Seroreactivity against PTEN-induced putative kinase 1 (PINK1) in Turkish patients with Behçet's disease.

BACKGROUND: Behçet's disease (BD) is a multisystem inflammatory disorder characterized by recurrent oral ulcers, genital ulcers and ocular inflammation, as well as skin, joint, vascular, pulmonary, central nervous system (CNS) and gastrointestinal tract manifestations. The etiopathogenesis of BD has not yet been identified; but it has generally been accepted that several environmental factors may induce an inflammatory attack in genetically susceptible individuals. In this study, we aimed to identify antigens that could elicit high-titer IgG responses by the serological analysis of recombinant expression of cDNA libraries method (SEREX). METHODS: We screened a human testis cDNA library with pooled sera obtained from 4 BD patients by SEREX. Antigens that were identified with the initial analysis were selected for seroreactivity analysis of a larger group of BD patients (n=78) and controls (n=66) by serological immunoscreening. RESULTS: We observed seroreactivity against 6 antigens using the pooled sera. These included rabaptin 5 (RABPT5), PTEN-induced putative kinase 1 (PINK1), switch associated protein 70 (SWAP70), interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), ankyrin repeat domain 20 family, member A1 (ANKRD20A1), and an unknown antigen. Eleven out of 82 (13.4%) BD patients were found to have antibodies elicited against PINK1 antigen, when none of the control sera showed reactivity (p=0.001). There was no significant difference in the frequency of other defined antigens between the patient and control groups. However, among BD clinical sub-groups, anti-SWAP70 antibodies were found to associate with vascular involvement. DISCUSSION: In this study, antibodies against PINK1 were found to specifically associate with BD while SWAP70 antibody was associated with clinical sub-groups of BD. Although variations in both genetic background and environmental factors may affect the outcome of serological responses, our results suggest that serological screening can be used to identify antigens that elicit antibody responses associated with BD.

Human lung cancer antigens recognized by autologous antibodies: definition of a novel cDNA derived from the tumor suppressor gene locus on chromosome 3p21.3.

Serological analysis of a recombinant lung cancer cDNA expression library with the autologous patient serum led to the isolation of 20 clones representing 12 different genes: 4 of these were known genes, and the other 8 were previously unknown genes. Of the four known genes, aldolase A (NY-LU-1), previously shown to be overexpressed in lung cancer, was most frequently isolated. The other three genes were annexin XI, human HIV Rev-interacting protein Rip-1, and the human homologue of the ATP-binding arsA component of the bacterial arsenite transporter, all of which are known to be widely expressed in human tissues. Among the eight unknown genes, of most interest was NY-LU-12. Cloning of full-length NY-LU-12 showed that this cDNA was derived from the same gene as g16, a partially sequenced gene that mapped to the lung cancer tumor suppressor gene locus on chromosome 3p21. The reported g16 sequence, however, was significantly shorter (2433 versus 3591 bp). As a result of alternate splicing and subsequent frameshift, the reported g16 protein is 603 amino acids shorter than the NY-LU-12 product (1123 residues) at its COOH terminus and would therefore lack the epitopes recognized by the autologous serum. Analysis of the putative NY-LU-12 protein sequence predicted that it is a nuclear zinc finger protein with two RNA-binding domains, and Southern analysis showed that this gene is partially deleted in the lung cancer line NCI-H740 but not in nine other lung cancer lines. Screening of normal and cancer patient sera showed anti-NY-LU-12 seroreactivity in 2 of 21 allogeneic lung cancer patients but not in 24 patients with other tumors or in 16 sera from healthy donors. Comparison of NY-LU-12 cDNA from Lu15 tumor and normal lung tissue by DNA sequencing and/or single-strand conformation polymorphism analysis showed no evidence of mutation. Considering the high frequency of 3p21 alterations in lung cancer and the fact that the tumor suppressor gene or genes in this locus have not been identified, additional studies on the NY-LU-12 gene and its product are warranted.

Identification of a tissue-specific putative transcription factor in breast tissue by serological screening of a breast cancer library.

