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Since its discovery in 2007, we have seen the lives of patients diagnosed with advanced anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancers (NSCLC) transform with the advent of molecular therapies with first-, second-, and third-generation ALK inhibitors now available in the clinic. Despite great gains in patient survival now measured in years and preserved quality of life with targeted therapies, drug resistance is unfortunately inevitably encountered in this rare and unique molecular subset of lung cancer, and patients will eventually succumb to the disease. As these patients are often young, fit, and never smokers, the clinical and scientific communities have aligned to expedite drug development and access. Drug resistance profiling and further strategies are being explored through clinical trials, including the evaluation of specific drug sequencing and combinations to overcome such resistance and promote patient longevity. The cases of this report focus on precision medicine and aim to portray the pertinent aspects to consider when treating ALK-rearranged NSCLC in 2020, an ever-shifting space. By way of case examples, this report offers valuable information to the treating clinician, including the evolution of systemic treatments and the management of oligo-progression and multisite drug resistance. With the maturation of real-world data, we are fortunate to be experiencing quality and length of life for patients with this disease surpassing prior expectations in advanced lung cancer. KEY POINTS: This report focuses on the importance of genetic analysis of serial biopsies to capture the dynamic therapeutic vulnerabilities of a patient's tumor, providing a perspective on the complexity of ALK tyrosine kinase inhibitor (ALKi) treatment sequencing. These case examples contribute to the literature on ALK-rearranged and oncogene addicted non-small cell lung cancer (NSCLC), providing a framework for care in the clinic. In oligo-progressive disease, local ablative therapy and continuation of ALKi postprogression should be considered with potential for sustained disease control. ALK G1202R kinase domain mutations (KDM), highly prevalent at resistance to second-generation ALKi resistances, may emerge in non-EML4-ALK variant 3 cases and is sensitive to third-generation lorlatinib. When in compound with one or more ALK KDMs, resistance to lorlatinib is expected. In the case of rampantly progressive disease, rebiopsy and redefining biology in a timely manner may be informative.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Quinasa de Linfoma Anaplásico/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Resistencia a Antineoplásicos/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Calidad de VidaRESUMEN
Inflammation and coagulation are two intertwined pathways with evolutionary ties being traced back to the hemocyte, a single cell type in invertebrates that has functions in both the inflammatory and coagulation pathways. These systems have functioned together throughout evolution to provide a solid defence against infection, damaged cells and irritants. While these systems work in harmony the majority of the time, they can also become dysregulated or corrupted by tumours, enhancing tumour proliferation, invasion, dissemination and survival. This review aims to give a brief overview of how these systems work in harmony and how dysregulation of these systems aids in the development and progression of cancer, using glioma as an example.
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Coagulación Sanguínea/genética , Glioma/genética , Inflamación/genética , Coagulación Sanguínea/inmunología , Progresión de la Enfermedad , Glioma/inmunología , Glioma/patología , Hemocitos/inmunología , Humanos , Inflamación/inmunología , Inflamación/patologíaRESUMEN
Radiation is an important component of cancer treatment with more than half of all patients receive radiotherapy during their cancer experience. While the impact of radiation on tumour morphology is routinely examined in the pre-clinical and clinical setting, the impact of radiation on the tumour microenvironment and more specifically the inflammatory/immune response is less well characterised. Inflammation is a key contributor to short- and long-term cancer eradication, with significant tumour and normal tissue consequences. Therefore, the role of radiation in modulating the inflammatory response is highly topical given the current wave of targeted and immuno-therapeutic treatments for cancer. This review provides a general overview of how radiation modulates the inflammatory and immune response-(i) how radiation induces the inflammatory/immune system, (ii) the cellular changes that take place, (iii) how radiation dose delivery affects the immune response, and (iv) a discussion on research directions to improve patient survival, reduce side effects, improve quality of life, and reduce financial costs in the immediate future. Harnessing the benefits of radiation on the immune response will enhance its maximal therapeutic benefit and reduce radiation-induced toxicity.
