Circulating antibodies to p40AIS in the sera of respiratory tract cancer patients

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CIRCULATING ANTIBODIES TO P40 AIS IN THE SERA OF RESPIRATORY TRACT CANCER PATIENTS K. YAMAGUCHI 1,2 , M. PATTURAJAN 1 , B. TRINK 1 , H. USADEL 1 , W. KOCH 1 , J. JEN 1 and D. SIDRANSKY 1 * 1 Division of Head and Neck Cancer Research, Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, MD, USA 2 Department of Otolaryngology, National Defense Medical College, Saitama, Japan Studies of immune recognition in cancer have defined sev- eral tumor antigens using autologous cytotoxic T lympho- cytes and by detection of serum antibodies to tumor-associ- ated products such as p53 and HER-2/neu. The AIS gene is a p53 homologue with multiple protein products (p40, p51, p63, p73L) on chromosomal arm 3q, frequently amplified and over-expressed in squamous-cell carcinoma of the respira- tory tract. We analyzed the humoral response to p40 AIS (a core domain of AIS products without the transactivation domain) by Western blot and ELISA using bacterially synthe- sized p40 AIS protein. Antibodies were detected in the sera of 17/94 (18%) HNSCCs and 13/76 (17%) lung cancers, including 5/18 (26%) squamous-cell carcinomas. Anti-p40 AIS antibodies were not associated with factors such as sex, age, histopatho- logical grading, extent or size of primary tumor, lymph node involvement and staging. Our results indicate that amplifica- tion and over-expression of p40 AIS may lead to antigen rec- ognition by an autologous host with cancer. AIS may thus represent a new group of developmentally regulated genes that are recognized as tumor antigens. Int. J. Cancer (Pred. Oncol.) 89:524 –528, 2000. © 2000 Wiley-Liss, Inc. Immune recognition of human cancer by an autologous host has helped to define several tumor antigens. Using autologous cyto- toxic T lymphocytes (CTLs), an array of auto-immunogenic tumor antigens have been identified in malignant melanoma, including melanocyte differentiation antigens (Coulie et al., 1994) and tes- ticular antigens aberrantly expressed in tumors (van der Bruggen et al., 1991; Gaugler et al., 1994). The p53 tumor-suppressor gene is also a target of tumor-directed T cells (Nijman et al.,1994; Ropke et al., 1995; Tilkin et al., 1995). Mutated p53 peptides can bind class I HLA haplotypes and generate CTLs capable of lysing neoplastic cells. However, elevation of antibodies through B-cell response has been demonstrated in cancer patients using several molecules, such as p53 (Crawford et al., 1982; Schlichthoiz et al., 1992) or oncogenic HER-2/neu proteins (Disis et al., 1997). An- tibodies directed against both mutated and wild-type p53 peptides have been found in the sera of patients with breast, lung, colon, head-and-neck and other cancers. Serum antibodies to p53 have also been suggested as early markers of lung cancer (Lubin et al., 1995). AIS (p40/p51/p63/p73L) has structural similarity to p53, espe- cially in the DNA-binding domain (Yang et al., 1998; Osada et al., 1998; Trink et al., 1998). The gene was amplified and over- expressed in squamous-cell carcinoma (SCC) of the head and neck and of the lung (AIS). Over-expression of the core 40 kDa domain (p40 AIS ) led to increased growth of rat 1A cells in soft agar and increased tumor growth in nude mice (Hibi et al., 2000). The gene is also critical for epidermal and limb differentiation, as shown in experiments on knockout mice (Mills et al., 1999; Yang et al., 1999); and mutations in this gene lead to the human EEC syn- drome, characterized by ectrodactyly, ectodermal dysplasia and facial clefts (Celli et al., 1999). Unlike p53, several splice variants of AIS are transcribed from 2 different promoters, yielding protein products with (TA) and without (DN) a transactivating domain. The immunogenicity of a 1 DN isotype (CUSP/DNp63a/ p68 AIS ) was demonstrated by Lee et al. (1999) in a unique clinical syndrome, chronic ulcerative stomatitis (CUS). In addition to the presence of auto-antibodies in sera of affected patients, direct immunofluorescence detected antibody deposits in a speckled pat- tern in the basal layer nuclei of their own stratified epithelia. The antigen was thought to be a DNA–protein complex because of a decrease in antigenic activity by DNase I or micrococcal nuclease treatments (Parodi et al., 1990). Our aim was to evaluate the incidence of humoral response against p40 AIS and to analyze the significance of the antibodies as serological markers in respiratory tract cancer patients. We tested the sera of 170 respiratory tract cancer patients and a limited number of controls by Western blotting and ELISA, using bacte- rially synthesized p40 AIS protein. Approximately 20% of patients with lung or head-and-neck cancer harbored high-titer antibodies to p40 AIS . MATERIAL AND METHODS Serum Serum samples from 94 consecutive head-and-neck SCC (HNSCC) and 76 lung-cancer patients were collected pre-opera- tively, and those from 16 age-matched controls free of cancer were obtained from our repository at Johns Hopkins School of Medi- cine. All sera were stored at – 80°C until use. Post-operative staging was recorded following the international TNM staging system (Sobin et al., 1997). Antigens The p40 AIS cDNA fragment (415 to 2,082 bp) including the entire open reading frame (436 to 1,503 bp) was subcloned into the bacterial expression plasmid pET 28c(1) (Novagen, Madison, WI), in which translation starts 109 bp before the cloned fragment. The plasmid was then transformed into BL21(DE3) cells. After induction of translation by 1 mM isopropyl-b-D-thiogalactopyr- anoside, the cell pellet was disrupted by sonication and the insol- uble fraction was solubilized in the presence of 6 M urea. The His-tagged p40 AIS protein was then eluted using metal-chelation chromatography, according to the manufacturer’s instructions (No- vagene). This resulted in production of a 75% pure protein of approximately 45 kDa, which was immunoreactive with a poly- clonal antibody directed against the p40 AIS protein (Hibi et al., 2000) and a polyclonal anti-His antibody (Santa-Cruz Biotechnol- ogy, Santa Cruz, CA) by Western blot analysis. GST-tagged p53 protein and GST protein were purchased from Santa Cruz Bio- technology and anti-p53 antibodies (Ab-6), from Labvision (Fre- mont, CA). Western blotting Eluted proteins (12 mg/plate) were resolved by 10% SDS-PAGE using a 20 cm gel apparatus and transferred to nitrocellulose filters. Transferred filters were cut into 3 mm strips, which were blocked with 5% non-fat dry milk in PBS plus 0.1% Tween-20 (PBST) at *Correspondence to: Division of Head and Neck Cancer Research, Johns Hopkins University, 818 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205-2196, USA. Fax: 1410-614-1411. E-mail: [email protected] Received 16 June 2000; Revised 5 October 2000; Accepted 9 October 2000 Int. J. Cancer (Pred. Oncol.): 89, 524 –528 (2000) © 2000 Wiley-Liss, Inc. Publication of the International Union Against Cancer