Application of SEREX (serological analysis of recombinant tumor cDNA expression libraries) to different tumor types has led to the identification of several categories of human tumor antigens. In this study, the analysis of a breast cancer library with autologous patient serum led to the isolation of seven genes, designated NY-BR-1 through NY-BR-7. NY-BR-1, representing 6 of 14 clones isolated, showed tissue-restricted mRNA expression in breast and testis but not in 13 other normal tissues tested. Among tumor specimens, NY-BR-1 mRNA expression was found in 21 of 25 breast cancers but in only 2 of 82 nonmammary tumors. Structural analysis of NY-BR-1 cDNA and the corresponding genomic sequences in the recently released working draft of human genome indicated that NY-BR-1 is composed of 37 exons and has an open reading frame of 4.0-4.2 kb, encoding a peptide of Mr 150,000-160,000. A bipartite nuclear localization signal motif indicates a nuclear site for NY-BR-1, and the presence of a bZIP site (DNA-binding site followed by leucine zipper motif) suggests that NY-BR-1 is a transcription factor. Additional structural features include five tandem ankyrin repeats, implying a role for NY-BR-1 in protein-protein interactions. NY-BR-1 thus represents a breast tissue-specific putative transcription factor with autoimmunogenicity in breast cancer patients. In addition to NY-BR-1, a homologous gene, NY-BR-1.1, was identified in this study. NY-BR-1.1 shares 54% amino acid homology with NY-BR-1 and also shows tissue-restricted mRNA expression. However, unlike NY-BR-1, NY-BR-1.1 mRNA is expressed in brain, in addition to breast and testis. The exon structure of NY-BR-1.1 remains to be defined. Using human genome database, NY-BR-1 was localized to chromosome 10p11-p12, and NY-BR-1.1 was tentatively localized to chromosome 9.

Serological cloning of a melanocyte rab guanosine 5'-triphosphate-binding protein and a chromosome condensation protein from a melanoma complementary DNA library.

Characterization of immunogenic human melanoma antigens has been a major focus of tumor immunologists over the past two decades, and a broad array of antigens recognized by antibodies and T cells in the autologous host has been defined. In the present study, a melanoma library was screened by SEREX (serological analysis of cDNA expression libraries), and 43 genes were isolated, 2 of which, NY-MEL-1 and NY-MEL-3, encode novel gene products with differential tissue expression. NY-MEL-1 encodes a new rab GTP-binding protein, rab38. Among >40 rab proteins, rab38 has a unique COOH terminus which would allow posttranslational farnesylation and palmitoylation, lipid modifications normally occurring in ras proteins but not in other rab proteins. It is also the only rab gene showing a predominant mRNA expression in melanocytes, a cell-specific expression pattern likely related to melanosomal transport and docking. Northern blot analysis showed no detectable expression in other normal tissues. Consistent with this lineage specificity, rab38 mRNA is expressed in 80-90% of melanoma (17 of 19), but rarely in nonmelanocytic malignancies (1 of 16). The second novel gene isolated, NY-MEL-3, encodes a mitotic protein highly homologous to the Xenopus chromosome condensation protein XCAP-G, designated hCAP-G. Analysis of hCAP-G mRNA expression showed highest expression in the testis among normal tissues and variable expression in tumor cells, reflecting the proliferative activity in these cells. This mitosis-related expression suggests hCAP-G as a possible proliferation marker and a potential prognostic indicator in cancer. These findings provide further support that SEREX can define biologically significant molecules in cancer.

Cancer-testis antigens and ING1 tumor suppressor gene product are breast cancer antigens: characterization of tissue-specific ING1 transcripts and a homologue gene.

SEREX (serological analysis of recombinant tumor cDNA expression libraries) has been applied to several different tumor types and has led to the identification of a wide range of tumor antigens. In this study, a breast cancer library and a normal testicular library were analyzed using autologous and allogeneic breast cancer sera. Thirty genes were isolated, including 27 known genes and 3 previously unknown genes. Among the known genes, two cancer-testis (CT) antigens, NY-ESO-1 and SSX2, previously defined by SEREX analysis, were found. In addition, ING1, a candidate breast cancer suppressor gene, was isolated. This ING1 gene product was also recognized by 2 of 14 allogeneic sera from breast cancer patients but not 12 normal adult sera. Comparison of ING1 cDNA from normal and tumor tissues showed no mutation in the index breast cancer case and revealed the presence of at least three different mRNA transcripts with variable transcription initiation sites and exon usage. Tissue-specific expression of these transcripts was found in normal tissues and tumor cell line mRNAs. Furthermore, a novel gene, designated as ING2, sharing 76% nucleotide homology with ING1 was identified in the breast cancer cDNA library. The basis of the immunogenicity of ING1 and the biological role of ING1 and ING2 need further exploration.

