Kristin Iversen

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.

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.

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.

Analysis of microphthalmia transcription factor expression in normal tissues and tumors, and comparison of its expression with S-100 protein, gp100, and tyrosinase in desmoplastic malignant melanoma.

Microphthalmia transcription factor (Mitf) is a nuclear protein involved in the development of melanocytes and the regulation of melanin synthesis. Recent studies have suggested that Mitf may be a more sensitive and specific melanocyte marker than S-100 protein and gp100. However, there is insufficient knowledge on the specificity of Mitf, and a systematic examination of its use for the recognition of desmoplastic melanoma has not yet been performed. In this study, we compared the expression of Mitf with S-100 protein, gp100, and tyrosinase in 20 desmoplastic melanomas by using the antibodies D5 (anti-Mitf), anti-S100P, HMB-45 (anti-gp100), and T311 (anti-tyrosinase). All 20 melanomas were positive for S-100 protein, 7 were positive for Mitf, 6 for gp100, and 11 for tyrosinase. To examine the specificity of Mitf, a panel of normal tissue and 386 samples of miscellaneous tumors, including dermal and subcutaneous spindle cell lesions relevant for the differential diagnosis of desmoplastic melanoma, were examined by immunohistochemistry. Furthermore, normal tissue samples were tested for Mitf mRNA by reverse transcriptase polymerase chain reaction (rt-PCR). Immunoreactivity for Mitf was seen not only in melanocytes of normal skin, but also in macrophages, lymphocytes, fibroblasts, Schwann cells, and smooth muscle cells at various sites, and tumors derived thereof. Our results indicate that the antibody D5 lacks sufficient sensitivity and specificity for widespread diagnostic use. Especially in re-excisions, when immunohistochemistry is often needed to distinguish an inflamed scar tissue from tumor, the presence of immunopositive inflammatory cells and fibroblasts limits the diagnostic use of D5.

Monophasic and biphasic synovial sarcomas abundantly express cancer/testis antigen ny‐eso‐1 but not mage‐a1 or ct7

Synovial sarcomas are high‐grade malignant mesenchymal tumors with biphasic (BSS) and monophasic (MSS) variants that carry a pathognomonic cytogenetic alteration, t(X;18), involving the SYT gene on chromosome 18 and one of several SSX genes on chromosome X, usually SSX1 or SSX2. Cancer/testis (CT) antigens are expressed in a variety of malignant neoplasms but, in normal tissues, are restricted to male germ cells. Previous analysis revealed a high incidence and homogeneous expression of MAGE CT antigen in synovial sarcomas. The present study was performed to analyze the expression of 3 CT antigens, NY‐ESO‐1, MAGE‐A1 and CT7, by immunohistochemistry with 3 monoclonal antibodies (MAbs), ES121 (anti‐NY‐ESO‐1), MA454 (anti‐MAGE‐A1) and CT7‐33 (anti‐CT7), in 25 synovial sarcomas (12 MSS, 13 BSS) typed for the t(X;18)‐derived fusion transcript by RT‐PCR (19 SYT‐SSX1, 6 SYT‐SSX2). NY‐ESO‐1 immunoreactivity was found in 20/25 (80%) cases, and antigen expression was homogeneous in 14/20 NY‐ESO‐1‐positive cases. Both morphologic variants and both translocation types were NY‐ESO‐1‐positive, whereas 5 SYT‐SSX1 tumors (1 MSS, 4 BSS) were NY‐ESO‐1‐negative. MAb MA454 was immunoreactive with 4/25 cases (2 MSS, 2 BSS; 3 SYT‐SSX1, 1 SYT‐SSX2), and MAb CT7‐33 was immunoreactive with only 2/25 cases (both BSS, SYT‐SSX1). Expression of MAGE‐A1 and CT7 was heterogeneous in all positive cases. Our study shows that NY‐ESO‐1 is highly expressed in a homogeneous pattern in synovial sarcomas of both morphologic variants and both translocation types, making these tumors an attractive target for NY‐ESO‐1 antigen‐based immunotherapy.

Expression of melanocytic differentiation markers in malignant melanomas of the oral and sinonasal mucosa.

Malignant melanomas of the oral and sinonasal mucosa are rare tumors. Amelanotic variants can, on occasion, be difficult to recognize by routine light microscopy. Immunohistochemical studies may be needed for a final diagnosis. A number of new monoclonal antibodies to melanocytic differentiation antigens have been studied recently on primary cutaneous and metastatic melanoma. However, little is known about these antibodies for the diagnosis of mucosal melanomas. In this study the authors analyzed 79 oral and sinonasal mucosal melanomas of 65 patients. A total of 35 tumors originated from the oral mucosa (21 primary tumors, eight local recurrences, and six metastases) and 44 melanomas were from the sinonasal tract (27 primary tumors, nine local recurrences, and eight metastases). Immunohistochemical studies were performed on paraffin-embedded tissues, using the following antibodies: anti-S-100 protein, T311 (anti-tyrosinase), A103 (anti-Mart-1/Melan-A), D5 (antimicrophthalmia-associated transcription factor), and HMB-45 (anti-gp100). Of 35 oral mucosal tumors, 34 (97%) were positive with anti-S-100 protein, 33 (94%) with T311, 30 (85%) with A103, 26 (74%) with D5, and 25 (71%) with HMB-45. All five desmoplastic melanomas of the oral mucosa were positive for S-100 protein, four for tyrosinase, and one each for HMB-45 and A103. No desmoplastic melanoma was positive with D5. All 44 sinonasal melanomas were positive for tyrosinase and Mart-1/Melan-A (100%). Forty-three (98%) were positive with HMB-45, 42 (95%) with anti-S-100 protein, and 40 (91%) with D5. These results reveal that T311 is the most sensitive marker for sinonasal melanomas and closely approaches the sensitivity of anti-S-100 protein for oral mucosal melanomas. For desmoplastic mucosal tumors, anti-S-100 protein remains the most sensitive marker.