Transcript of Circulating antibodies to p40AIS in the sera of respiratory tract cancer patients

Page 1: Circulating antibodies to p40AIS in the sera of respiratory tract cancer patients

CIRCULATING ANTIBODIES TO P40AIS IN THE SERA OF RESPIRATORYTRACT CANCER PATIENTSK. YAMAGUCHI

1,2, M. PATTURAJAN1, B. TRINK

1, H. USADEL1, W. KOCH

1, J. JEN1 and D. SIDRANSKY

1*1Division of Head and Neck Cancer Research, Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University,Baltimore, MD, USA2Department of Otolaryngology, National Defense Medical College, Saitama, Japan

Studies of immune recognition in cancer have defined sev-eral tumor antigens using autologous cytotoxic T lympho-cytes and by detection of serum antibodies to tumor-associ-ated products such as p53 and HER-2/neu. The AIS gene is ap53 homologue with multiple protein products (p40, p51,p63, p73L) on chromosomal arm 3q, frequently amplified andover-expressed in squamous-cell carcinoma of the respira-tory tract. We analyzed the humoral response to p40AIS (acore domain of AIS products without the transactivationdomain) by Western blot and ELISA using bacterially synthe-sized p40AIS protein. Antibodies were detected in the sera of17/94 (18%) HNSCCs and 13/76 (17%) lung cancers, including5/18 (26%) squamous-cell carcinomas. Anti-p40AIS antibodieswere not associated with factors such as sex, age, histopatho-logical grading, extent or size of primary tumor, lymph nodeinvolvement and staging. Our results indicate that amplifica-tion and over-expression of p40AIS may lead to antigen rec-ognition by an autologous host with cancer. AIS may thusrepresent a new group of developmentally regulated genesthat are recognized as tumor antigens. Int. J. Cancer (Pred.Oncol.) 89:524–528, 2000.© 2000 Wiley-Liss, Inc.

