Targeted therapy and immunotherapy: Emerging biomarkers in metastatic melanoma

Targeted therapy directed against oncogenic BRAF mutations and immune checkpoint inhibitors have transformed melanoma therapy over the past decade and prominently improved patient outcomes. However, not all patients will respond to targeted therapy or immunotherapy and many relapse after initially responding to treatment. This unmet need presents two major challenges. First, can we elucidate novel oncogenic drivers to provide new therapeutic targets? Second, can we identify patients who are most likely to respond to current therapeutic strategies in order to both more accurately select populations and avoid undue drug exposure in patients unlikely to respond? In an effort to evaluate the current state of the field with respect to these questions, we provide an overview of some common oncogenic mutations in patients with metastatic melanoma and ongoing efforts to therapeutically target these populations, as well as a discussion of biomarkers for response to immune checkpoint inhibitors—including tumor programmed death ligand 1 expression and the future use of neoantigens as a means of truly personalized therapy. This information is becoming important in treatment decision making and provides the framework for a treatment algorithm based on the current landscape in metastatic melanoma.     

Immune signatures in cutaneous melanoma correlate with survival independently of immunotherapy treatment

Immune checkpoint inhibitors (ICIs) have fundamentally improved survival from advanced cutaneous melanoma. Significant efforts have been made to understand the ICI response to identify ways to further improve outcomes. One such approach has been to investigate gene expression associated with response to ICI, which has identified various immune-related mRNA signatures, including a six-gene IFN-γ signature (IFN-γ⁶), an expanded immune signature (IFN-γ¹⁸), an effector T-cell gene signature (T_eff), and a T_eff-associated and IFN-γ-associated gene signature (T_eff + IFN-γ). Given that these signatures appear to reflect expression from T cells and the level of tumour-infiltrating immune cells has been associated with survival, we hypothesised that the prognostic value of the signatures is not limited to ICI treatment and investigated if they were associated with survival also in patients who never received ICI. The signatures were not present in melanoma cell lines when compared with tumour samples, confirming that the signatures were likely derived from the samples' non-tumour (immune) components. We acquired expression and survival data from five melanoma cohorts with a wide range of disease stages, treatments and metrics for survival, and correlated the expression signatures with survival. All four signatures were significantly associated (p < .05) with survival in four of five cohorts, with hazard ratios ranging from 0.69 to 0.92. We conclude that these immune signatures' association with survival is not specific to ICI-treated patients, but present in a number of settings.     

Molecular markers in malignant cutaneous melanoma: Gift horse or one‐trick pony?

The management of malignant cutaneous melanoma is problematic. Current clinical prognostic factors do not adequately predict disease recurrence and overall survival in a significant subset of patients. Adjuvant therapies for melanoma are notoriously toxic and associated with significant morbidity. Furthermore, it has been difficult to predict which patients will respond best to these treatments, if at all. DNA and RNA biomarkers have been developed to help overcome these problems. Biomarkers have been shown to upstage patients with melanoma, but are the assays sensitive and specific enough for clinical use as predictors of disease outcome or treatment response? We review our experience with DNA and RNA biomarkers in terms of their prognostic and predictive capabilities in malignant melanoma and outline their likely role in the future of melanoma staging, surveillance, and treatment. © 2005 Wiley‐Liss, Inc.     

Improving CRISPR/Cas9-mediated genome editing efficiency in Yarrowia lipolytica using direct tRNA-sgRNA fusions

