Yasmine M. Hijazi

Heterogeneous expression of melanoma-associated antigens and HLA-A2 in metastatic melanoma in vivo.

MART‐1/MelanA and Pmel17/gp100 are melanoma‐associated antigens (MAAs) that can be recognized by tumor‐infiltrating lymphocytes (TILs) capable of mediating successful adoptive therapy in vivo. Analysis of melanoma cell lines in vitro has demonstrated that heterogeneous antigen expression in the context of class I MHC is a significant co‐factor in determining the recognition of melanoma targets by cytotoxic lymphocytes (CTLs). In this study, 217 specimens from 103 patients with metastatic melanoma were examined for the expression of MART‐1/MelanA (monoclonal antibody \[MAb\] M27C10) and Pmel17/gp100 (HMB45 MAb) by immuno‐histochemistry. Marked heterogeneity in the expression of both MAAs was confirmed by analysis of the percentage of positively staining tumor cells or the average intensity of tumor staining. We also noted heterogeneity of expression among multiple lesions taken from different anatomic sites within a patient. A dissociation was noted in the detection of MART‐1 and gp100 in some lesions, with gp100 being undetectable in 24% of the lesions and MART‐1 being undetectable in 11%. In several cases, loss of one MAA was not associated with loss of the other MAA, suggesting that MART‐1 can represent a useful additional marker for the diagnosis of melanoma in gp100 (HMB45)‐negative lesions. Of the 217 specimens, 155 were obtained from HLA‐A*0201 patients, of which 6% were negative for HLA‐A2, 8% were negative for MART‐1/MelanA and 21% were negative for Pmel17/gp100. The potential significance of our findings is illustrated by a case study in which a patient with melanoma experienced rapid tumor progression in association with loss of either MAA or HLA expression in several lesions. Int. J. Cancer 75:517‐524, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Analysis of expression of the melanoma-associated antigens MART-1 and gp100 in metastatic melanoma cell lines and in in situ lesions

MART-1 and gp100 melanoma associated antigens (MAA) are expressed by cells of the melanocytic lineage and are recognized by the majority of HLA-A2 restricted tumor-infiltrating lymphocytes. Heterogeneity of expression of MAA in tumor deposits may affect the natural history or response to therapy of patients with melanoma. In this study, we evaluated the expression of these MAA with a new monoclonal antibody (mAb) directed against MART-1 (M2-7C10) and the commercially available HMB45 mAb directed against gp100. Expression was tested in vitro by intracellular fluorescence analysis and in vivo by immunophenotyping of tissue specimens. Nine melanoma cell lines and 25 tissue specimens from metastatic melanoma were analyzed. One cell line did not express MART-1 or gp100. The expression of both antigens was more heterogeneous and significantly reduced (p < 0.01) in melanoma cell lines compared with melanocytes, suggesting progressive loss of expression of MAA by neoplastic cells. None of the nonmelanoma cancer lines tested stained for MART-1 or gp100. Analysis of melanoma lesions by immunohistochemistry showed significant heterogeneity of expression of both MART-1 and gp100 MAA either as a percentage of cells expressing MAA or as intensity of expression. Ten of 25 frozen sections expressed MART-1 in < 50% of the cells. In 6 of 25 lesions, immunoreactivity for MART-1 was totally absent. Fine needle aspiration of metastatic lesions seemed to yield information accurately about amount and heterogeneity of expression of MAA in tumor lesions in vivo. Heterogeneity of expression of MAA may be one of several mechanisms leading to tumor escape from immune recognition, and pretreatment evaluation of tumor lesion for expression of these antigens may help in selecting patients best suited to antigen-specific vaccine therapies.

