Elisabeth Stockert

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.

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.

Humoral immune responses of cancer patients against "Cancer-Testis" antigen NY-ESO-1: correlation with clinical events.

Humoral immune responses against the “Cancer‐Testis” (CT) antigen NY‐ESO‐1 are frequently observed in patients with NY‐ESO‐1 expressing tumors. This is in contrast to other known tumor antigens (TA) defined by antibody or cytotoxic T cell (CTL) reactivity, i.e., MAGE‐1, MAGE‐3, SSX2, Melan A, and tyrosinase. No NY‐ESO‐1 antibody has been detected in healthy controls and patients with NY‐ESO‐1 negative tumors. In this study, we have assessed the NY‐ESO‐1 serum antibody response in patients with NY‐ESO‐1 positive tumors of different histological types and stages using Western blotting and an ELISA. Of the 12 patients analyzed, 10 had demonstrable NY‐ESO‐1 antibodies at the start of the study. All patients were followed for changes in NY‐ESO‐1 antibody titers during the course of tumor treatment and clinical evolution. In 4 patients, an increase of NY‐ESO‐1 antibody titer was observed with progression of disease or extensive tumor necrosis under treatment. One patient showed a stable NY‐ESO‐1 antibody titer over 3 years along with gradual regression of a large tumor mass. In 5 patients, a decrease of NY‐ESO‐1 antibody was detected: in 1 patient after curative tumor resection, in 3 patients with partial regression of metastatic disease under chemo‐ and immunotherapy, and in another patient with a NY‐ESO‐1 negative tumor relapse. Our results indicate that the induction and maintenance of NY‐ESO‐1 antibody is dependent on the presence of NY‐ESO‐1 expressing tumors. Furthermore, changes in NY‐ESO‐1 antibody titers correlate with the evolution of NY‐ESO‐1 positive disease. Int. J. Cancer (Pred. Oncol.) 84:506–510, 1999.

A103: An anti-melan-a monoclonal antibody for the detection of malignant melanoma in paraffin-embedded tissues.

Melan-A is a previously defined, melanocyte differentiation antigen, and an anti-Melan-A murine monoclonal antibody, A103, was recently developed by our group. In this study, we evaluated A103 immunoreactivity on formalin-fixed, paraffin-embedded tissues, exploring the potential of A103 in the diagnosis of metastatic melanoma. Seventy-five metastatic melanomas, 10 primary melanomas, and 10 benign melanocytic nevi were tested. The reactivity of A103 was compared with HMB-4, an anti-gp100 antibody. Results showed that all nevi were A103 positive, and most primary melanomas were A103 and HMB45 positive. Of 75 metastatic melanomas, 61 (81%) were A103 positive, and 56 (75%) were HMB45 positive. Of 19 HMB45-negative lesions, 8 were A103 positive; of 14 A103-negative lesions, 3 were HMB45 positive. Eleven metastatic lesions, as well as 2 of 10 primary melanomas, were dual negative. These negative cases consisted mainly of the spindle cell and desmoplastic variants. Of the positive cases, A103 showed homogeneous staining in a significantly higher proportion of cases than HMB45 (72% versus 52%). In addition, focal staining with less than 5% reactive tumor cells was seen more frequently in HMB45 (12 of 56) than in A103 (5 of 61). These results indicated that A103 can be used as a first-line antibody in the diagnosis of metastatic melanoma. Our results also showed that A103 reacted with angiomyolipoma, which is known to be HMB45 positive. Of normal tissues, unexpected A103 reactivity was observed in the adrenal cortex, granulosa and theca cells of the ovary, and Leydig cells of the testis. This A103 immunoreactivity in benign and neoplastic tissues of nonmelanocytic origin, the basis of which is unclear, could also be of potential diagnostic value.

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.

Immunoreactivity for A103, an antibody to melan-A (Mart-1), in adrenocortical and other steroid tumors.

The Melan-A (MART-1) gene encodes an antigen recognized by cytotoxic T cells. It has been said to be restricted in its expression to melanocytes. However, here we report the presence of immunoreactivity for A103, an antibody to Melan-A, in five adrenocortical adenomas, 16 primary and 13 metastatic adrenocortical carcinomas, four Leydig cell tumors of the testis, and three Sertoli-Leydig cell tumors of the ovary. To evaluate the potential diagnostic role of this antibody, we studied immunoreactivity for A103 in 111 carcinomas, 40 germ cell tumors, and 33 miscellaneous nonmelanocytic epithelioid tumors. All of them were negative for A103. Our findings suggest that once melanoma is excluded, A103 can aid in the recognition of steroid hormone-producing tumors and may be particularly useful in the diagnosis of adrenocortical carcinoma. The presence of immunoreactivity for A103 practically excludes any other carcinoma that may enter into the differential diagnosis of adrenocortical tumors.

