Elisabeth Laine

Integrative analysis of the melanoma transcriptome

Global studies of transcript structure and abundance in cancer cells enable the systematic discovery of aberrations that contribute to carcinogenesis, including gene fusions, alternative splice isoforms, and somatic mutations. We developed a systematic approach to characterize the spectrum of cancer-associated mRNA alterations through integration of transcriptomic and structural genomic data, and we applied this approach to generate new insights into melanoma biology. Using paired-end massively parallel sequencing of cDNA (RNA-seq) together with analyses of high-resolution chromosomal copy number data, we identified 11 novel melanoma gene fusions produced by underlying genomic rearrangements, as well as 12 novel readthrough transcripts. We mapped these chimeric transcripts to base-pair resolution and traced them to their genomic origins using matched chromosomal copy number information. We also used these data to discover and validate base-pair mutations that accumulated in these melanomas, revealing a surprisingly high rate of somatic mutation and lending support to the notion that point mutations constitute the major driver of melanoma progression. Taken together, these results may indicate new avenues for target discovery in melanoma, while also providing a template for large-scale transcriptome studies across many tumor types.

Interleukin-10 is a growth factor for human melanoma cells and down-regulates HLA class-I, HLA class-II and ICAM-1 molecules.

IL‐10 is a cytokine which shows various effects including inhibition of T‐cell proliferation or HLA‐dependent antigen presentation. In this study, we analysed the effects of exogenous or autocrine IL‐10 on proliferation and expression of immunocritical surface molecules. Fourteen cultures of human melanoma cells were established from primary melanomas, locoregional lymph‐node or distant metastases. In 5 melanoma cell cultures, proliferation in the presence of IL‐10, anti‐IL‐10 antibodies (Ab) or control Ab was assessed with colorimetric and [3H]thymidine uptake assays. Flow cytometric analysis was used to quantify the expression of human leukocyte antigen (HLA) class‐I, HLA class‐II and intercellular adhesion molecule (ICAM)‐I and the IL‐10 receptor (IL‐10R). IL‐10 production of melanoma cells was documented by RT‐PCR and IL‐10 protein was detected in the supernatants by means of ELISA. IL‐10 enhanced proliferation and prolonged survival of melanoma cells in 5 out of 5 cultures. Anti‐IL‐10 Ab decreased proliferation. IL‐10R expression was found in 12 out of 14 (86%) melanoma cell cultures. The expression of HLA‐I, HLA‐II and ICAM‐I on all melanoma cells that were positive for IL‐10R showed a reduction of 10‐60% by IL‐10, whereas the surface levels of HLA‐I, HLA‐II and ICAM‐I in 5 out of 5 cell cultures revealed an increase of 10‐170% by anti‐IL‐10 Ab. These findings suggest that IL‐10 is an autocrine growth factor with significant impact on immunocritical molecules in melanoma. IL‐10 effects have to be considered when planning therapeutic immunointerventions in melanoma patients. Int. J. Cancer 71:630‐637, 1997.

Imiquimod Treatment Induces Expression of Opioid Growth Factor Receptor: A Novel Tumor Antigen Induced by Interferon-α?