Isoforms of the human PDZ-73 protein exhibit differential tissue expression.

Patients with renal and colon cancer frequently develop IgG autoantibodies toward the NY-CO-38/PDZ-73 antigen, a protein of 652 amino acids (73 kDa) which contains three copies of the PDZ protein-protein interaction domain. The gene encoding PDZ-73 mapped to chromosome 11p15.4-p15.1. Additional tissue-specific isoforms were identified: PDZ-45, which lacks the third PDZ domain and the putative PEST protein degradation motif, is expressed in kidney, colon, small intestine, brain and testis; PDZ-54 and PDZ-59, which also lack the third PDZ domains, have unique carboxyl terminal amino acids and are expressed in brain, kidney, bladder, colon cancer and renal cancer; and a putative PDZ-37 isoform, containing only the third PDZ domain, that is expressed in the central nervous system. Immunohistochemical staining with anti-PDZ 73 monoclonal antibodies showed strong cytoplasmic reactivity in epithelial cells of the small intestine, colon and kidney tubules, with a prominent apical staining pattern in cells of the small intestine. The reactivity pattern of the antibodies with various tissues correlated with the mRNA expression pattern of the PDZ-45 isoform. The existence of multiple PDZ-73 isoforms with variations in tissue distribution, PDZ domains, protein degradation sequences and carboxyl terminal structure indicate that these isoforms have distinct tissue-specific functions.

Humoral immunity to human breast cancer: antigen definition and quantitative analysis of mRNA expression.

The ability of the immune system to recognize structurally altered, amplified or aberrantly expressed proteins can be used to identify molecules of etiologic relevance to cancer and to define targets for cancer immunotherapy. In the current study, ninety-four distinct antigens reactive with serum IgG from breast cancer patients were identified by immunoscreening breast cancer-derived cDNA expression libraries (SEREX). A serological profile was generated for each antigen on the basis of reactivity with allogeneic sera from normal individuals and cancer patients, and mRNA expression profiles for coding sequences were assembled based upon the tissue distribution of expressed sequence tags, Northern blots and real-time RT-PCR. Forty antigens reacted exclusively with sera from cancer patients. These included well-characterized tumor antigens, e.g. MAGE-3, MAGE-6, NY-ESO-1, Her2neu and p53, as well as newly-defined breast cancer antigens, e.g. kinesin 2, TATA element modulatory factor 1, tumor protein D52 and MAGE D, and novel gene products, e.g. NY-BR-62, NY-BR-75, NY-BR-85, and NY-BR-96. With regard to expression profiles, two of the novel gene products, NY-BR-62 and NY-BR-85, were characterized by a high level of testicular mRNA expression, and were overexpressed in 60% and 90% of breast cancers, respectively. In addition, mRNA encoding tumor protein D52 was overexpressed in 60% of breast cancer specimens, while transcripts encoding SNT-1 signal adaptor protein were downregulated in 70% of these cases. This study adds to the growing list of breast cancer antigens defined by SEREX and to the ultimate objective of identifying the complete repertoire of immunogenic gene products in human cancer (the cancer immunome).

Expression of cancer-testis antigens in lung cancer: definition of bromodomain testis-specific gene (BRDT) as a new CT gene, CT9.

In an effort to define new cancer-testis (CT) genes, we investigated whether BRDT, a testis-restricted member of the RING3 family of transcriptional regulators, is also expressed in cancer. Standard RT-PCR expression analysis detected BRDT transcripts in 12 of 47 cases of non-small cell lung cancer and single cases of both squamous cell carcinoma of the head and neck (1/12) and esophagus (1/12) but not in melanoma or in cancers of the colon, breast, kidney and bladder. Typing of 33 non-small cell lung cancers for coexpression of a panel of CT antigens revealed a high incidence (60%) of MAGE-3 mRNA expression, followed by MAGE-1 (36%), CT7/MAGE-C1 (30%), CT10 (30%), SSX4 (23%), BRDT (21%), NY-ESO-1 (21%) and HOM-MEL-40/SSX2 (15%). The coexpression pattern of these antigens provides a foundation for developing a polyvalent lung cancer vaccine.