Comparative analysis of the in vivo expression of tyrosinase, MART-1/Melan-A and gp100 in metastatic melanoma lesions: Implications for immunotherapy

A variety of human melanoma-associated antigens (MAA) have been identified that can be recognized by T lymphocytes in a major histocompatibility complex-restricted fashion. Among them, tyrosinase, MART-1/Melan- A, and gp100 are derived from nonmutated melanocyte lineage-specific antigens (Ag). These Ag can be recognized by CD8+ and, in the case of tyrosinase, CD4+ T cells. The in situ expression of these MAA may be a significant cofactor in determining the recognition of melanoma targets by Ag-specific T cells. In this study, we examined the patterns of expression of these MAA using immunohistochemical methods on 30 metastatic tumor deposits derived from 25 patients. MAA expression was heterogeneous among the 30 specimens and also within individual lesions. Of note, 23% of the samples examined failed to express the gp100 protein, and 17% of samples had no detectable expression of MART-1. In contrast, all lesions demonstrated some degree of tyrosinase expression even in cases where both gp100 and MART-1 were not detectable. In addition, 60% of samples (18 of 30) showed strong positivity for tyrosinase (> 75% of cells staining) compared with 40% for gp100 and 36% for MART-1. Currently, a number of experimental immunotherapies for melanoma are directed against the MAA tyrosinase, MART-1, and gp100. Although threshold levels of Ag required for T-cell recognition have not yet been defined, tumor-associated Ag expressed in high density, such as tyrosinase, may be better targets for future immunotherapy trials.

Neuroectodermal differentiation in Ewing's sarcoma family of tumors does not predict tumor behavior

The observation that neuroectodermal differentiation imparts a worse prognosis to the Ewing family of tumors has been suggested by some studies and refuted by others. To assess whether the diagnosis of Ewings sarcoma versus peripheral primitive neuroectodermal tumor (PNET) affects prognosis, we analyzed tumors from 63 analogously treated pediatric and young adult patients from the National Cancer Institute and St Jude Childrens Research Hospital and retrospectively compared the results with clinical outcomes. The tumors were assessed using standard light microscopy and immunohistochemical stains for neuron-specific enolase, CD57, S100 protein, neurofilament protein, and synaptophysin with or without antigen retrieval. Ultrastructural evaluation was also performed in 39 tumors. Classification was performed using Kiel criteria as well as a modified classification. Kaplan-Meier analyses, with Mantel-Haenzel evaluation of the significance of the differences, were performed separately for localized or metastatic tumors. Using the Kiel classification on a subset of 60 cases, 39 tumors qualified as PNET and 21 as Ewings sarcoma. Using the modified classification on a subset of 61 cases, 14 were classified as PNET, 21 as atypical Ewings sarcoma, and 26 as Ewings sarcoma. The addition of electron microscopy to the diagnostic armamentarium significantly increased the likelihood of identifying PNET. No significant differences in event-free or overall survival were seen using either the modified or Kiel classification, regardless of the ancillary diagnostic techniques employed. In this exploratory analysis, neuroectodermal differentiation did not play a role in clinical outcome. Confirmation of this finding will require a larger, separate study of similarly treated patients, and it may not apply to older patients.

Immunocytochemical detection of MART-1 in fresh and paraffin-embedded malignant melanomas

MART-1 is a transmembrane protein that appears to be melanocyte specific. This study evaluated the use of a new monoclonal antibody, directed against MART-1, in fresh and paraffin-embedded specimens. Twenty-three paraffin-embedded tumors were evaluated for MART-1 immunoreactivity both before and after microwave-based antigen retrieval. After this comparison, 53 fine-needle aspirates from 43 patients with malignant melanoma were assessed for MART-1 immunoreactivity. Immunocytochemistry was performed on both cytospins and paraffin-embedded tissues that were pretreated with antigen retrieval. Seventeen (74%) of 23 tumors evaluated for immunoreactivity before and after antigen retrieval showed a significant increase in both staining intensity and the number of cells stained. When cytospins and antigen-retrieved cell blocks from 53 fine-needle aspirates were compared. 38 (72%) showed good correlation. In 13 (25%) of 53 tumors, MART-1 immunoreactivity was more intense in the cytospins, although the differences were marked in only two cases: Microwave-based antigen retrieval renders paraffin-embedded tissues nearly as sensitive as fresh material for use in the immunocytochemical detection of MART-1. This technique will allow the evaluation of MART-1 immunoreactivity in archival malignant melanomas.