Immune recognition of human cancer by an autologous host hashelped to define several tumor antigens. Using autologous cyto-toxic T lymphocytes (CTLs), an array of auto-immunogenic tumorantigens have been identified in malignant melanoma, includingmelanocyte differentiation antigens (Coulieet al., 1994) and tes-ticular antigens aberrantly expressed in tumors (van der Bruggenetal., 1991; Gaugleret al.,1994). Thep53 tumor-suppressor gene isalso a target of tumor-directed T cells (Nijmanet al.,1994; Ropkeet al., 1995; Tilkin et al., 1995). Mutated p53 peptides can bindclass I HLA haplotypes and generate CTLs capable of lysingneoplastic cells. However, elevation of antibodies through B-cellresponse has been demonstrated in cancer patients using severalmolecules, such as p53 (Crawfordet al.,1982; Schlichthoizet al.,1992) or oncogenic HER-2/neu proteins (Disiset al., 1997). An-tibodies directed against both mutated and wild-type p53 peptideshave been found in the sera of patients with breast, lung, colon,head-and-neck and other cancers. Serum antibodies to p53 havealso been suggested as early markers of lung cancer (Lubinet al.,1995).

AIS (p40/p51/p63/p73L)has structural similarity top53, espe-cially in the DNA-binding domain (Yanget al.,1998; Osadaet al.,1998; Trink et al., 1998). The gene was amplified and over-expressed in squamous-cell carcinoma (SCC) of the head and neckand of the lung (AIS). Over-expression of the core 40 kDa domain(p40AIS) led to increased growth of rat 1A cells in soft agar andincreased tumor growth in nude mice (Hibiet al.,2000). The geneis also critical for epidermal and limb differentiation, as shown inexperiments on knockout mice (Millset al., 1999; Yanget al.,1999); and mutations in this gene lead to the human EEC syn-drome, characterized by ectrodactyly, ectodermal dysplasia andfacial clefts (Celliet al.,1999). Unlikep53,several splice variantsof AISare transcribed from 2 different promoters, yielding proteinproducts with (TA) and without (DN) a transactivating domain.

The immunogenicity of a 1DN isotype (CUSP/DNp63a/p68AIS) was demonstrated by Leeet al. (1999) in a unique clinicalsyndrome, chronic ulcerative stomatitis (CUS). In addition to thepresence of auto-antibodies in sera of affected patients, direct

immunofluorescence detected antibody deposits in a speckled pat-tern in the basal layer nuclei of their own stratified epithelia. Theantigen was thought to be a DNA–protein complex because of adecrease in antigenic activity by DNase I or micrococcal nucleasetreatments (Parodiet al., 1990).

Our aim was to evaluate the incidence of humoral responseagainst p40AIS and to analyze the significance of the antibodies asserological markers in respiratory tract cancer patients. We testedthe sera of 170 respiratory tract cancer patients and a limitednumber of controls by Western blotting and ELISA, using bacte-rially synthesized p40AIS protein. Approximately 20% of patientswith lung or head-and-neck cancer harbored high-titer antibodiesto p40AIS.

MATERIAL AND METHODS

SerumSerum samples from 94 consecutive head-and-neck SCC

(HNSCC) and 76 lung-cancer patients were collected pre-opera-tively, and those from 16 age-matched controls free of cancer wereobtained from our repository at Johns Hopkins School of Medi-cine. All sera were stored at –80°C until use. Post-operativestaging was recorded following the international TNM stagingsystem (Sobinet al., 1997).