SOX1 antibodies are markers of paraneoplastic Lambert-Eaton myasthenic syndrome.

BACKGROUND/OBJECTIVE: We reported that 43% of patients with Lambert-Eaton myasthenic syndrome (LEMS) and small cell lung cancer (SCLC) had an antibody called anti-glial nuclear antibody (AGNA), defined by the immunoreaction with the nuclei of the Bergmann glia of the cerebellum. This study was undertaken to identify the antigen recognized by AGNA and to confirm the association with paraneoplastic LEMS in a larger series. METHODS: We probed a fetal brain cDNA library with AGNA-positive sera. The presence of antibodies against the isolated antigen was detected by immunoblot of phage plaques from two positive clones. We studied 105 patients with LEMS (55 with SCLC), 50 with paraneoplastic neurologic syndromes, SCLC, and Hu antibodies, and 50 with only SCLC. RESULTS: Probing of the fetal brain expression library with AGNA sera resulted in the isolation of SOX1, a highly immunogenic tumor antigen in SCLC. IgG eluted from SOX1 clones produced the same cerebellar immunoreactivity as of AGNA sera. SOX1 antibodies were present in 64% of patients with LEMS and SCLC but in none of the 50 with idiopathic LEMS (p < 0.0001). Compared with paraneoplastic LEMS, the frequency of SOX1 antibodies was significantly lower in patients with Hu antibodies (32%, p = 0.002) and in those with only SCLC (22%). CONCLUSIONS: SOX1 is the antigen recognized by anti-glial nuclear antibody-positive sera. The detection of SOX1 antibodies in patients with Lambert-Eaton myasthenic syndrome (LEMS) predicts the presence of small cell lung cancer and may be used to follow more closely those LEMS patients with no evidence of cancer at the initial workup.

Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library.

Cancer/testis (CT) antigens-immunogenic protein antigens that are expressed in testis and a proportion of diverse human cancer types-are promising targets for cancer vaccines. To identify new CT antigens, we constructed an expression cDNA library from a melanoma cell line that expresses a wide range of CT antigens and screened the library with an allogeneic melanoma patient serum known to contain antibodies against two CT antigens, MAGE-1 and NY-ESO-1. cDNA clones isolated from this library identified four CT antigen genes: MAGE-4a, NY-ESO-1, LAGE-1, and CT7. Of these four, only MAGE-4a and NY-ESO-1 proteins had been shown to be immunogenic. LAGE-1 is a member of the NY-ESO-1 gene family, and CT7 is a newly defined gene with partial sequence homology to the MAGE family at its carboxyl terminus. The predicted CT7 protein, however, contains a distinct repetitive sequence at the 5' end and is much larger than MAGE proteins. Our findings document the immunogenicity of LAGE-1 and CT7 and emphasize the power of serological analysis of cDNA expression libraries in identifying new human tumor antigens.

SSX: a multigene family with several members transcribed in normal testis and human cancer.

Analysis of t(X;18) translocation in synovial sarcoma had previously led to the definition of the SSX2 gene, the fusion partner on chromosome X. Subsequent screening of testicular cDNA libraries identified 2 highly homologous genes, SSX1 and SSX3. Among these 3 genes, SSX2 has been found to be identical to HOM-MEL-40, which codes for an immunogenic tumor antigen expressed in various human cancers. SSX2 thus belongs to the family of cancer/testis (CT) antigens, i.e., immunogenic protein antigens with characteristic mRNA expression in normal testis and in cancer. To define additional CT antigens, we have immuno-screened a testicular cDNA expression library with an allogeneic serum from a melanoma patient, and both SSX2 and SSX3 were isolated. Further studies using testicular cDNA and SSX probes defined 2 new members of this gene family, SSX4 and SSX5, while a shorter cDNA variant of SSX4 was also identified. All 5 members of the SSX family shared strong sequence homology, with nucleotide homology ranging from 88 to 95% and amino acid homology ranging from 77 to 91%. Genomic cloning of a prototype SSX gene (SSX2) showed that its coding region is encoded by 6 exons, and the shortened form of SSX4 cDNA represents an alternatively spliced product lacking the 5th exon. Analysis of SSX mRNA expression by gene-specific RT-PCR confirmed that all 5 SSX genes are expressed in testis. In addition, analysis of a panel of 12 melanoma cell lines showed strong mRNA expression of either SSX1 (3/12), SSX2 (3/12), SSX4 (1/12), or SSX5 (1/12), indicating variable activation of the genes in malignant cells.

