbance being 1.112. Next, we correlated the occurrence of NY-ESO-I autoantibodies with histology (Table 2A). Whereas patients with SCLC rarely have antibodies to this antigen (9%), NY-ESO-I reactivity is significantly more frequent in those diagnosed with NSCLC (23%, Fisher test PZ0.049) (Table 2A). NSCLC is constituted of three major histological subtypes, which according to our results also influence eliciting of NY-ESO-I antibodies. The prevalence of autoantibodies correlates with the histological subtype of NSCLC (Fisher test PZ0.043), in that 15 and 29% of patients with adenocarcinoma and with squamous cell cancer, respectively display NY-ESO-I seroreactivity, whereas autoantibody was significantly more frequent in patients with undifferentiated tumors (40%) (Table 2A). Even though highest values were observed in adenocarcinoma patients with an average absorbance of 1.104G1.219 (nZ9), whereas absorbance in squamous cell cancer (nZ16) and undifferentiated tumors (nZ6) are 0.369G0.588 and 0.462G0.385, statistical analysis failed to show significance of differences in subtype-associated absorbance levels. Noteworthy, also patients with small primary tumors (T1, T2) have autoantibodies to NY-ESO-I, indicating that these may be elicited early in disease (Table 2B). In 16 % of patients with manifestation of distant metastasis (stage IV), but in 26 % of patients with stage !IV we observed seroreactivity towards NY-ESO-I (Table 2C). However, statistical testing did not support significance of this observations. Titration of sera from selected individuals was performed to deduce levels of serum-titers from absorbance values. Fig. 3 illustrates this for patients 063 and 033. Both were typed as NY-ESO-I autoantibody positive upon admission to our study. Sera from different time-points were titered. Our findings indicate that titers of spontaneously occurring

¨ . Tu¨reci et al. / Cancer Letters 236 (2006) 64–71 O

69

Table 2 Correlation between prevalence of NY-ESO-1 autoantibodies and (A) histological subtype of lung cancer, (B) size of primary tumor as well as (C) stage of disease

(A)

SCLC NSCLC NSCLC NSCLC NSCLC Total

(B)

(C)

Adenocarcinoma Squamous Cell Cancer Undifferentiated

Positive

Tested

Proportion (%)

4 31 9 16

43 132 62 55

9 23 15 29

6 35

15 175

40 20

Primary

Positive

Tested

Proportion (%)

T1 T2 T3 T4

2 3 2 26

3 21 21 127

67 14 10 20

Stage

Positive

Tested

Proportion (%)

I,II IIIA IIIB IV

1 4 14 16

6 9 57 103

17 44 25 16

circulating anti-NY-ESO-I antibody in cancer patients were between 1:4,000 and 1:25,600.

2.0

Patient 033

We have studied the prevalence of antibodies against NY-ESO-I, a widely occurring tumor-associated protein, in sera from lung cancer patients. Using an enzyme linked immuno sorbent assay and parametrical statistical analysis, we show that an anti-NY-ESO-I antibody response is detected in 20% of lung cancer patients. Specificity of NY-ESO-I recognition was confirmed by lack of reactivity with an unrelated recombinant tumor-associated protein prepared and tested using the same procedure. Moreover, positive sera were confirmed by a complementary lambda phage assay. The patient population subjected to our study was unselected. In particular, patients have not been typed for NY-ESO-I antigen status of their tumors. However, it is established that stable expression of this antigen in the tumor is a prerequisite for generation and maintenance of the associated autoantibody response [17]. Previous expression studies of

06/07/99 08/30/00 09/14/00 10/12/00 Cut-off

1.0 0.5 0.0 1:400

1:1600

1:6400

1:25600

1:102400

Serum dilution 2.0

Patient 063 06/17/99 08/09/99 11/10/99 01/18/00 Cut-off

1.5

A 405nm

4. Discussion

A 405nm

1.5

1.0 0.5 0.0 1:400

1:1600

1:6400

1:25600

1:102400

Serum dilution

Fig. 3. Representative results of ELISA reactivity with sera from patients 033 and 063 against recombinant NY-ESO-1 titered in 4fold dilutions. Patients’ sera were tested at different time points.