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Inmunidad Innata/efectos de la radiación , Inflamación/radioterapia , Neoplasias/radioterapia , Análisis Costo-Beneficio , Relación Dosis-Respuesta en la Radiación , Humanos , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Inflamación/inmunología , Inflamación/patología , Neoplasias/inmunología , Neoplasias/patología , Calidad de Vida , Dosis de Radiación , Análisis de SupervivenciaRESUMEN
The discovery of a number of viruses with the ability to induce tumours in animals and transform human cells has vastly impacted cancer research. Much of what is known about tumorigenesis today regarding tumour drivers and tumour suppressors has been discovered through experiments using viruses. The SV40 virus has proven extremely successful in generating transgenic models of many human cancer types and this review provides an overview of these models and seeks to give evidence as to their relevance in this modern era of personalised medicine and technological advancements.
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Antígenos Transformadores de Poliomavirus/genética , Neoplasias Experimentales/virología , Animales , Humanos , Ratones Transgénicos , Virus 40 de los Simios/genéticaRESUMEN
Given the large socio-economic burden of cancer, there is an urgent need for in vivo animal cancer models that can provide a rationale for personalised therapeutic regimens that are translatable to the clinic. Recent developments in establishing mouse models that closely resemble human lung cancers involve the application of genetically engineered mouse models (GEMMs) for use in drug efficacy studies or to guide patient therapy. Here, we review recent applications of GEMMs in non-small cell lung cancer research for drug development and their potential in aiding biomarker discovery and understanding of biological mechanisms behind clinical outcomes and drug interactions.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Ingeniería Genética , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/tratamiento farmacológico , Ratones Transgénicos , Terapia Molecular Dirigida , Neoplasias Experimentales/sangre , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Medicina de PrecisiónRESUMEN
Brain cancer is a devastating and life-changing disease. Biomarkers are becoming increasingly important in addressing clinical issues, including in monitoring tumour progression and assessing survival and treatment response. The goal of this study was to identify prognostic biomarkers associated with glioma progression. Discovery proteomic analysis was performed on a small cohort of astrocytomas that were diagnosed as low-grade and recurred at a higher grade. Six proteins were chosen to be validated further in a larger cohort. Three proteins, CA9, CYFIP2, and LGALS3BP, were found to be associated with glioma progression and, in univariate analysis, could be used as prognostic markers. However, according to the results of multivariate analysis, these did not remain significant. These three proteins were then combined into a three-protein panel. This panel had a specificity and sensitivity of 0.7459 for distinguishing between long and short survival. In silico data confirmed the prognostic significance of this panel.
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BACKGROUND: Radiotherapy (RT) is a mainstay of treatment for patients with glioblastoma (GB). Early clinical trials show that short course hypofractionation showed no survival benefit compared to conventional regimens with or without temozolomide chemotherapy (TMZ) but reduces the number of doses required. Concerns around delayed neurological deficits and reduced cognition from short course hypofractionated RT remain a concern. The aim of this study was to evaluate the effect of increased interfractional time using two different radiation fractionation regimens on GB. METHODS: The radiobiological effect of increasing doses 0-20 Gy x-ray photon RT on Gl261 and CT2A GB cell lines was compared by colony forming assay, DNA damage by alkaline comet assay, oxidative stress, DNA damage, cell cycle, and caspase-3/7 by MUSE® flow cytometric analyses, and protein expression by western blot analyses. Conventional (20 Gy/10 fractions) and hypofractionated (20 Gy/4 fractions spaced 72 h apart) RT regimens with and without TMZ (200 mg/kg/day) were performed in syngeneic Gl261 and CT2A intracranial mouse models using the Small Animal Radiation Research Platform (Xstrahl Inc.). RESULTS: X-ray photon radiation dose-dependently increased reactive oxygen species, DNA damage, autophagy, and caspase 3/7-mediated apoptotic cell death. While the conventional fractionated dose regimen of 20 Gy/10 f was effective at inducing cell death via the above mechanism, this was exceeded by a 20 Gy/4 f regimen which improved median survival and histopathology in Gl261-tumor bearing mice, and eradicated tumors in CT2A tumors with no additional toxicity. CONCLUSIONS: Spacing of hypofractionated RT doses 72 h apart showed increased median survival and tumor control via increased activation of RT-mediated cell death, with no observed increased in radiotoxicity. This supports further exploration of differential RT fractionated regimens in GB clinical trials to reduce delayed neurological radiotoxicity.