A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening.

Serological analysis of recombinant cDNA expression libraries (SEREX) using tumor mRNA and autologous patient serum provides a powerful approach to identify immunogenic tumor antigens. We have applied this methodology to a case of esophageal squamous cell carcinoma and identified several candidate tumor targets. One of these, NY-ESO-1, showed restricted mRNA expression in normal tissues, with high-level mRNA expression found only in testis and ovary tissues. Reverse transcription-PCR analysis showed NY-ESO-1 mRNA expression in a variable proportion of a wide array of human cancers, including melanoma, breast cancer, bladder cancer, prostate cancer, and hepatocellular carcinoma. NY-ESO-1 encodes a putative protein of Mr 17,995 having no homology with any known protein. The pattern of NY-ESO-1 expression indicates that it belongs to an expanding family of immunogenic testicular antigens that are aberrantly expressed in human cancers in a lineage-nonspecific fashion. These antigens, initially detected by either cytotoxic T cells (MAGE, BAGE, GAGE-1) or antibodies [HOM-MEL-40(SSX2), NY-ESO-1], represent a pool of antigenic targets for cancer vaccination.

Expression of SSX genes in human tumors.

The HOM-MEL-40 antigen which is encoded by the SSX-2 gene was originally detected as a tumor antigen recognized by autologous IgG antibodies in a melanoma patient. Expression analysis demonstrated that SSX-2 is a member of the recently described cancer/testis antigen (CTA) class as it is expressed in a variety of different human neoplasms, but not in normal tissues with the exception of testis and a weak expression in the thyroid. Further studies demonstrated that SSX-2 belongs to a gene family consisting of at least 5 homologous genes. We now report the analysis of the expression of all 5 SSX genes in 325 specimens of human neoplasms from various histological origins, using reverse transcription polymerase chain reaction (RT-PCR). SSX-1, -2, and -4 were found to be expressed in 8%, 15% and 15%, of the tumors, respectively, while the expression of the SSX-5 gene was rare (7/325), and SSX-3 expression was not detected. For defined tumor types, expression of at least one of the SSX family members was most frequently observed in head and neck cancer (75%), followed by ovarian cancer (50%), malignant melanoma (43%), lymphoma (36%), colorectal cancer (27%) and breast cancer (23%), while leukemias and the few cases of leiomyosarcomas, seminomas and thyroid cancers were found not to express any SSX gene.

Characterization of human colon cancer antigens recognized by autologous antibodies.

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) has proven to be a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. In the present study, 48 distinct antigens (NY-CO-1-NY-CO-48) reactive with autologous IgG were identified by SEREX analysis in 4 patients with colon cancer. Sequencing analysis showed that 17 of the cDNA clones were previously uncharacterized molecules and 31 represented known gene products. The individual cDNA clones were analyzed in the following manner: a search for mutations or other structural changes; an analysis of mRNA expression in a panel of normal tissues; and a frequency analysis of the antibody response to the expressed product in the sera of colon cancer patients and normal individuals. The initial analysis showed NY-CO-13 to be a mutated version of the p53 tumor suppressor gene. Three of the 48 antigens showed a differential pattern of mRNA expression, with NY-CO-27 (galectin-4) expressed primarily in gastrointestinal tract, and NY-CO-37 and -38 showing a pattern of tissue-specific isoforms. With regard to immunogenicity, 20 of the 48 antigens were detected by allogeneic sera; 14 of these were reactive with sera from both normal donors and cancer patients, and 6 other clones (NY-CO-8, -9, -13, -16, -20 and -38) reacted exclusively with sera from colon cancer patients (ranging from 14% to 27%). Our results on colon cancer illustrate both the complexity and the potential of the SEREX approach for analysis of the humoral immune response against human cancer.

Serological identification of embryonic neural proteins as highly immunogenic tumor antigens in small cell lung cancer.