70

¨ . Tu¨reci et al. / Cancer Letters 236 (2006) 64–71 O

NY-ESO-I antigen in lung cancer performed by us and others have detected expression of this antigen in 17–21% of NSCLC specimen tested [7,22,23]. Since in our study 23% of patients with NSCLC have NY-ESO-I antibody, this would suggest that more than 50% of patients with antigen-positive tumors develop associated antibodies. Noteworthy, we found significant differences in prevalence of NY-ESO-I antibody depending on the histological subtype with undifferentiated NSCLC having the highest probability of eliciting an antibody response as compared to adeno- and squamous cell carcinoma. This may well reflect, that expression of NY-ESO-I antigen in NSCLC depends on the histological differentiation of the cancer cells. In fact, analysis of antigen expression in 33 adenocarcinoma and 9 squamous cell cancer of the lung [24] has unraveled differences in NY-ESO-I positivity in these subtypes. Extended studies on a larger panel of tissue specimen including matched tumor/serum samples from individual patients will allow a direct testing of this relationship. Due to the therapeutic procedure in SCLC favoring chemotherapy over surgery, availability of fresh frozen tissue from this patient population for expression analysis is limited. Studies in SCLC cell lines suggest also high frequency of NY-ESO-I antigen in this type of lung cancer [20]. However, we observe that antibody responses are rare in SCLC. It is not clear why only a subset of patients with a tumor type develop a humoral immune response to a particular antigen, which is a consistent finding for all antigen-associated antibody responses in tumors investigated up to now. Immunogenicity may depend on a multitude of factors, among them the level of expression, posttranslational modification but also the MHC class II alleles carried by the individual patients. Anti-NY-ESO-I antibodies have a higher prevalence in lung cancer patients than anti-p53 antibodies which are also described and extensively studied in this tumor entity [25,26]. Most importantly, we detected NY-ESO-I antibody also in patients with small primary tumors and more frequently before distant metastasis occurred. This suggests that generation of NY-ESO-I antibody is an early event. Thus, even though its overall prevalence in an unselected cohort of lung cancer patients is not high, NY-ESO-I is attractive as one among several components of a multi-antigen ELISA we are

currently developing. Our intention is to increase the sensitivity of a serological tumor screening combining several lung cancer associated antigens with high cumulative prevalence. Anti-p53 antibodies have been reported to be detectable several years before the clinical manifestation of lung cancer [27,28]. It is an intriguing question, whether anti-NY-ESO-I antibodies could also serve as an early predictive marker in patients at high risk of developing tumors. This is currently being investigated by assessing sera from patients collected at various time points before and after diagnosis of lung cancer.

Acknowledgements This study was supported by the 5th framework program of the European Commission and by the Deutsche Forschungsgemeinschaft (projects D1 and A14 of the combined project grant SFB 432 as well as Heisenberg scholarship TU 115/3). We wish to thank Christoph Huber for continuous support.

References [1] E. De Plaen, C. Lurquin, B. Lethe, P. van der Bruggen, V. Brichard, J.C. Renauld, et al., Identification of genes coding for tumor antigens recognized by cytolytic T lymphocytes, Methods 12 (1997) 125–142. [2] U. Sahin, O. Tureci, H. Schmitt, B. Cochlovius, T. Johannes, R. Schmits, et al., Human neoplasms elicit multiple specific immune responses in the autologous host, Proc. Natl. Acad. Sci. USA 92 (1995) 11810–11813. [3] U. Sahin, O. Tureci, M. Pfreundschuh, Serological identification of human tumor antigens, Curr. Opin. Immunol. 9 (1997) 709–716. [4] T. Boon, L.J. Old, Cancer Tumor antigens, Curr. Opin. Immunol. 9 (1997) 681–683. [5] P. van der Bruggen, C. Traversari, P. Chomez, C. Lurquin, E. De Plaen, B. van den Eynde, et al., A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma, Science 254 (1991) 1643–1647. [6] O. Tureci, U. Sahin, I. Schobert, M. Koslowski, H. Schmitt, H.J. Schild, et al., The SSX-2 gene, which is involved in the t(X;18) translocation of synovial sarcomas, codes for the human tumor antigen HOM-MEL-40, Cancer Res. 56 (1996) 4766–4772.