AntigensThe p40AIS cDNA fragment (415 to 2,082 bp) including the

entire open reading frame (436 to 1,503 bp) was subcloned into thebacterial expression plasmid pET 28c(1) (Novagen, Madison,WI), in which translation starts 109 bp before the cloned fragment.The plasmid was then transformed into BL21(DE3) cells. Afterinduction of translation by 1 mM isopropyl-b-D-thiogalactopyr-anoside, the cell pellet was disrupted by sonication and the insol-uble fraction was solubilized in the presence of 6 M urea. TheHis-tagged p40AIS protein was then eluted using metal-chelationchromatography, according to the manufacturer’s instructions (No-vagene). This resulted in production of a 75% pure protein ofapproximately 45 kDa, which was immunoreactive with a poly-clonal antibody directed against the p40AIS protein (Hibi et al.,2000) and a polyclonal anti-His antibody (Santa-Cruz Biotechnol-ogy, Santa Cruz, CA) by Western blot analysis. GST-tagged p53protein and GST protein were purchased from Santa Cruz Bio-technology and anti-p53 antibodies (Ab-6), from Labvision (Fre-mont, CA).

Western blottingEluted proteins (12mg/plate) were resolved by 10% SDS-PAGE

using a 20 cm gel apparatus and transferred to nitrocellulose filters.Transferred filters were cut into 3 mm strips, which were blockedwith 5% non-fat dry milk in PBS plus 0.1% Tween-20 (PBST) at

*Correspondence to: Division of Head and Neck Cancer Research, JohnsHopkins University, 818 Ross Research Building, 720 Rutland Avenue,Baltimore, MD 21205-2196, USA. Fax:1410-614-1411.E-mail: [email protected]

Received 16 June 2000; Revised 5 October 2000; Accepted 9 October2000

Int. J. Cancer (Pred. Oncol.):89, 524–528 (2000)© 2000 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

Page 2: Circulating antibodies to p40AIS in the sera of respiratory tract cancer patients

4°C overnight. Strips were immersed with each patient’s serum(1:200 dilution) in blocking buffer at room temperature (RT) for30 min. After a washing step, strips were incubated with a1:10,000 dilution of horseradish peroxidase–conjugated goat anti-human IgA1IgG1IgM (H1L) antibody (Santa Cruz Biotechnol-ogy) at RT for 1 hr. The reaction was detected by enhancedchemiluminescence (Amersham, Arlington Heights, IL) after afinal washing step (consisting of 3 changes of PBST buffer for 30min). Each experiment included positive controls incubated withanti-his antibody, serum from tumor H96 and a negative controlserum from a normal individual. Six positive sera were alsoincubated with transferred bacterial cell lysate without the p40plasmid, and no band was seen at the same level as the p40AIS

protein (data not shown).

ELISAWe further purified the eluted p40 protein by gel electrophore-

sis. Five milligrams of protein were separated by SDS-PAGEusing a 20 cm gel apparatus. After incubation with 0.3 M CuCl2,a band corresponding to p40AIS was cut from the negativelystained gel. p40AIS protein was eluted for 2 hr at 37°C in PBScontaining 100 mM EDTA, and the protein was then aliquoted andkept at –80°C until used. The same volume of the gel underneaththe band (with non-specific protein) was treated the same way andused as control extract. The protein was heat-denatured and incu-bated for 2 hr at room temperature with PBS containing 6 M urea.

Each well of the high-binding polystyrene microtiter plates(Corning, Corning, NY) was coated with 50ml of either p40AIS (10mg/ml), control extract, purified GST-p53 (3mg/ml) or GST (3mg/ml) at RT overnight. After 6 extensive washing steps withPBST, plates were blocked with 100ml of PBST and 5% non-fatdry milk for 1 hr at RT. After this washing step, 100ml of sera(diluted 1:100 in blocking buffer) were tested. Plates were incu-bated for 1 hr at RT, followed by another washing step. The samesecondary antibody used for Western blotting was diluted 1:1,000in 100ml of blocking buffer and incubated for 1 hr at RT. After thefinal washing step, plates were developed using 2,29-azino-di-[3-ethylbenzthiazoline sulfonate] diammonium salt (Roche, IN) at RTfor 20 min and optical density (OD) was measured in an ELISAplate reader at 405 nm. Rabbit anti-His antibody (Santa CruzBiotechnology) or anti-p53 antibody (Ab-6) diluted 1:10,000 wereused as positive controls.

A reactivity score (R) for each serum was calculated as net ODrelative to that of a positive control as follows:

R 5 ~ODp40 2 ODcontrol extract!serum/

~ODp40 2 ODcontrol extract!positive control

All samples, including positive controls, were run in duplicate, andmean values of reactivity were used.

Statistical analysisFisher’s test or Pearson’sx2 test were used to evaluate the

correlation between antibodies detected by ELISA and clinico-

pathological parameters such as sex, age, tumor site (HNSCConly), histology, differentiation, extent and size of primary tumor,lymph node involvement and stage.

