Toni B. Simonis

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.

Common expression of melanoma tumor-associated antigens recognized by human tumor infiltrating lymphocytes: analysis by human lymphocyte antigen restriction.

Summary: Major histocompatibility complex (MHC) class I antigens (Ag), particularly human lymphocyte antigen (HLA)-A2, have been shown to function as restriction elements in human cytotoxic T lymphocyte recognition of tumor. This study was undertaken to determine the function of non-A2 MHC class I Ag in tumor recognition by tumor-infiltrating lymphocytes (TILs) cultured from six melanomas, and to find evidence for shared or unique tumorassociated Ag. Four predominantly CD8+ and two mixed CD4+, CD8+ population TIL cultures were tested for lysis in short-term 51Cr-release assays against a panel of targets including 29 fresh melanomas, 2 fresh sarcomas, 11 cultured melanoma lines, and 14 nonmelanoma cell lines derived from HLA-typed patients. All six melanoma TILs lysed the autologous melanoma. Two of three TILs from HLA-A2+ patients lysed allogeneic melanomas matched for HLA-A2, giving evidence for shared tumor Ag; one of these TILs also used HLA-B44 as a restriction element. The third HLA-A2+ TIL lysed autologous melanoma but not autologous Epstein-Barr virus-transformed B cells nor 14 HLA-A2 matched allogeneic melanomas, suggesting the possibility of a unique tumor Ag in this system. The three HLA-A2- TILs each lysed multiple HLA-matched melanomas, using HLA-A24, HLA-A31, and HLA-Cw7 as restriction elements. Blocking of autologous and allogeneic melanoma lysis by TILs with mAb w6/32 (anti-MHC class I) and anti-CD3, as well as cold target inhibition assays, confirmed that specific interaction of the T-cell receptor with MHC class I Ag and the relevant tumor Ag on the target cell surface is required for tumor lysis. These data provide evidence for specific recognition of shared melanoma Ag by human TILs.

HLA allele distribution distinguishes sporadic inclusion body myositis from hereditary inclusion body myopathies

We studied the HLA class II associations in patients with sporadic inclusion body myositis (s-IBM) and hereditary inclusion body myopathies (h-IBM) and attempted to distinguish these myopathies on the basis of HLA allele assignments. Forty-five patients, 30 with s-IBM and 15 with h-IBM, underwent HLA class II allele-specific typing using polymerase chain reaction sequence-specific primers for 71 alleles contained in the DRbeta1, DRbeta3-5, and DQbeta1 loci. In s-IBM, we found a high (up to 77%) frequency of DRbeta1*0301, DRbeta3*0101 (or DRbeta3*0202) and DQbeta1*0201 alleles. No significant association with alleles in the DR and DQ haplotypes was found among the 15 h-IBM patients. The strong association of prominent alleles with s-IBM, but not h-IBM, suggests that s-IBM is a distinct disorder with an immunogenetic background that differs from h-IBM.

HLA associations in the antitumor response against malignant melanoma

In this study we analyzed the human leukocyte antigen (HLA) pattern of North American Caucasian patients with metastatic melanoma as compared with the North American Caucasian (NAC) population. We also investigated whether the HLA type of melanoma patients had an effect on their tolerance and response to interleukin-2 (IL-2)-based therapy. Four hundred twelve serologic phenotypes of Caucasian melanoma patients referred to the National Cancer Institute, National Institutes of Health, from February 1989 through December 1993 were collected by typing the patients peripheral blood lymphocytes. Furthermore, 74 melanoma patients were typed for HLA class II by high-resolution sequence specific primer-polymerase chain reaction. Response rate and treatment-related toxicity in those patients receiving IL-2-based treatment (N = 272) were compared with HLA serologic types. The frequency of four HLA-B alleles was significantly different in the melanoma compared with the NAC population: Of these, HLA-B5, -B8, and -B15 had a frequency falling between the NAC and the Northern European population. No other significant differences between melanoma patients and NAC population were noted for other HLA loci. A correlation was noted between HLA-DR3 and -DR4 alleles and decreased tolerance to IL-2, whereas homozygosity for HLA-DR decreased the chance of response. There were no significant associations between HLA type and response. It is unlikely that the associations noted between some HLA-B alleles and melanoma bear significantly on the etiology of the disease. The differences seen between American melanoma patients and the NAC population are probably best explained by geographical ancestry. The association between HLA-DR and tolerance to IL-2 therapy noted in this study may offer insight toward the understanding of mechanisms regulating the cascade of events after the systemic administration of IL-2.