A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) has attracted considerable attention as a target for cancer therapy. Wild-type (wt)EGFR is amplified/overexpressed in a number of tumor types, and several mutant forms of the coding gene have been found, with ΔEGFR, a deletion mutation lacking exons 2–7 of the external domain, being the most common and particularly associated with glioblastoma. We generated monoclonal antibodies (mAbs) against NR6ΔEGFR (mouse fibroblast line NR6 transfected with ΔEGFR). mAb 806 with selective reactivity for NR6ΔEGFR in mixed hemadsorption assays, fluorescence-activated cell sorting, Western blot, and immunohistochemistry was analyzed in detail and compared with mAbs 528 (anti-wtEGFR) and DH8.3 (anti-ΔEGFR). In xenograft tumors and molecularly pretyped glioblastomas, the reactivity pattern was as follows: 528 reactive with amplified and nonamplified wtEGFR; DH8.3 reactive with ΔEGFR; and 806 reactive with amplified/overexpressed wtEGFR (with or without ΔEGFR). In normal tissues, 528 but not DH8.3 or 806 was widely reactive with many organs, e.g., liver expressing high EGFR levels. In glioblastoma and non-CNS tumor panels, 806 was reactive with a high proportion of glioblastomas and a substantial number of epithelial cancers of lung and of head and neck. DH8.3 reactivity was restricted to ΔEGFR-positive glioblastoma. Thus, 806 represents a category of mAbs that recognizes tumors with EGFR amplification/overexpression but not normal tissues or tumors with normal EGFR levels. Our study also indicates that ΔEGFR is restricted to glioblastoma, in contrast to other reports that this mutation is found in tumors outside the brain.

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.

Asparagine Synthetase Activity of Mouse Leukemias

Various transplanted leukemias and normal tissues of the mouse were tested for asparagine synthetase activity. Leukemias susceptible to suppression by asparaginase have little or no synthetase activity. In contrast, leukemias insensitive to asparaginase exhibit substantial and often very high asparagine synthetase activity. Asparaginase-resistant variants of sensitive leukemias also have considerable synthetase activity. Thus the requirement by certain malignant cells of exogenous asparagine, which entails sensitivity to asparaginase, may be ascribed to lack of asparagine synthetase. Development of asparaginase-resistant variants from asparaginase-sensitive lines is consistently associated with acquisition of asparagine synthetase activity.

Identification of NY-ESO-1 Peptide Analogues Capable of Improved Stimulation of Tumor-Reactive CTL

Expression of NY-ESO-1 in a high proportion of different human tumors makes this protein a very attractive vaccine target. NY-ESO-1 peptides, recognized by HLA-A2-restricted CTL, have recently been described. However, it remains unclear how efficiently tumors generate these epitopes, and whether peptide analogues can be used for optimal expansion and activation of NY-ESO-1-specific HLA-A2-restricted CTL. By generating unique CTL clones, we demonstrate that NY-ESO-1-positive tumor cells are efficiently killed by HLA-A2-restricted CTL specific for the peptide epitope NY-ESO-1 157–165. Presentation of this epitope is not affected by the presence or absence of the proteasome subunits low molecular proteins 2 and 7 and is not blocked by proteasome inhibitors, while it is impaired in the TAP-deficient cell line LBL 721.174. NY-ESO-1 157–165 peptide analogues were compared for their antigenicity and immunogenicity using PBL from melanoma patients. Three peptides, containing the carboxyl-terminal cysteine substituted for either valine, isoleucine, or leucine, were recognized at least 100 times more efficiently than the wild-type peptide by specific CTL. Peptide analogues were capable of stimulating the expansion of NY-ESO-1-specific CTL from PBL of melanoma patients much more efficiently than wild-type peptide. These findings define the processing requirements for the generation of the NY-ESO-1 157–165 epitope. Identification of highly antigenic NY-ESO-1 peptide analogues may be important for the development of vaccines capable of expanding NY-ESO-1-specific CTL in cancer patients.