Patricia Fetsch

ABCG2: A perspective☆

ABCG2, or breast cancer resistance protein (BCRP), is an ABC transporter that has been the subject of intense study since its discovery a decade ago. With high normal tissue expression in the brain endothelium, gastrointestinal tract, and placenta, ABCG2 is believed to be important in the protection from xenobiotics, regulating oral bioavailability, forming part of the blood-brain barrier, the blood-testis barrier, and the maternal-fetal barrier. Notably, ABCG2 is often expressed in stem cell populations, where it likely plays a role in xenobiotic protection. However, clues to its epigenetic regulation in various cell populations are only beginning to emerge. While ABCG2 overexpression has been demonstrated in cancer cells after in vitro drug treatment, endogenous ABCG2 expression in certain cancers is likely a reflection of the differentiated phenotype of the cell of origin and likely contributes to intrinsic drug resistance. Notably, research into the transporters role in cancer drug resistance and its development as a therapeutic target in cancer has lagged. Substrates and inhibitors of the transporter have been described, among them chemotherapy drugs, tyrosine kinase inhibitors, antivirals, HMG-CoA reductase inhibitors, carcinogens, and flavonoids. This broad range of substrates complements the efficiency of ABCG2 as a transporter in laboratory studies and suggests that, while there are redundant mechanisms of xenobiotic protection, the protein is important in normal physiology. Indeed, emerging studies in pharmacology and toxicology assessing polymorphic variants in man, in combination with murine knockout models have confirmed its dynamic role. Work in pharmacology may eventually lead us to a greater understanding of the physiologic role of ABCG2.

Immune selection after antigen-specific immunotherapy of melanoma

BACKGROUND Melanoma antigen (MA)-specific vaccination strongly enhances antitumor reactivity in vivo and is capable of producing strong cytotoxic T lymphocyte responses in vitro. Furthermore, specific human leukocyte antigen-restricted T cell activation is hypothesized to occur in response to peptide-based immunotherapy, which may lead to the preferential killing of tumor cells bearing the relevant MA. The development of melanoma antigen-loss variants may subsequently occur in vivo. METHODS Analysis of 532 melanoma lesions from 204 patients was performed on fine-needle aspiration biopsy specimens. Lesions were graded for the expression of the MAs gp100 and MART-1 with use of immunocytochemistry. A total of 351 melanoma lesions were divided into cohorts on the basis of the treatment received. The pretreatment group (n = 175) consisted of lesions obtained before any form of gp100 immunotherapy, with the posttreatment group (n = 176) consisting of lesions obtained after vaccination with a modified gp100 epitope, gp209-2M +/- interleukin 2 (IL-2). RESULTS The percentage of lesions not expressing the gp100 antigen is greater than the percentage not expressing MART-1 (26% vs 14%). The frequency of lesions with high expression (> 75%) of gp100 significantly decreased with therapy (47% vs 34%) and conversely negative lesions increased (18% vs 29%). Treatment of lesions with peptide alone (no IL-2) revealed a significant decrease in gp100 expression (47% vs 32%), enhanced with the addition of IL-2 to therapy (47% vs 35%). The expression of MART-1 remained essentially unchanged unless IL-2 was added (54% vs 54%, MART-1 peptide alone, 54% vs 43%, MART-1 peptide + IL-2). Of 94 patients (181 lesions) assessed for gp100 expression before treatment, 10 patients responded to therapy. Pretreatment lesions in responding patients expressed some level of gp100 in all cases compared with 27% of nonresponding lesions, which were negative for gp100 expression. CONCLUSIONS. This study indirectly demonstrates that vaccination with an MA-derived peptide can result in immune selection in vivo. Furthermore, it provides strong immunologic evidence for the specificity of MA vaccines and to the relevance of MA expression in predicting the response to vaccination.

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.

Analysis of the matrix metalloproteinase family reveals that MMP8 is often mutated in melanoma

A mutational analysis of the matrix metalloproteinase (MMP) gene family in human melanoma identified somatic mutations in 23% of melanomas. Five mutations in one of the most commonly mutated genes, MMP8, reduced MMP enzyme activity. Expression of wild-type but not mutant MMP8 in human melanoma cells inhibited growth on soft agar in vitro and tumor formation in vivo, suggesting that wild-type MMP-8 has the ability to inhibit melanoma progression.

Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2

Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N-linked glycosylation was also obtained using tunicamycin and N-glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport.

Characteristics of nine newly derived mesothelioma cell lines

This report characterizes nine new cell lines derived from patients with malignant pleural mesothelioma. The lines were initiated between July 1990 and July 1992 from solid tumors (5 lines) or effusions (4 lines) and had proliferated for a period of at least 2 months without senescence. They were characterized by cell size, doubling time, immunohistochemical analyses, electron microscopy, and chromosomal karyotyping. Growth factor/cytokine elaboration was determined using enzyme-linked immunoassays. The established lines were similar in morphology to their parent tumor (ie, epithelial or sarcomatoid). Cell sizes ranged from 59 to 81 microns, and the doubling times varied from 31 to 65 hours. The lines stained with cytokeratin and showed expected negative staining for adenomarkers including B72.3 and carcinoembryonic antigen. All cell lines exhibited aneuploidy, with modal chromosome numbers between 40 and 81 and had multiple chromosomal aberrations. Significant production of granulocyte-monocyte colony-stimulating factor, leukemia inhibitory factor, platelet-derived growth factor, and interleukin-6 was seen. These new cell lines derived from human mesotheliomas can now be used to aid in the design of innovative treatment strategies.

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.

Sequential gene profiling of basal cell carcinomas treated with imiquimod in a placebo-controlled study defines the requirements for tissue rejection

BackgroundImiquimod is a Toll-like receptor-7 agonist capable of inducing complete clearance of basal cell carcinoma (BCC) and other cutaneous malignancies. We hypothesized that the characterization of the early transcriptional events induced by imiquimod may provide insights about immunological events preceding acute tissue and/or tumor rejection.ResultsWe report a paired analysis of adjacent punch biopsies obtained pre- and post-treatment from 36 patients with BCC subjected to local application of imiquimod (n = 22) or vehicle cream (n = 14) in a blinded, randomized protocol. Four treatments were assessed (q12 applications for 2 or 4 days, or q24 hours for 4 or 8 days). RNA was amplified and hybridized to 17.5 K cDNA arrays. All treatment schedules similarly affected the transcriptional profile of BCC; however, the q12 × 4 days regimen, associated with highest effectiveness, induced the most changes, with 637 genes unequivocally stimulated by imiquimod. A minority of transcripts (98 genes) confirmed previous reports of interferon-α involvement. The remaining 539 genes portrayed additional immunological functions predominantly involving the activation of cellular innate and adaptive immune-effector mechanisms. Importantly, these effector signatures recapitulate previous observations of tissue rejection in the context of cancer immunotherapy, acute allograft rejection and autoimmunity.ConclusionThis study, based on a powerful and reproducible model of cancer eradication by innate immune mechanisms, provides the first insights in humans into the early transcriptional events associated with immune rejection. This model is likely representative of constant immunological pathways through which innate and adaptive immune responses combine to induce tissue destruction.

Short-Term Kinetics of Tumor Antigen Expression in Response to Vaccination

The melanoma patient’s immune response to tumor has been extensively studied. Yet, the frequently observed coexistence of tumor-associated Ag (TAA)-specific T cells with their target cells in vivo remains unexplained. Loss of TAA expression might contribute to this paradox. We studied TAA expression in metastases by obtaining fine-needle aspirations from 52 tumor lesions in 30 patients with melanoma before and soon after immunotherapy. Limitations due to low amounts of starting material were overcome with a high fidelity antisense RNA amplification method. TAA expression was measured by quantitative real-time PCR of anti-sense RNA. Decrease in gp100/Pmel-17 TAA preceded tumor disappearance in several instances and could be best explained by immune selection because most patients had received gp100/Pmel-17-specific vaccination. Conversely, immune selection was absent in nonregressing lesions. These observations suggest that vaccination, when successful, triggers a broad inflammatory reaction that can lead to tumor destruction despite immune selection. Additionally, lack of clinical response might be attributed to lack of this initiating event rather than immune escape. This study provides an insight into the natural history of tumors and defines a strategy for the characterization of gene expression in tumors during therapy.