Yarrowia lipolytica is an important oleaginous yeast currently used in the production of specialty chemicals and has a great potential for further applications in lipid biotechnology. Harnessing the full potential of Y. lipolytica is, however, limited by its inherent recalcitrance to genetic manipulation. In contrast to Saccharomyces cerevisiae, Y. lipolytica is poor in homology-mediated DNA repair and thus in homologous recombination, which limits site-specific gene editing in this yeast. Recently developed CRISPR/Cas9-based methods using tRNA-sgRNA fusions succeeded in editing some genomic loci in Y. lipolytica. Nonetheless, the majority of other tested loci either failed editing or editing was achieved but at very low efficiency using these methods. Using tools of secondary RNA structure prediction, we were able to improve the design of the tRNA-sgRNA fusions used for the expression of single guide RNA (sgRNA) in such methods. This resulted in high efficiency CRISPR/cas9 gene editing at chromosomal loci that failed gene editing or were edited at very low efficiencies with previous methods. In addition, we characterized the gene editing performance of our newly designed tRNA-sgRNA fusions for both chromosomal gene integration and deletion. As such, this study presents an efficient CRISPR/Cas9-mediated gene-editing tool for efficient genetic engineering of Yarrowia lipolytica.     

Validation study of existing gene expression signatures for anti-TNF treatment in patients with rheumatoid arthritis

So far, there are no means of identifying rheumatoid arthritis (RA) patients who will fail to respond to tumour necrosis factor blocking agents (anti-TNF), prior to treatment. We set out to validate eight previously reported gene expression signatures predicting therapy outcome. Genome-wide expression profiling using Affymetrix GeneChip Exon 1.0 ST arrays was performed on RNA isolated from whole blood of 42 RA patients starting treatment with infliximab or adalimumab. Clinical response according to EULAR criteria was determined at week 14 of therapy. Genes that have been reported to be associated with anti-TNF treatment were extracted from our dataset. K-means partition clustering was performed to assess the predictive value of the gene-sets. We performed a hypothesis-driven analysis of the dataset using eight existing gene sets predictive of anti-TNF treatment outcome. The set that performed best reached a sensitivity of 71% and a specificity of 61%, for classifying the patients in the current study. We successfully validated one of eight previously reported predictive expression profile. This replicated expression signature is a good starting point for developing a prediction model for anti-TNF treatment outcome that can be used in a daily clinical setting. Our results confirm that gene expression profiling prior to treatment is a useful tool to predict anti-TNF (non) response.     

Isolation of the melanoma-associated antigen p23 using antibody phage display

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma-associated Ags in patients. In this study, we isolated a recombinant phage-Fab clone (A10-5) from a phage-Fab library derived from the B cells of a melanoma patient in remission after immunotherapy. Purified A10-5 Fab bound at high levels to cultured melanoma cell lines and to tissue sections of metastatic and vertical growth phase primary melanoma, but not to radial growth phase primary melanoma, nevi, or normal skin. A10-5 Fab bound to both the surface and the cytoplasm of cultured melanoma cells, but only to the cytoplasm of cultured fibroblasts. Western blot analysis revealed A10-5 Fab reactivity with a 33- and a 23-kDa glycoprotein under nonreducing conditions, and with a 23-kDa protein only under reducing conditions. A cDNA with an open reading frame predicted to encode a 23-kDa protein was cloned by screening a melanoma cell cDNA library with A10-5 Fab. This protein (p23) is the human homologue of the murine tumor transplantation Ag P198 that interacts with the cytoplasmic domain of ErbB-3 expressed by melanoma cells. Thus, the Ab phage display method has identified a novel, stage-specific melanoma-associated Ag that may have therapeutic and diagnostic value.     

Active specific immunotherapy with hapten-modified autologous melanoma cell vaccine