RESULTS

By Western blotting, a strong positive band was seen in the seraof 20/41 (48.7%) patients with HNSCC and 8/30 (29.7%) withlung cancer (Table I, Fig. 1). However, a faint positive band wasalso detected in 2/16 (12.5%) healthy volunteers. We then em-ployed a more quantitative ELISA after further purification of thep40AIS protein. Analysis of the data obtained from Western blot-ting and ELISA showed an overall agreement of 78% between the2 methods when a cut-off value of 0.34 (mean6 1.1 SD ofnormal) was used for ELISA.

ELISA was then used to detect p40AIS antibodies in the sera of121 additional patients (total 170 patients) with respiratory tractcancer (Fig. 2). A positive test for antibodies directed againstp40AIS using ELISA was defined as a reactivity score.2.7 SDabove the mean value of all healthy controls. Anti-p40AIS antibod-ies were observed in 17/94 (18.1%) patients with HNSCC and13/76 (17.1%) with lung cancer. All normal controls were negativeby this assay.

Table II shows the relationship between positivity of p40AIS

auto-antibodies by ELISA and various clinical/pathological char-acteristics. Higher positivity was observed in SCC and broncho-alveolar carcinoma than adenocarcinomas of lung. The absence ofbroncho-alveolar growth features further decreased the positivityof p40AIS antibodies in adenocarcinoma of the lung (p 5 0.097).Anti-p40AIS antibodies were not significantly associated with fac-tors such as sex, age, site of HNSCC, histopathological grading(G), extent or size of primary tumor, lymph node involvement (N)or staging. However, there was a trend toward a higher frequencyof antibodies in HNSCC patients with advanced stage (p 5 0.085)and in older lung-cancer patients (.65 years) (p 5 0.076). Re-markably, 10/45 (22%) stage I lung-cancer patients were positivefor circulating antibodies to p40.

Because of the structural similarity between p40 and p53, wetested all patient sera with a similar ELISA for p53. Eleven of 94(12%) HNSCC patients and 3/76 (17%) lung-cancer patients dem-onstrated p53 antibodies. There was minimal cross-reactivity insera to these proteins (Table III). All normal controls were nega-tive for p53.

DISCUSSION

TheAIS(p63) gene locus on chromosome 3q27-8 yields severaltranscripts by alternative splicing. We examined antibodies to p40(the core domain ofDN isotypes) in the sera of respiratory tractcancer patients. Antibodies to p40AIS were detected in approxi-mately 20% of HNSCCs and lung cancers. Most patients withp40AIS antibodies harbored SCCs. Over-expression through AISamplification was found to be common in HNSCCs and SCCs of

FIGURE 1 – Detection of anti-p40AIS auto-antibodies by Western blotting. Sera from patients with respiratory tract cancer were tested usingbacterially expressed His-tagged p40AIS protein as antigen for the presence of anti-p40AIS antibodies (see Material and Methods). Eachexperiment included a positive control incubated with anti-His antibody and a negative control with serum from a healthy individual.

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the lung, and forced over-expression in rat 1A cells increasedgrowth in soft agar and tumorigenicity in nude mice (Hibiet al.,2000). Lung adenocarcinomas rarely over-express AIS proteins,and in agreement with this observation, only a small minority ofthese patients demonstrate serum antibodies. The high prevalenceof p40AIS antibodies in patients with broncho-alveolar carcinoma(BAC) is interesting; although the status of the AIS locus in thesetumors has not been studied, it is tempting to speculate that AISproteins may be elevated in BACs at a level similar to thatobserved for SCCs.

Normal epithelial tissues dominantly expressDN AIS/p63 iso-types, and expression typically occurs in the nucleus of basal cells(Yanget al.,1998). Over-expressed genes are thought to elicit animmune response by over-riding thresholds critical for the main-tenance of tolerance (Viola and Lanzavecchia, 1996). In a sero-logical analysis of recombinant cDNA expression (SEREX) onSCC of the lung (Brasset al., 1999), most isolated cDNA cloneswere mapped to the distal arm of chromosome 3 (3q25-qter),where AIS is located. This region is also the most frequentlyover-represented region in SCC of the respiratory tract (Speicheretal., 1995; Bockmu¨hl et al.,1996; Balsaraet al.,1997; Petersenetal., 1997). BecauseAIS is rarely mutated in human cancer (Tanietal., 1999; Hagiwaraet al., 1999; Hibi et al., 2000), genetic am-plification and/or over-expression represent possible mechanismsfor eliciting the humoral response to p40 in respiratory tractcancer. Genetic or epigenetic alterations ofAIS in BAC of the lungremains to be shown. The lack of p53 expression or mutation inthis subtype of lung cancer compared with conventional adenocar-cinoma of the lung has been suggested as an alternative tumori-genic pathway (Wanget al., 1995; Ahrendtet al., 1999).