Use of recombinant poxviruses to stimulate anti-melanoma T cell reactivity

AbstractBackground: Dendritic cells (DC) are potent professional antigen-presenting cells that can activate naive T lymphocytes and initiate cellular immune responses. As adjuvants, DC may be useful for enhancing immunogenicity and mediating tumor regression. Endogenous expression of antigen by DC could offer the potential advantage of allowing prolonged constitutive presentation of endogenously processed epitopes and exploitation of multiple restriction elements for the presentation of the same antigen. Methods: DC were prepared from the peripheral blood of HLA A*0201 patients with metastatic melanoma in the presence of IL-4 (1000 IU/mL) and GMCSF (1000 IU/mL). Recombinant vaccinia and fowlpox viruses encoding the hMART-1 gene were constructed and used to infect DC. The efficiency of infection and expression of the MART-1 antigen were assessed by immunohistochemistry and intracellular FACS analyses. Cytotoxic lymphocytes (CTL) were generated by the stimulation of CD8+ T cells, with DC expressing the recombinant gene. Reactivity of the CTL was determined at weeks 1 and 2 by the amount of IFN-γ released. Results: DC were infected with recombinant poxviruses and demonstrated specific melanoma antigen expression by immunohistochemistry, immunofluorescence, and intracellular FACS analysis. The expression by DC of MART-1 MAA after viral infection was sufficient to generate CD8+ T lymphocytes that recognized naturally processed epitopes on tumor cells in 10 of 11 patients. Conclusions: Human DC are receptive to infection by recombinant poxviruses encoding MAA genes and are capable of efficiently processing and presenting these MAA to cytotoxic T cells. The potential advantage of this approach is the ability to present specific antigen independent of the identification of the epitope or the MHC restriction element. This strategy may be useful for the identification of relevant epitopes for a diverse number of HLA alleles and for active immunization in patients.

Mantle cell lymphoma: a cytopathological and immunocytochemical study.

Mantle cell lymphoma (MCL) is a rare type of non‐Hodgkins lymphoma which is thought to derive from the cuff of the lymphoid follicle. The histopathologic and immunophenotypic features of MCL are well described. The literature contains few cytopathologic studies of collected cases of MCL.

Medullary carcinoma of the thyroid as a cause of Cushing's syndrome : a case with ectopic adrenocorticotropin secretion characterized by double enzyme immunostaining

A case of a medullary carcinoma of the thyroid gland that secreted both calcitonin and adrenocorticotropin (ACTH) is reported. The patient was a 32-year-old man who was referred to the Clinical Center of the National Institutes of Health with radiologic evidence of intrathoracic and hepatic masses accompanied by florid Cushings syndrome. Serum levels of calcitonin and ACTH were elevated. The thoracic and hepatic masses were resected. The histologic findings were typical of medullary carcinoma of the thyroid with extensive metastases to the liver. The neoplasm had a predominantly solid pattern, and the neoplastic cells were either round or spindled, many with cytologic atypia. Immunohistochemical analysis of fixed, paraffin-embedded sections demonstrated chromogranin, calcitonin, and ACTH in the neoplastic cells. The immunostaining for chromogranin was intense in all of the cells, whereas weaker staining for calcitonin and ACTH was present in scattered cells. Electron microscopy revealed sparse secretory granules in the majority of tumor cells; a minority of neoplastic cells contained numerous granules. We further characterized this neoplasm by performing dual immunohistochemical analysis. This technique clearly demonstrated the presence of ACTH and calcitonin within the same neoplastic cells. Thus, the medullary carcinoma of the thyroid in this patient was the source of ectopic ACTH secretion causing Cushings syndrome. In addition, this report highlights the value of using double immunostaining to localize both the ACTH and calcitonin within the same cells.

Cytologic effects of photodynamic therapy in body fluids

Photodynamic therapy (PDT) has been used in phase I clinical trials at the National Institutes of Health for the treatment of malignancies disseminated within the peritoneal and pleural cavities. Review of records revealed 18 patients who were treated with PDT between April 1988–June 1993. Sixty‐five pleural and peritoneal fluids, 22 pre‐ and 43 post‐PDT, were available for evaluation.