Purpose: Imiquimod represents a synthetic local immune response modifier that has demonstrated efficacy in clearing basal cell carcinoma. Via interaction with Toll-like receptor 7 on immune cells, imiquimod induces local production of cytokines, such as interferon (IFN)-α. Experimental Design: To more closely define and elucidate mechanisms leading to basal cell carcinoma clearance in vivo, we examined gene expression profiles of skin basal cell carcinoma before and after treatment with 5% imiquimod cream (Aldara) by using high-density oligonucleotide arrays. Results: We show that imiquimod predominantly induces genes involved in different aspects of immune response. In addition to effects on immunity, imiquimod treatment modulates the expression of genes involved in the control of apoptosis and oncogenesis. Array data indicated that imiquimod treatment induces expression of opioid growth factor receptor, a molecule recently reported to be a target for antitumor antibody responses. Immunohistochemistry revealed in vivo up-regulation of opioid growth factor receptor protein on tumor and on infiltrating cells after treatment. By using basal cell carcinoma cell lines treated with IFN-α or imiquimod, we show that opioid growth factor receptor up-regulation is IFN-α-mediated, rather then directly imiquimod-mediated. By using tissue microarray containing 52 basal cell carcinomas, we demonstrate opioid growth factor receptor expression in almost half of the cases. Expression of opioid growth factor receptor correlated with a longer recurrence-free period in basal cell carcinoma that recurred after radiotherapy (Kaplan-Meier analysis, P = 0.041). Conclusions: In addition to its immunomodulatory and antiproliferative activity, opioid growth factor receptor seems to have a prognostic significance in basal cell carcinoma patients. Our data add to the growing list of basal cell carcinoma-associated tumor antigens.

Genotypic, phenotypic and functional analysis of CD4+CD7+ and CD4+CD7- T lymphocyte subsets in Sézary syndrome

Abstract  The expansion of CD4 + CD7 – T cells in the peripheral blood of Sézary syndrome (SS) is well known. It remains unclear whether this population contains the dominant T cell clone. Peripheral blood mononuclear cells (PBMC) of five SS patients were sorted by fluorescence-activated cell sorting into CD4 + CD7 – and CD4 + CD7 + populations. These populations were analysed separately for clonality of the T cell receptor γ chain (TCR-γ) by PCR-DGGE. The cytokine profile of both populations was investigated by RT-PCR ELISA for IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13 and IL-15. In three other patients with known Vβ-usage, the dominant T cell clones were phenotypically characterized by double staining. PCR-DGGE of TCR-γ demonstrated that all patients had a clonal population in their blood and that this population was present in CD4 + CD7 – and CD4 + CD7 + populations. Concerning mRNA cytokine transcription, the two populations did not show any consistent differences. In three patients with identified clones (Vβ 3.1, 5.3 and 6.7), double staining revealed positivity for CD2, CD3, CD4, CD5, CD45RO and CD7 in a significant proportion (at least 35%). We conclude that the CD4 + CD7 – population does not represent the dominant T cell clone in patients with SS. An increase in this population of PBMC in SS might account for deviations in the T cell functions of the patients.

Green tea extract reduces induction of p53 and apoptosis in UVB-irradiated human skin independent of transcriptional controls

Abstract:  Ultraviolet (UV) irradiation plays a pivotal role in human skin carcinongenesis. Preclinically, systemically and topically applied green tea extract (GTE) has shown reduction of UV‐induced (i) erythema, (ii) DNA damage, (iii) formation of radical oxygen species and (iv) downregulation of numerous factors related to apoptosis, inflammation, differentiation and carcinogenesis. In humans, topical GTE has so far only been tested in limited studies, with usually very high GTE concentrations and over short periods of time. Both chemical stability of GTE and staining properties of highly concentrated green tea polyphenols limit the usability of highly concentrated green tea extracts in cosmetic products. The present study tested the utility of stabilized low‐dose GTE as photochemopreventive agents under everyday conditions. We irradiated with up to 100 mJ/cm2 of UVB light skin patches which were pretreated with either OM24®‐containing lotion or a placebo lotion. Biopsies were taken from both irradiated and un‐irradiated skin for both immunohistochemistry and DNA microarray analysis. We found that while OM24® treatment did not significantly affect UV‐induced erythema and thymidine dimer formation, OM24® treatment significantly reduced UV‐induced p53 expression in keratinocytes. We also found that OM24® treatment significantly reduced the number of apoptotic keratinocytes (sunburn cells and TUNEL‐positive cells). Carefully controlled DNA microarray analyses showed that OM24® treatment does not induce off‐target changes in gene expression, reducing the likelihood of unwanted side‐effects. Topical GTE (OM24®) reduces UVB‐mediated epithelial damage already at low, cosmetically usable concentrations, without tachyphylaxis over 5 weeks, suggesting GTE as suitable everyday photochemopreventive agents.