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Antineoplásicos Alquilantes/uso terapéutico , Glioblastoma/tratamiento farmacológico , Glioblastoma/radioterapia , Hipofraccionamiento de la Dosis de Radiación , Temozolomida/uso terapéutico , Animales , Ratones , Radioterapia/métodos , Factores de Tiempo , Resultado del TratamientoRESUMEN
Mesothelioma is an aggressive cancer with limited treatment options and a poor prognosis. Phytocannabinoids possess anti-tumour and palliative properties in multiple cancers, however their effects in mesothelioma are unknown. We investigated the anti-cancer effects and potential mechanisms of action for several phytocannabinoids in mesothelioma cell lines. A panel of 13 phytocannabinoids inhibited growth of human (MSTO and H2452) and rat (II-45) mesothelioma cells in vitro, and cannabidiol (CBD) and cannabigerol (CBG) were the most potent compounds. Treatment with CBD or CBG resulted in G0/G1 arrest, delayed entry into S phase and induced apoptosis. CBD and CBG also significantly reduced mesothelioma cell migration and invasion. These effects were supported by changes in the expression of genes associated with the cell cycle, proliferation, and cell movement following CBD or CBG treatment. Gene expression levels of CNR1, GPR55, and 5HT1A also increased with CBD or CBG treatment. However, treatment with CBD or CBG in a syngeneic orthotopic rat mesothelioma model was unable to increase survival. Our data show that cannabinoids have anti-cancer effects on mesothelioma cells in vitro and alternatives of drug delivery may be needed to enhance their effects in vivo.
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BACKGROUND: High grade gliomas (HGG) are incapacitating and prematurely fatal diseases. To overcome the poor prognosis, novel therapies must overcome the selective and restricted permeability of the blood-brain barrier (BBB). This study critically evaluated whether in vitro human normal BBB and tumor BBB (BBTB) are suitable alternatives to "gold standard" in vivo models to determine brain permeability. METHODS: A systematic review utilizing the PRISMA guidelines used English and full-text articles from the past 5 years in the PubMed, Embase, Medline and Scopus databases. Experimental studies employing human cell lines were included. RESULTS: Of 1335 articles, the search identified 24 articles for evaluation after duplicates were removed. Eight in vitro and five in vivo models were identified with the advantages and disadvantages compared within and between models, and against patient clinical data where available. The greatest in vitro barrier integrity and stability, comparable to in vivo and clinical permeability data, were achieved in the presence of all cell types of the neurovascular unit: endothelial cells, astrocytes/glioma cells, pericytes and neurons. CONCLUSIONS: In vitro co-culture BBB models utilizing stem cell-derived or primary cells are a suitable proxy for brain permeability studies in order to reduce animal use in medical research.
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Thioredoxin reductase (TrxR) is an evolutionary diverse enzyme. In higher eukaryotes, TrxR contains a selenocysteine in the active site which confers high activity and a broad substrate range. The parasitic nematode Haemonchus contortus contains two TrxRs, a cytoplasmic enzyme HcTrxR1 with a selenocysteine in the active site (Gly-Cys-SeCys-Gly), similar to the mammalian TrxR, and a mitochondrial enzyme HcTrxR2 with a Gly-Cyc-Cys-Gly active site which is unique to nematodes. Both enzymes were cloned and expressed and their activity compared to rat TrxR. Although the expressed HcTrxR1 contained a cysteine to replace the selenocycteine, it showed broad activity with thioredoxins from Escherichia coli, sheep, and H. contortus similar to the rat selenoenzyme. However, HcTrxR2 had high activity only with the mitochondrial H. contortus thioredoxin. HcTrxR2 was also active with the commonly used substrate 5,5'-dithiobis(2-nitrobenzoate) and sodium selenite and was inhibited by auranofin and the natural product compounds curcumin and theaflavin. This demonstrates that the non-seleno-enzyme HcTrxR2 is as active as the rat seleno-containing enzyme when reacting with the H. contortus thioredoxin.