Serological analysis of expression cDNA libraries (SEREX) derived from two small cell lung cancer (SCLC) cell lines using pooled sera of SCLC patients led to the isolation of 14 genes, including 4 SOX group B genes (SOX1, SOX2, SOX3, and SOX21) and ZIC2. SOX group B genes and ZIC2 encode DNA-binding proteins; SOX group B proteins regulate transcription of target genes in the presence of cofactors, whereas ZIC2 is also suspected to be a transcriptional regulator. These genes are expressed at early developmental stages in the embryonic nervous system, but are down-regulated in the adult. Although SOX2 mRNA can be detected in some adult tissues, ZIC2 is expressed only in brain and testis, and SOX1, SOX3, and SOX21 transcripts are not detectable in normal adult tissues. Of SCLC cell lines tested, 80% expressed ZIC2 mRNA, and SOX1, SOX2, and SOX3 expression was detected in 40%, 50%, and 10%, respectively. SOX group B and ZIC2 antigens elicited serological responses in 30-40% of SCLC patients in this series, at titers up to 1:10(6). In sera from 23 normal adults, no antibody was detected against SOX group B or ZIC2 proteins except for one individual with low-titer anti-SOX2 antibody. Seroreactivity against SOX1 and 2 was consistently higher titered than SOX3 and 21 reactivity, suggesting SOX1 and/or SOX2 as the main antigens eliciting anti-SOX responses. Although paraneoplastic neurological syndromes have been associated with several SCLC antigens, neurological symptoms have not been observed in patients with anti-SOX or anti-ZIC2 antibodies.

CT10: a new cancer-testis (CT) antigen homologous to CT7 and the MAGE family, identified by representational-difference analysis.

Assays relying on humoral or T-cell-based recognition of tumor antigens to identify potential targets for immunotherapy have led to the discovery of a significant number of immunogenic gene products, including cancer-testis (CT) antigens predominantly expressed in cancer cells and male germ cells. The search for cancer-specific antigens has been extended via the technique of representational-difference analysis and SK-MEL-37, a melanoma cell line expressing a broad range of CT antigens. Using this approach, we have isolated CT antigen genes, genes over-expressed in cancer, e. g., PRAME and KOC, and genes encoding neuro-ectodermal markers. The identified CT antigen genes include the previously defined MAGE-A6, MAGE-A4a, MAGE-A10, CT7/MAGE-C1, as well as a novel gene designated CT10, which shows strong homology to CT7/MAGE-C1 both at cDNA and at genomic levels. Chromosome mapping localized CT10 to Xq27, in close proximity to CT7/MAGE-C1 and MAGE-A genes. CT10 mRNA is expressed in testis and in 20 to 30% of various human cancers. A serological survey identified 2 melanoma patients with anti-CT10 antibody, demonstrating the immunogenicity of CT10 in humans.

Antigens recognized by autologous antibody in patients with renal-cell carcinoma.

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) is a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. Sixty-five distinct antigens (NY-REN-1 to NY-REN-65) reactive with autologous IgG were identified by SEREX analysis of 4 renal cancer patients and were characterized in terms of cDNA sequence, mRNA expression pattern, and reactivity with allogeneic sera. REN-9, -10, -19, and -26 have a known association with human cancer. REN-9 (LUCA-15) and REN-10 (gene 21) map to the small cell lung cancer tumor suppressor gene locus on chromosome 3p21.3. REN-19 is equivalent to LKB1/STK11, a gene that is defective in Peutz-Jeghers syndrome and cancer. REN-26 is encoded by the bcr gene involved in the [t(9:22)] bcr/abl translocation. Genes encoding 3 of the antigens in the series showed differential mRNA expression; REN-3 displays a pattern of tissue-specific isoforms, and REN-21 and REN-43 are expressed at a high level in testis in comparison to 15 other normal tissues. The other 62 antigens were broadly expressed in normal tissues. With regard to immunogenicity, 20 of the 65 antigens reacted only with autologous sera. Thirty-three antigens reacted with sera from normal donors, indicating that their immunogenicity is not restricted to cancer. The remaining 12 antigens reacted with sera from 5-25% of the cancer patients but not with sera from normal donors. Seventy percent of the renal cancer patients had antibodies directed against one or more of these 12 antigens. Our results demonstrate the potential of the SEREX approach for the analysis of the humoral immune response against human cancer.