Type I IFN innate immune response to adenovirus-mediated IFN-γ gene transfer contributes to the regression of cutaneous lymphomas

The fact that adenoviral vectors activate innate immunity and induce type I IFNs has not been fully appreciated in the context of cancer gene therapy. Type I IFNs influence different aspects of human immune response and are believed to be crucial for efficient tumor rejection. We performed transcriptional profiling to characterize the response of cutaneous lymphomas to intralesional adenovirus-mediated IFN-gamma (Ad-IFN-gamma) gene transfer. Gene expression profiles of skin lesions obtained from 19 cutaneous lymphoma patients before and after treatment with Ad-IFN-gamma revealed a distinct gene signature consisting of IFN-gamma- and numerous IFN-alpha-inducible genes (type II- and type I-inducible genes, respectively). The type I IFN response appears to have been induced by the vector itself, and its complexity, in terms of immune activation, was potentiated by the IFN-gamma gene insert. Intralesional IFN-gamma expression together with the induction of a combined type I/II IFN response to Ad-IFN-gamma gene transfer seem to underlie the objective (measurable) clinical response of the treated lesions. Biological effects of type I IFNs seem to enhance those set in motion by the transgene, in our case IFN-gamma. This combination may prove to be of therapeutic importance in cytokine gene transfer using Ads.

IL-12 directly up-regulates the expression of HLA class I, HLA class II and ICAM-1 on human melanoma cells: a mechanism for its antitumor activity?

IL‐12 enhances cytolytic activity and proliferation of NK and T cells, and induces cytokines such as IFN‐γ. No direct effects on non‐hematopoietic cells have been shown. This study investigates the effects of IL‐12 on melanoma cells in vitro. We analyzed 15 melanoma cell cultures and 1 melanoma cell line. Out of 16 samples 13 expressed the β chain of the IL‐12 receptor (IL‐12Rβ). Preincubation with IL‐12 increased the surface levels of human leukocyte antigen (HLA) class I, HLA class II and intercellular adhesion molecule (ICAM)‐1 of those cultures with IL‐12Rβ expression. The effects of IL‐12 on HLA class I could be blocked by an IL‐12‐neutralizing monoclonal antibody (mAb), but not by an mAb against IFN‐γ. Melanoma cells transduced with IL‐12 expressed enhanced levels of HLA class I, HLA class II and ICAM‐1 compared to controls. Co‐incubation of the melanoma cells with allogeneic peripheral blood mononuclear cells (PBMC) resulted in enhanced proliferation and increased production of IL‐2 and IFN‐γ after pretreatment with IL‐12. IL‐12 pretreatment increased the susceptibility of melanoma cells to lysis by prestimulated autologous PBMC. Since IL‐12 induced immunocritical surface molecules on melanoma cells, it might be beneficial during immune interventions in melanoma patients.

Loss of single HLA Class I allospecificities in melanoma cells due to selective genomic abbreviations