CT7 (MAGE‐C1) antigen expression in normal and neoplastic tissues

CT7 (MAGE‐C1) is a member of the cancer testis (CT) antigen family. The present study describes the generation of CT7‐33, a monoclonal antibody (MAb) to CT7, and the preliminary protein expression analysis of CT7 in normal tissues and in a limited number of neoplastic lesions. CT7‐33 was effective in frozen as well as formalin‐fixed, paraffin‐embedded tissues, and immunohistochemistry/reverse transcriptase polymerase chain reaction (RT‐PCR) co‐typing demonstrated antibody specificity. CT7‐33 immunoreactivity in normal adult tissues is restricted to testicular germ cells. In neoplastic lesions, CT7‐33 immunostaining is confined to tumor cells, and the frequency of CT7 protein expression mostly parallels previous mRNA analyses. Whereas colorectal and renal cell carcinomas, as well as sarcomas, exhibit poor or no CT7‐33 staining, carcinomas of the mammary gland and ovary, nonsmall cell lung carcinoma and metastatic melanomas exhibit a high incidence of CT7 protein expression. However, as seen in previous analyses of other CT antigens, the expression pattern is mostly heterogeneous, and tumors with more than 50% of tumor staining are only infrequently encountered. In summary, our study presents a new serologic reagent for the analysis of CT7 on a protein level and confirms what is known with regard to the expression pattern of other CT antigens in tumors: frequent heterogeneity of antigen expression.

T311--an anti-tyrosinase monoclonal antibody for the detection of melanocytic lesions in paraffin embedded tissues.

Tyrosinase is a key enzyme in melanin biosynthesis and represents a marker of melanocytic differentiation. We previously generated T311, a murine monoclonal antibody to the tyrosinase recombinant protein. This study was performed to evaluate T311 as a diagnostic immunohistochemical reagent for use on formalin-fixed paraffin-embedded pathological material. We analyzed the specificity of the antibody on a panel of normal and neoplastic tissues, and we assessed its sensitivity in a large number of metastatic and primary malignant melanomas, nevi, three angiomyolipomas, and two vitiligo specimens. T311 revealed intense reactivity on paraffin-embedded material. Immunoreactivity was limited to cells of melanocytic differentiation and no immunostaining was present in unrelated normal tissues and tumors. Eighty-four percent of metastatic malignant melanomas were immunoreactive with T311 and showed predominantly a homogeneous expression pattern. However, in primary melanomas of the desmoplastic/spindle cell type, T311 revealed a poor immunoreactivity. Nevi showed intense staining at the junctional zone, while the dermal component revealed decreasing reactivity towards deeper areas. Only one angiomyolipoma was focally immunoreactive with T311. Vitiligo specimens were immunonegative. We conclude that T311 is a specific and sensitive marker for the detection of melanocytic lesions in formalin-fixed paraffin-embedded tissues and a useful serological reagent for diagnostic pathology.

Immunoreactivity with the Anti-MAGE Antibody 57B in Malignant Melanoma: Frequency of Expression and Correlation with Prognostic Parameters

The melanoma-associated antigen (MAGE) family consists of a number of antigens initially recognized by cytotoxic T lymphocytes, which are currently being investigated for immunotherapy of patients with metastatic melanoma and other tumor types. Expression of MAGE mRNA in melanocytic tumors is said to be restricted to invasive malignant tumors and absent in nevi. Recently, a monoclonal antibody (57B) has become available to examine MAGE protein expression in archival material. In this study, we performed immunohistochemical analysis on 132 melanocytic nevi and 205 melanomas (85 primary cutaneous melanomas and 120 metastatic tumors) to determine the frequency of MAGE expression and to explore a potential correlation with various prognostic parameters. None of the melanocytic nevi and none of the 20 in situ melanomas was immunopositive with the antibody 57B. Immunoreactivity was present in 17 of 65 (26%) primary invasive melanomas of the skin and in 30 of 120 (25%) metastatic tumors. Positive immunostaining did not correlate with tumor stage (P =.66), Breslow thickness (P =.39), Clark level (P =.5), or the histologic type of melanoma (P =.23) but was associated with a brisk infiltrate of lymphocytes involving the vertical growth phase of melanomas (P =.01). Because tumor-infiltrating lymphocytes in melanoma are associated with longer survival, our findings suggest a potential prognostic role for MAGE. Furthermore, the seeming restriction of immunopositivity to invasive malignant tumors suggests a potential diagnostic role for the antibody 57B in confirming the malignant potential of a melanocytic tumor.