Differences in frequency distribution of HLA-A2 subtypes between North American and Italian white melanoma patients: Relevance for epitope specific vaccination protocols

Cytotoxic T lymphocytes (CTL) associated in vivo with tumor regression recognize the product of nonmutated genes expressed by most melanoma cells as peptides bound to human leukocyte antigen (HLA) molecules. Multiple HLA-A*0201 restricted peptides derived from melanoma associated antigens (MAA) have been described, and peptide-based vaccination protocols against melanoma are being developed worldwide for the treatment of HLA-A2 melanoma patients based on the assumption that most serologically typed HLA-A2+ individuals will be suitable for such vaccinations. Serologic typing of HLA-A2, however, encompasses a family of at least 17 related alleles recognized by molecular typing techniques and differing at one or more functional residues of the HLA class I molecule. We have recently shown that naturally occurring single-residue variants of HLA-A*0201 are responsible for significant differences in CTL response to MAA-peptide stimulation. Existing data for HLA-A*02 subtype frequencies among whites (who are most affected by melanoma) derive from analyses of Northern European and North American populations that are of similar heritage and predict an exceedingly rare (< 5%) frequency of non-HLA-A*0201 alleles. Melanoma however, affects other white populations in which the prevalence of HLA-A*02 alleles could be more variable. This study was done to identify HLA-A*02 subtypes and their prevalence in two ancestrally different white melanoma populations. HLA-A*02 subtype frequencies were compared by polymerase chain reaction between serologically HLA-A2+ melanoma patients referred for treatment to the Istituto Nazionale Tumori of Milan (n = 93), Italy or the National Cancer Institute, Bethesda, MD, U.S.A. (n = 100). This analysis demonstrated differences in subtype specificity and distribution between the two populations, with a significantly higher percentage of non HLA-A*0201 subtypes in the Italian population. Only 2% of serologically HLA-A2+ Northern American white melanoma patients did not express HLA-A*0201. In contrast, 15% of HLA-A2+ Italian patients were not HLA-A*0201 (p2 value = 0.001). As allele-specific/peptide-based vaccination protocols are presently pursued at several institutions, a proportion of patients might be inappropriately enrolled basing their eligibility on serologically defined HLA-typing.

Locus-Specific Analysis of Human Leukocyte Antigen Class I Expression in Melanoma Cell Lines

Surface expression of human leukocyte antigen (HLA) class I antigens on melanoma lines was evaluated by locus-specific monoclonal antibodies (mAbs) with three different techniques: Fluorescence-activated cell sorting (FACS), immunohistochemistry with cytospin preparation (ICP), and complement-mediated cytotoxicity (CMC). Eleven HLA class I-expressing cell lines developed from metastases were used. Specific expression of HLA loci was examined under routine culture conditions and after 48-h incubation in interferon-gamma (IFN-gamma; 500 U/ml). Loss of allelic expression was seen in one line (586-MEL): Products of genes coding for HLA-A29 and -B44, in strong linkage disequilibrium, were not detectable. HLA-A antigens were consistently detected by all methodologies and minimally affected by pretreatment with IFN-gamma. HLA-B antigens were detectable in 8 of 11 lines by ICP and 3 of 11 lines by CMC. By FACS the supratypic specificity HLA-Bw6 was expressed at low levels in most lines (mean fluorescence 47.2 +/- 13.4 and rose to 259.8 +/- 45.9 after incubation with IFN-gamma; p < 0.001). HLA-Cw antigen detection by CMC correlated with HLA-B (p < 0.01), suggesting that down-regulation and sensitivity to IFN-gamma are shared by the two loci. This low expression of the HLA-B antigens may play a role in the evasion of the host immune response and its up-regulation may be useful in allowing tumor antigen recognition.

Combination therapy with interferon-γ and interleukin-2 for the treatment of metastatic melanoma

The toxicity and clinical response to treatment with the combination of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) in patients with metastatic melanoma was evaluated. From May 1993 through February 1994, 20 patients were treated with 24 courses of IFN-gamma with or without IL-2. A 7-day course of subcutaneous IFN-gamma alone was administered to cohorts of two or three patients each at doses of 0.1, 0.2, or 0.3 mg/m2. Thirteen patients received escalating doses of IFN-gamma between 0.2 and 0.5 mg/m2 followed by the intravenous (i.v.) administration of IL-2 (720,000 IU/kg) given three times a day. A treatment course consisted of two cycles (maximum of 15 doses of IL-2 per cycle) separated by a 10-day interval. Five additional patients were treated with five courses of IFN-gamma, IL-2, and tumor-infiltrating lymphocytes (TILs). All patients treated had the diagnosis of metastatic melanoma. The maximal tolerated dose of subcutaneous IFN-gamma was established at 0.3 mg/m2 with dose-limiting hepatotoxicity. Immunohistochemistry analyses showed detectable upregulation of MHC class I alleles in one (8%) of 12 patients. Two of 20 patients who received the combination of IFN-gamma and IL-2 had responses, one partial and one complete response. The duration of response was 7 months for the partial response and 12 months for the complete response. IFN-gamma was tolerated with minimal side effects of nausea, vomiting, malaise, and decreased hematopoiesis. No increased toxicities were found with the combination treatment, as compared with IL-2 alone. One death occurred on the third day of treatment with IFN-gamma alone from hemorrhage into brain metastases. There were no responders in the five patients who received the combination treatment of TIL, IL-2, and IFN-gamma. From these findings, we conclude that further studies looking at this combination treatment are not warranted.