Expression of human leucocyte antigen (HLA) Class I molecules is essential for the recognition of malignant melanoma (MM) cells by CD8+ T lymphocytes. A complete or partial loss of HLA Class I molecules is a potent strategy for MM cells to escape from immunosurveillance. In 2 out of 55 melanoma cell cultures we identified a complete phenotypic loss of HLA allospecificities. Both patients have been treated unsuccessfully with HLA‐A2 peptides. To identify the reasons underlying the loss of single HLA‐A allospecificities, we searched for genomic alterations at the locus for HLA Class I α‐chain on chromosome 6 in melanoma cell cultures established from 2 selected patients with MM in advanced stage. This deficiency was associated with alterations of HLA‐A2 gene sequences as determined by polymerase chain reaction‐sequence specific primers (PCR‐SSP). Karyotyping revealed a chromosomal loss in Patient 1, whereas melanoma cell cultures established from Patient 2 displayed 2 copies of chromosome 6. Loss of heterozygosity (LOH) using markers located around position 6p21 was detected in both cases. By applying group‐specific primer‐mixes spanning the 5′‐flanking region of the HLA‐A2 gene locus the relevant region was amplified by PCR and subsequent sequencing allowed alignment with the known HLA Class I reference sequences. Functional assays using HLA‐A2‐restricted cytotoxic T‐cell clones were performed in HLA‐A2 deficient MM cultures and revealed a drastically reduced susceptibility to CTL lysis in HLA‐A2 negative cells. We could document the occurrence of selective HLA‐A2 deficiencies in cultured advanced‐stage melanoma metastases and identify their molecular causes as genomic alterations within the HLA‐A gene locus.

Impact of interferons on the expression of melanoma-associated antigens in melanoma short-term cell cultures.

Some immunotherapeutic approaches based on melanoma-associated antigens rely on in vitro cultivation of melanoma cells. A beneficial effect of interferons has been shown in melanoma. This study aimed to determine whether stimulation of patient-derived melanoma short-term cell cultures using interferon-α and -γ changes the expression pattern of melanoma-associated antigens. Lymph node, skin and brain metastases were cultivated for up to 3 weeks and treated with interferon-α, interferon-γ or mock stimulation. Expression of the melanoma-associated antigens MAGE-3, MelanA/MART-1 and tyrosinase was determined by flow cytometry and compared with the expression pattern of HLA class I molecules. We found consistently enhanced expression of HLA class I molecules, whereas the melanoma-associated antigens showed mixed responses, with moderate induction, suppression or no visible effect. The reaction to interferon stimulation was similar for all the antigens examined within a single melanoma cell culture. In contrast to the HLA class I molecules, which showed induced expression with interferon, the melanoma-associated antigens showed a varied response to interferon stimulation. Differential reaction to interferon stimulation is of importance to immunotherapeutic modalities and might influence progression of the disease. We therefore suggest that evaluation of variation in melanoma-associated antigen expression in the clinical setting may help to identify patients who would profit from adjuvant interferon therapy.

Upregulation of interleukin-12 receptor on peripheral blood mononuclear cells and HLA class I, HLA class II or ICAM-1 on melanoma cells by B7.1 and interleukin-12: a mechanism for immunostimulatory impact of melanoma cells adenovirally transfected with B7.1 and IL12?

Melanoma is an immunogenic tumour and may express both HLA class I and class II molecules. These can be recognized by cytotoxic T-cells. Melanoma cells can evade immunosurveillance due to the lack of co-stimulatory molecules such as B7.1 or B7.2. Interleukin-12 (IL12) exerts antitumour effects, and B7.1 and IL12 synergistically induce effective antitumour immunity. We investigated the immunostimulatory potential of melanoma cells adenovirally transduced with B7.1, IL12 or B7.1 plus IL12. We observed that: (i) melanoma cells transduced with B7.1 plus IL12 can elicit a strong proliferative response from peripheral blood mononuclear cells (PBMCs); (ii) a high level of TH1 cytokine production from PBMCs was induced by melanoma cells transduced with Adv-B7.1 plus Adv-IL12; (iii) the expression of HLA class I antigens, HLA class II antigens or ICAM-1 antigens was higher on melanoma cells transduced with Adv-IL12 or Adv-B7.1 plus IL12 than those transduced with Adv-LacZ or wild-type melanoma cells; and (iv) the expression of IL12 receptors on PBMCs was upregulated by melanoma cells transfected with Adv-IL12 or Adv-B7.1 plus IL12. Thus, melanoma cells transduced with both Adv-IL12 and B7.1 may represent another clinical approach for antimelanoma gene therapy.