Barbara Williamson

Immunohistochemical analysis of NY‐ESO‐1 antigen expression in normal and malignant human tissues

NY‐ESO‐1, a member of the CT (cancer/testis) family of antigens, is expressed in normal testis and in a range of human tumor types. Knowledge of NY‐ESO‐1 expression has depended on RT‐PCR detection of mRNA and there is a need for detecting NY‐ESO‐1 at the protein level. In the present study, a method for the immunochemical detection of NY‐ESO‐1 in paraffin‐embedded tissues has been developed and used to define the expression pattern of NY‐ESO‐1 in normal tissues and in a panel of human tumors. No normal tissue other than testis showed NY‐ESO‐1 reactivity, and expression in testis was restricted to germ cells particularly spermatogonia. In human tumors, the frequency of NY‐ESO‐1 antigen expression corresponds with past analysis of NY‐ESO‐1 mRNA expression e.g., 20–30% of lung cancers, bladder cancers and melanoma, and no expression in colon and renal cancer. Co‐typing of NY‐ESO‐1 antigen and mRNA expression in a large panel of lung cancers showed a good correlation. There is great variability in NY‐ESO‐1 expression in individual tumors, ranging from an infrequent homogeneous pattern of staining to highly heterogeneous antigen expression.

Antitumor Monoclonal Antibodies Enhance Cross-Presentation of Cellular Antigens and the Generation of Myeloma-specific Killer T Cells by Dendritic Cells

The mechanism of antitumor effect of monoclonal antibodies (mAbs) is not fully understood. Here we show that coating myeloma cells with anti–syndecan-1 antibody promotes cross-presentation of cellular antigens by dendritic cells (DCs) to autologous T cells from healthy donors. The tumor cells treated with anti–syndecan-1 or isotype-matched control antibody were fed to HLA-mismatched monocyte-derived immature DCs. Tumor cell–loaded mature DCs induced a strong CD8+ T cell response that was specific for the cancer-testis (C-T) antigens expressed in the tumor. The CD8+ T cells killed peptide-pulsed targets, as well as myeloma tumor cells. Importantly, mAbs-coated tumor-loaded DCs were consistently superior to DCs loaded with peptides or dying cells for eliciting tumor-specific killer T cells. This enhanced cross-presentation was not due to enhanced tumor cell uptake or to DC maturation. When mixtures of NY-Eso-1-positive and -negative myeloma cells were captured by DCs, the anti–syndecan-1 antibody had to be on the NY-Eso-1-positive cells to elicit NY-Eso-1–specific response. Cross-presentation was inhibited by pretreatment of DCs with Fcγ receptor blocking antibodies. Targeting of mAb-coated tumors to DCs may contribute to the efficacy of tumor-reactive mAb and offers a new strategy for immunotherapy.

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. Int. J. Cancer76:652–658, 1998.© 1998 Wiley‐Liss, Inc.

Cancer-testis genes are coordinately expressed and are markers of poor outcome in non-small cell lung cancer.

Purpose: Cancer-testis genes mapping to the X chromosome have common expression patterns and show similar responses to modulators of epigenetic mechanisms. We asked whether cancer-testis gene expression occurred coordinately, and whether it correlated with variables of disease and clinical outcome of non–small cell lung cancer (NSCLC). Experimental Design: Tumors from 523 NSCLC patients undergoing surgery were evaluated for the expression of nine cancer-testis genes (NY-ESO-1, LAGE-1, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, CT7/MAGE-C1, SSX2, and SSX4) by semiquantitative PCR. Clinical data available for 447 patients were used to correlate cancer-testis expression to variables of disease and clinical outcome. Results: At least one cancer-testis gene was expressed by 90% of squamous carcinoma, 62% of bronchioloalveolar cancer, and 67% of adenocarcinoma samples. Statistically significant coexpression was observed for 34 of the 36 possible cancer-testis combinations. Cancer-testis gene expression, either cumulatively or individually, showed significant associations with male sex, smoking history, advanced tumor, nodal and pathologic stages, pleural invasion, and the absence of ground glass opacity. Cox regression analysis revealed the expression of NY-ESO-1 and MAGE-A3 as markers of poor prognosis, independent of confounding variables for adenocarcinoma of the lung. Conclusions: Cancer-testis genes are coordinately expressed in NSCLC, and their expression is associated with advanced disease and poor outcome.

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. Int. J. Cancer 83:456–464, 1999.

Vaccine-Induced CD4+ T Cell Responses to MAGE-3 Protein in Lung Cancer Patients

MAGE-3 is the most commonly expressed cancer testis Ag and thus represents a prime target for cancer vaccines, despite infrequent natural occurrence of MAGE-3-specific immune responses in vivo. We report in this study the successful induction of Ab, CD8+, and CD4+ T cells in nonsmall cell lung cancer patients vaccinated with MAGE-3 recombinant protein. Two cohorts were analyzed: one receiving MAGE-3 protein alone, and one receiving MAGE-3 protein with adjuvant AS02B. Of nine patients in the first cohort, three developed marginal Ab titers and another one had a CD8+ T cell response to HLA-A2-restricted peptide MAGE-3 271–279. In contrast, of eight patients from the second cohort vaccinated with MAGE-3 protein and adjuvant, seven developed high-titered Abs to MAGE-3, and four had a strong concomitant CD4+ T cell response to HLA-DP4-restricted peptide 243–258. One patient simultaneously developed CD8+ T cells to HLA-A1-restricted peptide 168–176. The novel monitoring methodology used in this MAGE-3 study establishes that protein vaccination induces clear CD4+ T cell responses that correlate with Ab production. This development provides the framework for further evaluating integrated immune responses in vaccine settings and for optimizing these responses for clinical benefit.