HLA antigens in childhood onset schizophrenia

Evidence of immune system abnormalities in adult schizophrenia has prompted examination of the human leukocyte antigen (HLA) system. Childhood onset schizophrenia offers a unique opportunity to test neurodevelopmental hypotheses of schizophrenia, including those which implicate components of the immune system. In the present study, class I and II HLA antigens were typed using sequence-specific primers and the polymerase chain reaction in 28 childhood onset schizophrenics and 51 ethnically matched healthy subjects. Groups were compared for frequencies of HLA antigens reported to be associated with schizophrenia and/or autoimmune disorders. We hypothesized that antigen frequencies would differ between schizophrenic and healthy children, suggesting that some dimension of the neurodevelopmental disturbance experienced by these children may be mediated by subtle abnormalities of immune function. There were no significant differences between schizophrenic and healthy subjects in the frequency of any antigen tested. These findings do not support HLA-associated pathology in childhood onset schizophrenia.

High throughput HLA sequence-based typing (SBT) utilizing the ABI Prism 3700 DNA Analyzer.

Aims and background The genetic complexity of the human major histocompatibility complex (MHC) has required the development of various molecular typing methods. The purpose of this paper is to compare the results of two of these molecular methods: sequenced based typing (SBT) and polymerase chain reaction (PCR) using sequence specific primers (PCR-SSP). Methods The SBT method described utilizes an ABI Prism® 3700 DNA Analyzer, which has been designed fro high throughput production of sequence data through highly automated operation with significant walk-away time. The ABI Prism® 3700 DNA Analyzer is a 96-capillary electrophoresis instrument with the capability of running four 96-well plates black to back in a sixteen-hour period. Potentially, data from this machine can produce Class I sequences for A or B loci for 64 samples in this time frame. The SBT method encompassed exons 2, 3, and 4 with forward and reverse sequence orientation reactions using the PE Biosystems HLA-A and HLA-B Sequenced Based Typing Kits (PE Applied Biopsystems/Perkin-Elmer, Foster City, CA, USA). Most SBT methods previously employed only gather data from exons 2 and 3 which distinguishes most of the polymorphism necessary to identify the majority of alleles in the HLA region. However, in an effort to discern numerous null alleles in the HLA region, exon 4 data is also included. The PCR-SSP method utilized consists of one 96 well tray, with 95 primer mixes and one negative control, per sample designed to produce an intermediate/high resolution HLA-A, B typing. Results Data from one 96-well capillary run on the ABI Prism® 3700 DNA Analyzer, which consists of results from 16 samples for HLA-A or HLA-B loci, was compared to data derived from sixteen HLA-A and HLA-B PCR-SSP typings. 75% of loci tested achieved a higher resolution HLA typing by the SBT method. Discussion The ability to provide allele level HLA typing results can have significant functional implications for the bone marrow transplant community and numerous vaccine studies.

Frequency of human leukocyte antigen-A 24 alleles in patients with melanoma determined by human leukocyte antigen-A sequence-based typing.

The analysis of immune responses of patients with melanoma has led to the identification of melanoma-associated antigens targeted by T cells. Cytotoxic T lymphocytes recognize peptides from melanoma-associated antigens presented on the cancer cell surface in the context of HLA class I molecules. Immunodominant melanoma-associated antigen epitopes are being evaluated for their ability to immunize patients with advanced melanoma. However, these vaccination efforts are limited by the extensive polymorphism of the HLA class I heavy chain, which occurs in functional domains of the molecule. Patients with melanoma with the HLA-A-24 phenotype were recruited for vaccination with the peptide AFLPWHRLF from the melanoma-associated antigen tyrosinase. This peptide is recognized in association with HLA-A*2402. The HLA-A24 family includes at least 15 alleles whose frequency and ability to present the same peptide are unknown. The distribution of HLA-A24 alleles was studied in a melanoma population for the practical purpose of identifying patients suitable for vaccination with HLA-A*2402 epitopes. An HLA-A locus-specific polymerase chain reaction method followed by sequencing was developed to determine the HLA-A alleles in genomic DNA. HLA-A 24 was also typed in healthy persons of various ethnic backgrounds to further explore the HLA-A24 family. In white persons, the HLA-A*2402 allele was most common (in 85% of white persons and in 97% of the patients with melanoma). Fewer persons carried the HLA-A*2403 allele (13% in all samples, 3% in melanoma patients). Finally, two new alleles, HLA-A*2422 and HLA-A*24 null, were identified. These results suggest that vaccination with HLA-A*2402–associated epitopes has the potential for broad use in this patient population.