CUS is a syndrome defined as chronic ulcerative stomatitis withIgG auto-antibodies to keratinocyte nuclei. ADNAIS isotype(CUSP/p68AIS) protein was isolated as an auto-antigen in CUSpatients, and all 9 CUS patients tested had antibody against theprotein (Leeet al., 1999). Investigators confirmed the absence ofthese auto-antibodies by Western blotting and immunoprecipita-tion in control sera from 74 controls consisting of 10 healthyindividuals, 24 patients with recurrent aphthous stomatitis, 6 pa-tients with recurrent aphthomatitis, 6 patients with oral lichenplanus, 2 patients with dermatomyositis, 15 patients with subacutecutaneous lupus, 15 patients with discoid lupus and 2 patients withsystemic lupus erythematosus without cutaneous lesions. Theseresults confirm the virtual absence of high-titer antibodies top40AIS in the limited number of normals tested in our study.Because AIS proteins appear to be essential for epithelial devel-opment and regeneration, the presence of these antibodies in CUSraises the issue of whether they may be functional, potentiallyleading to poor wound healing and chronic ulcerations. Interest-ingly, many cancer patients also demonstrate poor wound healingand chronic ulcerations in clinical practice.

p53 antibodies predict a poor outcome, including increasedfrequency of recurrence in HNSCC patients (Ralhanet al.,1998). We found some cross-reactivity between p53 and p40 inthese patient samples. It is interesting that all 7 patients whoharbored antibodies to p53 and p40 had SCC tumors (6HNSCCs and 1 lung).p53 mutations andp40 amplification arecommon in SCCs, and it is possible that both genetic alterationscould elicit an independent immune response in a subset ofpatients. Serum antibodies to p53 and other developmentalproteins have also been useful in monitoring disease and de-

TABLE I – DETECTION OF ANTI-P40 ANTIBODIES IN RESPIRATORY CANCER PATIENTS BY WESTERN BLOTTING

HNSCCLung cancer

NormalSCC Adeno1 Others Total

Positive 20 3 3 1 8 22

Total 41 7 18 3 30 1648.7% 42.8% 16.7% 33.3% 29.7% 12.5%

1Adeno, adenocarcinoma.–2Minimally positive faint bands.

FIGURE 2 – Detection of serum antibodies to p40AIS in respiratory tract cancer patients by ELISA. p40AIS protein was further purified forELISA as described. A reactivity score was calculated as net OD relative to that for the positive control (see Material and Methods).

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tecting cancers before the onset of clinical symptoms. Antibod-ies to AIS may also become useful in the detection, prognosisand monitoring of patients with SCC.

Antibodies to tumor antigens are usually detected in only smallnumbers (10% to 30%) of patients with cancer. It is plausible thatthe capacity to produce antibodies against tumor antigens dependson the individual status of human leukocyte antigen (HLA) com-plexes. Certain DQ and DR alleles have been frequently found inpatients who produced serum p53 antibodies, and mutatedp53

generated new epitopes that bound HLA complexes (unpublisheddata). Likewise, the ability to produce p40AIS antibodies maydepend on individual HLA status. Human cancer serology is nowadvancing with the use of new panning approaches such asSEREX, providing a way to analyze more thoroughly the humoralimmune response to intracellular cancer antigens (Chenet al.,1997; Old and Chen, 1998). An accumulating array of cancerantigens may eventually provide better diagnostic and therapeuticapproaches for cancer patients.

REFERENCES

AHRENDT, S.A., HALACHMI , S., CHOW, J.T., WU L., HALACHMI , N., YANG,S.C., WEHAGE, S., JEN, J. and SIDRANSKY, D., Rapid p53 sequence analysisin primary lung cancer using an oligonucleotide probe array.Proc. nat.Acad. Sci. (Wash.),96, 7282–7287 (1999).