Identification of cancer/testis genes by database mining and mRNA expression analysis.

Cancer/testis (CT) antigens are immunogenic proteins expressed predominantly in gametogenic tissue and cancer; they are considered promising target molecules for cancer vaccines. The identification of new CT genes is essential to the development of polyvalent cancer vaccines designed to overcome tumor heterogeneity and antigen loss. In the current study, a search for new CT genes was conducted by mining the Unigene database for gene clusters that contain expressed sequence tags derived solely from both normal testis and tumor‐derived cDNA libraries. This search identified 1,325 different cancer/testis‐associated Unigene clusters. The mRNA expression pattern of 73 cancer/testis‐associated Unigene clusters was assessed by reverse transcriptase polymerase chain reaction. Three gene products, CT15/Hs.177959, CT16/Hs.245431 and CT17/Hs.178062, were detected only in testis and in tumor tissue. CT15 is equivalent to ADAM2/fertilin‐β. CT16, an uncharacterized gene product, has homology (30–50%) to members of the GAGE gene family and is 89% identical to CT16.2/Hs.293317, indicating that CT16 and CT16.2 are members of a new GAGE gene family. The uncharacterized gene product, CT17, has homology (30%) to phospholipase A1. RT‐PCR analysis showed that CT15 is expressed exclusively in renal cancer, whereas CT16 and CT17 are expressed in a range of human cancers. Real‐time RT‐PCR analysis of newly defined CT genes and the prototype CT antigens, MAGE‐3 and NY‐ESO‐1, revealed low levels (less than 3% of the level detected in testis) of CT15, CT16 and NY‐ESO‐1 in a limited range of normal, non‐gametogenic tissues. This study demonstrates the merits of database mining with respect to the identification of tissue‐restricted gene products expressed in cancer.

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.

Immunomic analysis of human sarcoma

The screening of cDNA expression libraries from human tumors with serum antibody (SEREX) has proven to be a powerful method for identifying the repertoire of tumor antigens recognized by the immune system of cancer patients, referred to as the cancer immunome. In this regard, cancer/testis (CT) antigens are of particular interest because of their immunogenicity and restricted expression patterns. Synoivial sarcomas are striking with regard to CT antigen expression, with >80% of specimens homogeneously expressing NY-ESO-1 and MAGE-A3. In the present study, 54 sarcoma patients were tested for serum antibodies to NY-ESO-1, SSX2, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, CT7, and CT10. Two patients had detectable antibodies to CT antigens, and this seroreactivity was restricted to NY-ESO-1. Thus, although highly expressed in sarcoma, CT antigens do not induce frequent humoral immune responses in sarcoma patients. Sera from these two patients were used to immunoscreen cDNA libraries from two synovial sarcoma cell lines and normal testis, resulting in the identification of 113 distinct antigens. Thirty-nine antigens were previously identified by SEREX analysis of other tumor types, and 23/39 antigens (59%) had a serological profile that was not restricted to cancer patients, indicating that only a proportion of SEREX-defined antigens are cancer-related. A novel CT antigen, NY-SAR-35, mapping to chromosome Xq28 was identified among the cancer-related antigens, and encodes a putative extracellular protein. In addition to testis-restricted expression, NY-SAR-35 mRNA was expressed in sarcoma, melanoma, esophageal cancer, lung cancer and breast cancer. NY-SAR-35 is therefore a potential target for cancer vaccines and monoclonal antibody-based immunotherapies.

NY-ESO-1 Expression and Immunogenicity in Malignant and Benign Breast Tumors

NY-ESO-1 is a cancer/testis antigen expressed in normal adult tissues solely in the testicular germ cells of normal adults and in various cancers. It induces specific humoral and cellular immunity in patients with NY-ESO-1-expressing cancer. The aim of this study was to determine the frequency of NY-ESO-1 mRNA and protein expression in malignant and benign breast tumors. NY-ESO-1 mRNA expression was detected by conventional reverse transcription-PCR and real-time PCR, and that of the protein expression by immunohistochemistry and Western blot analysis. Expression of NY-ESO-1 mRNA was detected in 37 of 88 (42%) cancer specimens, whereas that of the NY-ESO-1 protein was detected only in 1 mRNA-positive specimen. In the latter case, expression level of NY-ESO-1 mRNA relative to that in the testis was relatively high (75% of testicular expression) and to the other among breast cancer specimens. In benign breast lesions, 21 of 31 (68%) specimens expressed low levels of NY-ESO-1 mRNA. In 1 case of fibroadenoma, NY-ESO-1 mRNA was 8% of the testicular level, and protein was detected by Western blot analysis. Only 1 breast cancer patient had detectable antibody at time of surgery, which disappeared within 2 years. Tumor specimen from this patient was both NY-ESO-1 mRNA and protein positive, and NY-ESO-1-specific CD8 T cells were detected in this patient by IFN-γ enzyme-linked immunospot assay using NY-ESO-1 recombinant adeno and vaccinia virus. A higher rate of NY-ESO-1 expression was noted in breast cancer with high histological grade and negative hormone receptor status, suggesting NY-ESO-1 as a potential tumor antigen for immunotherapy in patients with breast cancer and poor prognosis.