 We have developed a novel approach to cancer immunotherapy – an autologous whole-cell vaccine modified with the hapten dinitrophenyl (DNP). This approach elicits significant inflammatory responses in metastatic sites and some objective tumor responses. Post-surgical adjuvant immunotherapy with DNP-modified melanoma vaccine in a setting of micrometastatic disease produces significant survival prolongation in stage III melanoma patients. Histologically, the inflammatory responses of the tumor consist of infiltration by lymphocytes, the majority of which are CD8⁺, HLA-DR⁺ T cells. T cells from these lesions tend to have mRNA for interferon γ. T cell receptor analysis suggests that the tumor-infiltrating T cells are clonally expanded. DNP-modified vaccine also induces T cells in the peripheral blood, which respond to DNP-modified autologous cells in a hapten-specific, MHC-restricted manner. Moreover, a T cell line generated from these lymphocytes responded to only a single HPLC fraction of MHC-associated, DNP-modified tumor peptides. Since inflammatory responses in metastases were not consistently associated with dramatic tumor regression, we considered the possibility of immunosuppression at the tumor site. We found that mRNA for the anti-inflammatory cytokine, interleukin-10 (IL-10) is expressed in most metastatic melanoma tissues and subsequently demonstrated that IL-10 protein is produced by melanoma cells. Thus the efficacy of DNP vaccine could be further enhanced by inhibition of IL-10 production or binding. Finally, we expect these results obtained with melanoma to be applicable to other human cancers. Received: 6 August 1996 / Accepted: 20 September 1996     

The OSUMMER lines: A series of ultraviolet-accelerated NRAS-mutant mouse melanoma cell lines syngeneic to C57BL/6

An increasing number of cancer subtypes are treated with front-line immunotherapy. However, approaches to overcome primary and acquired resistance remain limited. Preclinical mouse models are often used to investigate resistance mechanisms, novel drug combinations, and delivery methods; yet most of these models lack the genetic diversity and mutational patterns observed in human tumors. Here we describe a series of 13 C57BL/6J melanoma cell lines to address this gap in the field. The Ohio State University-Moffitt Melanoma Exposed to Radiation (OSUMMER) cell lines are derived from mice expressing endogenous, melanocyte-specific, and clinically relevant Nras driver mutations (Q61R, Q61K, or Q61L). Exposure of these animals to a single, non-burning dose of ultraviolet B accelerates the onset of spontaneous melanomas with mutational patterns akin to human disease. Furthermore, in vivo irradiation selects against potent tumor antigens, which could prevent the outgrowth of syngeneic cell transfers. Each OSUMMER cell line possesses distinct in vitro growth properties, trametinib sensitivity, mutational signatures, and predicted antigenicity. Analysis of OSUMMER allografts shows a correlation between strong, predicted antigenicity and poor tumor outgrowth. These data suggest that the OSUMMER lines will be a valuable tool for modeling the heterogeneous responses of human melanomas to targeted and immune-based therapies.     

The evolution of chloroplast RNA editing

RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria.     

A 15-year follow-up of AJCC stage III malignant melanoma patients treated postsurgically with Newcastle disease virus (NDV) oncolysate and determination of alterations in the CD8 T cell repertoire.

BACKGROUND: The development of effective adjuvant therapies for the treatment of high-risk melanoma patients is critical for the prevention of metastatic disease and improvement of patient survival. Active specific immunotherapy has been tested as an adjuvant treatment in numerous clinical trials with overall limited, but occasionally promising, success rates. Newcastle disease virus (NDV) oncolysate has been utilized as an adjunctive immunotherapeutic agent in the postsurgical management of these patients. A phase II study initiated in 1975 using adjuvant vaccine therapy composed of allogeneic and autologous human melanoma cells infected with live NDV (NDV oncolysate) in patients with AJCC stage III melanoma following therapeutic lymph node dissection has shown >60% survival rate at 10 years with no adverse effects. Continued long-term analysis of trials with promising early results as well as assessment of immunologic responses generated in these patients may result in improved therapeutic decisions for clinical trials in the future. MATERIALS AND METHODS: We analyzed the 15-year survival of patients treated postsurgically with NDV oncolysate in the phase II study described above. In an attempt to understand the immunological effects of this treatment, we have also carried out a comprehensive analysis of the peripheral blood T cell repertoire in these patients. RESULTS: The overall 15-year survival of this group of patients is 55%. Previous studies have suggested that improved outcome in patients undergoing immunotherapy is correlated with increased numbers of CD8(+)CD57(+) cells. In surviving patients, we observed a striking oligoclonality in the CD8(+) T cell population in peripheral blood, which reflects clonal expansions in the CD8(+)CD57(+) subset. CONCLUSIONS: The data suggest that adjuvant vaccination with NDV oncolysates is associated with prolonged survival of patients with lymph node-positive malignant melanoma and that CD8(+) T cells may be an important component of therapeutic efficacy.     