BALSARA, B.R., SONODA, G.,DE MANOIR, S., SIEGFRIED, J.M., GABRIELSON,E. and TESTA, J.R., Comparative genomic hybridization analysis detectsfrequent, often high-level, overrepresentation of DNA sequences at 3q, 5p,7p, and 8q in human non-small cell carcinomas.Cancer Res.,57, 2116–2120 (1997).

BOCKMUHL, U., SCHWENDEL, A., DIETEL, M. and PETERSEN, I., Distinctpatterns of chromosomal alterations in high- and low-grade head and necksquamous cell carcinomas.Cancer Res.,56, 5325–5329 (1996).

BRASS, N., RACZ, A., BAUER, C., HECKEL, D., SYBRECHT, G. and MEESE, E.,Role of amplified genes in the production of autoantibodies.Blood, 93,2158–2166 (1999).

CELLI, J. and 17OTHERS, Heterozygous germline mutations in the p53homologue p63 are the cause of EEC syndrome.Cell, 99,143–153 (1999).

CHEN, Y.T., SCANLAN, M.J., SAHIN, U., TURECI, O., GURE, A.O., TSANG, S.,WILLIAMSON , B., STOCHERT, E., PFREUNDSCHUH, M. and OLD, L., A testic-ular antigen aberrantly expressed in human cancers detected by autologousantibody screening.Proc. nat. Acad. Sci. (Wash.),94, 1914–1918 (1997).

COULIE, P.G., BRICHARD, V., VAN PEL, A., WOLFEL, T., SCHNEIDER, J.,TRAVERSARI, C., DE PLAEN, E., LURQUIN C., RAUNALD , C.J. and BOON, T.,A new gene coding for a differentiation antigen recognized by autologous

TABLE II – CLINICAL CHARACTERISTICS OF PATIENTS AND ASSOCIATION WITH ANTI-P40 ANTIBODIES

HNSCC Lung cancer

Characteristic Number ofpatients

p40 antibodyCharacteristic Number of

patientsp40 antibody

Positive % p Positive % p

Sex SexMale 69 13 19 Male 36 7 20Female 25 4 16 1.0001 Female 40 6 15 0.6072

Age (years) Age (years),64 52 9 17 ,64 35 9 26.65 42 8 19 0.8282 .65 41 4 10 0.0761

Site HistologyOral cavity 54 9 17 SCC 18 5 28 0.0891,3

Larynx 30 7 23 Ad5 29 2 7Pharynx 8 1 13 BAL6 19 5 26 0.0971,4

Nose 1 0 0 Other 10 1 10Differentiation Differentiation

G1-2 55 9 16 G1-2 46 9 20G3-4 27 4 15 1.0001 G3-4 23 4 17 1.0001

Extent of tumor Extent of tumorT1-2 52 6 12 T1-2 63 11 17T3-4 42 11 26 0.0852 T3-4 11 2 18 1.0001

Tumor size(cm)

Tumor size(cm)

,3 43 5 12 ,3 37 4 11$3 45 10 22 0.2591 $3 37 9 24 0.1431

Lymph node Lymph nodeN0 51 8 16 N0 53 11 21N1 43 9 21 0.5112 N1 20 1 5 0.1601

Stage StageI–II 33 4 12 I 45 10 22III–IV 61 13 21 0.4011 II–IV 29 3 10 0.2271

1Fisher’s exact test.–2x2 test.–3p value for SCCvs. adenocarcinoma.–4p value for BAL vs. adenocarcinoma.–5Ad, adenocarcinoma.–6BAL,bronchoalveolar carcinoma or bronchoalveolar growth features.

TABLE III – COMPARISON OF CIRCULATING ANTI-P53 AND ANTI-P60AIS ANTIBODIES

H & NAnti-p53

LungAnti-p53

1 2 Total 1 2 Total

Anti-p40 Anti-p401 6 11 17 1 11 12 132 5 77 77 2 22 61 63Total 11 83 94 Total 3 73 76

p3 0.0027 p3 0.43511One sCC was positive for both anti-p40 and anti-p53 antibodies.–2An adenocarcinoma and a small-cell carcinoma had anti-p53 antibodies

but not anti-p40 antibodies.–3Fisher’s exact test.

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