¨ . Tu¨reci et al. / Cancer Letters 236 (2006) 64–71 O [7] Y.T. Chen, M.J. Scanlan, U. Sahin, O. Tureci, A.O. Gure, S. Tsang, et al., A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening, Proc. Natl. Acad. Sci. U.S.A 94 (1997) 1914–1918. [8] L.J. Old, Cancer/testis (CT) antigens—a new link between gametogenesis and cancer, Cancer Immun. 1 (2001) 1. [9] E. Jager, D. Jager, J. Karbach, Y.T. Chen, G. Ritter, Y. Nagata, et al., Identification of NY-ESO-1 epitopes presented by human histocompatibility antigen (HLA)-DRB4*0101-0103 and recognized by CD4(C) T lymphocytes of patients with NY-ESO-1-expressing melanoma, J. Exp. Med. 191 (2000) 625–630. [10] G. Zeng, X. Wang, P.F. Robbins, S.A. Rosenberg, R.F. Wang, CD4(C) T cell recognition of MHC class IIrestricted epitopes from NY-ESO-1 presented by a prevalent HLA DP4 allele: association with NY-ESO-1 antibody production, Proc. Natl. Acad. Sci. USA 98 (2001) 3964–3969. [11] H.M. Zarour, B. Maillere, V. Brusic, K. Coval, E. Williams, S. Pouvelle-Moratille, et al., NY-ESO-1 119-143 is a promiscuous major histocompatibility complex class II T-helper epitope recognized by Th1- and Th2-type tumorreactive CD4C T cells, Cancer Res. 62 (2002) 213–218. [12] R.F. Wang, S.L. Johnston, G. Zeng, S.L. Topalian, D.J. Schwartzentruber, S.A. Rosenberg, A breast and melanoma-shared tumor antigen: T cell responses to antigenic peptides translated from different open reading frames, J. Immunol. 161 (1998) 3598–3606. [13] E. Jager, Y.T. Chen, J.W. Drijfhout, J. Karbach, M. Ringhoffer, D. Jager, et al., Simultaneous humoral and cellular immune response against cancer-testis antigen NYESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes, J. Exp. Med. 187 (1998) 265–270. [14] E. Jager, S. Gnjatic, Y. Nagata, E. Stockert, D. Jager, J. Karbach, et al., Induction of primary NY-ESO-1 immunity: CD8C T lymphocyte and antibody responses in peptidevaccinated patients with NY-ESO-1C cancers, Proc. Natl. Acad. Sci. USA 97 (2000) 12198–12203. [15] E. Stockert, E. Jager, Y.T. Chen, M.J. Scanlan, I. Gout, J. Karbach, et al., A survey of the humoral immune response of cancer patients to a panel of human tumor antigens, J. Exp. Med. 187 (1998) 1349–1354. [16] T. Kurashige, Y. Noguchi, T. Saika, T. Ono, Y. Nagata, A. Jungbluth, et al., Ny-ESO-1 expression and immunogenicity associated with transitional cell carcinoma: correlation with tumor grade, Cancer Res. 61 (2001) 4671–4674.

71

[17] E. Jager, E. Stockert, Z. Zidianakis, Y.T. Chen, J. Karbach, D. Jager, et al., Humoral immune responses of cancer patients against “Cancer-Testis” antigen NY-ESO-1: correlation with clinical events, Int. J. Cancer 84 (1999) 506–510. [18] M. Marchand, P. Weynants, E. Rankin, F. Arienti, F. Belli, G. Parmiani, et al., Tumor regression responses in melanoma patients treated with a peptide encoded by gene MAGE-3, Int. J. Cancer 63 (1995) 883–885. [19] S.K. Huang, T. Okamoto, D.L. Morton, D.S. Hoon, Antibody responses to melanoma /melanocyte autoantigens in melanoma patients, J. Invest. Dermatol. 111 (1998) 662–667. [20] L. Lee, R.F. Wang, X. Wang, A. Mixon, B.E. Johnson, S.A. Rosenberg, D.S. Schrump, NY-ESO-1 may be a potential target for lung cancer immunotherapy, Cancer J. Sci. Am. 5 (1999) 20–25. [21] O. Tureci, H. Schmitt, N. Fadle, M. Pfreundschuh, U. Sahin, Molecular definition of a novel human galectin which is immunogenic in patients with Hodgkin’s disease, J. Biol. Chem. 272 (1997) 6416–6422. [22] O. Tureci, U Luxemburger, H. Heinen, U. Mack, G.W. Sybrecht, C. Huber, U. Sahin, CrELISA: a fast and robust enzyme-linked immunosorbent assay bypassing the need for purification of recombinant protein, J. Immunol. Methods 289 (2004) 191–199. [23] B. Lethe, S. Lucas, L. Michaux, C. De Smet, D. Godelaine, A. Serrano, et al., LAGE-1, a new gene with tumor specificity, Int. J. Cancer 76 (1998) 903–908. [24] M.J. Scanlan, N.K. Altorki, A.O. Gure, B. Williamson, A. Jungbluth, Y.T. Chen, L.J. Old, Expression of cancer-testis antigens in lung cancer: definition of bromodomain testisspecific gene (BRDT) as a new CT gene, CT9, Cancer Lett. 150 (2000) 155–164. [25] A. Jungbluth, Y.T. Chen, E. Stockert, K.J. Busam, D. Kolb, K. Iversen, et al., Immuno-histochemical analysis of NY-ESO1 antigen expression in normal and malignant human tissues, Int. J. Cancer 92 (2001) 856–860. [26] K. Angelopoulou, E.P. Diamandis, D.J. Sutherland, J.A. Kellen, P.S. Bunting, Prevalence of serum antibodies against the p53 tumor suppressor gene protein in various cancers, Int. J. Cancer 58 (1994) 480–487. [27] U. Mack, D. Ukena, M. Montenarh, G.W. Sybrecht, Serum anti-p53 antibodies in patients with lung cancer, Oncol. Rep. 7 (2000) 669–674. [28] R. Lubin, G. Zalcman, L. Bouchet, J. Tredanel, Y. Legros, D. Cazals, et al., Serum p53 antibodies as early markers of lung cancer, Nat. Med. 1 (1995) 701–702.