Emerging strategies to treat rare and intractable subtypes of melanoma

Melanoma is the deadliest form of skin cancer, possessing a diverse landscape of subtypes with distinct molecular signatures and levels of aggressiveness. Although immense progress has been achieved therapeutically for patients with the most common forms of this disease, little is known of how to effectively treat patients with rarer subtypes of melanoma. These subtypes include acral lentiginous (the rarest form of cutaneous melanoma; AL), uveal, and mucosal melanomas, which display variations in distribution across (a) the world, (b) patient age‐groups, and (c) anatomic sites. Unfortunately, patients with these relatively rare subtypes of melanoma typically respond worse to therapies approved for the more common, non‐AL cutaneous melanoma and do not have effective alternatives, and thus consequently have worse overall survival rates. Achieving durable therapeutic responses in these high‐risk melanoma subtypes represents one of the greatest challenges of the field. This review aims to collate and highlight effective preclinical and/or clinical strategies against these rare forms of melanoma.     

The Use of Sensitive Chemical Antibodies for Diagnosis: Detection of Low Levels of Epcam in Breast Cancer

EpCAM is expressed at low levels in a variety of normal human epithelial tissues, but is overexpressed in 70–90% of carcinomas. From a clinico-pathological point of view, this has both prognostic and therapeutic significance. EpCAM was first suggested as a therapeutic target for the treatment of epithelial cancers in the 1990s. However, following several immunotherapy trials, the results have been mixed. It has been suggested that this is due, at least in part, to an unknown level of EpCAM expression in the tumors being targeted. Thus, selection of patients who would benefit from EpCAM immunotherapy by determining EpCAM status in the tumor biopsies is currently undergoing vigorous evaluation. However, current EpCAM antibodies are not robust enough to be able to detect EpCAM expression in all pathological tissues. Here we report a newly developed EpCAM RNA aptamer, also known as a chemical antibody, which is not only specific but also more sensitive than current antibodies for the detection of EpCAM in formalin-fixed paraffin-embedded primary breast cancers. This new aptamer, together with our previously described aptamer, showed no non-specific staining or cross-reactivity with tissues that do not express EpCAM. They were able to reliably detect target proteins in breast cancer xenograft where an anti-EpCAM antibody (323/A3) showed limited or no reactivity. Our results demonstrated a more robust detection of EpCAM using RNA aptamers over antibodies in clinical samples with chromogenic staining. This shows the potential of aptamers in the future of histopathological diagnosis and as a tool to guide targeted immunotherapy.     

基于转录组测序数据识别黑猩猩RNA编辑位点

使用转录组测序(RNA-Seq)数据识别黑猩猩RNA编辑位点,探索了RNA编辑的识别机制以及潜在的功能影响．基于黑猩猩RNA-Seq数据与基因组序列的比对信息发现RNA-DNA错配位点,并构建编辑位点候选集．从中滤除基因组或转录组测序质量低的位点,其他的过滤条件包括3′端测不准、覆盖度、SNP位点以及估算的编辑水平．构建二项分布统计模型和Bonferroni多重检验滤除候选集中的随机错误,得到RNA编辑位点．选取落在已知基因上的编辑位点进行功能分析,并用Two Sample Logo软件分析编辑位点上下游序列的特征．识别出黑猩猩12种碱基替换型RNA编辑位点8 334个,其中有41个编辑位点改变原有的氨基酸,另有3个编辑位点落在microRNA(miRNA)潜在靶基因的种子结合区．统计学分析表明,分别有640和872个RNA编辑位点存在组织和性别差异．上下游碱基频率分析表明,多种类型的编辑位点紧邻碱基具有显著偏好．结果显示, RNA编辑在黑猩猩体内大量存在,且潜在具有重要的生物学功能,为进一步深入研究灵长类RNA编辑的机制奠定了基础．