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Haemonchus/enzimología , Proteínas del Helminto/metabolismo , Proteínas Mitocondriales/metabolismo , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Animales , Auranofina/farmacología , Biflavonoides/farmacología , Catequina/farmacología , Clonación Molecular , Curcumina/farmacología , ADN de Helmintos/química , ADN de Helmintos/genética , Ácido Ditionitrobenzoico/metabolismo , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Expresión Génica , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Selenito de Sodio/metabolismo , Especificidad por SustratoRESUMEN
Glioblastoma, the most aggressive form of glioma, has a 5-year survival rate of <5%. While radiation and immunotherapies are routinely studied in the murine Gl261 glioma model, little is known about its inherent immune response. This study quantifies the temporal and spatial localization of immune cell populations and mediators during glioma development. Eight-week old male C57Bl/6 mice were orthotopically inoculated with 1x106 Gl261 cells and tumor morphology, local and systemic immune cell populations, and plasma cytokines/chemokines assessed at day 0, 1, 3, 7, 14, and 21 post-inoculation by magnetic resonance imaging, chromogenic immunohistochemistry, multiplex immunofluorescent immunohistochemistry, flow cytometry and multiplex immunoassay respectively. From day 3 tumors were distinguishable with >30% Ki67 and increased tissue vascularization (p<0.05). Increasing tumor proliferation/malignancy and vascularization were associated with significant temporal changes in immune cell populations within the tumor (p<0.05) and systemic compartments (p = 0.02 to p<0.0001). Of note, at day 14 16/24 plasma cytokine/chemokines levels decreased coinciding with an increase in tumor cytotoxic T cells, natural killer and natural killer/T cells. Data derived provide baseline characterization of the local and systemic immune response during glioma development. They reveal that type II macrophages and myeloid-derived suppressor cells are more prevalent in tumors than regulatory T cells, highlighting these cell types for further therapeutic exploration.
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Glioma/inmunología , Inmunidad Innata , Células Asesinas Naturales/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Linaje de la Célula/inmunología , Proliferación Celular/genética , Quimiocinas/sangre , Quimiocinas/inmunología , Citocinas/sangre , Citocinas/inmunología , Progresión de la Enfermedad , Citometría de Flujo , Glioma/sangre , Glioma/patología , Humanos , Células Asesinas Naturales/metabolismo , Ratones , Linfocitos T Citotóxicos/metabolismoRESUMEN
We discuss the molecular evolution of gliosarcoma, a mesenchymal type of glioblastoma (GBM), using the case of a 37-yr-old woman who developed two recurrences and an extracranial metastasis. She was initially diagnosed with isocitrate dehydrogenase (IDH) wild-type gliosarcoma in the frontal lobe and treated with surgery followed by concurrent radiotherapy with temozolomide. Five months later the tumor recurred in the left frontal lobe, outside the initially resected area, and was treated with further surgery and radiotherapy. Six months later the patient developed a second left frontal recurrence and was again treated with surgery and radiotherapy. Six weeks later, further recurrence was observed in the brain and bone, and biopsy confirmed metastases in the pelvic bones. To understand the clonal relationships between the four tumor instances and the origin of metastasis, we performed whole-genome sequencing of the intracranial tumors and the tumor located in the right iliac bone. We compared their mutational and copy-number profiles and inferred the clonal phylogeny. The tumors harbored shared alterations in GBM driver genes, including mutations in TP53, NF1, and RB1, and CDKN2A deletion. Whole-genome doubling was identified in the first recurrence and the extracranial metastasis. Comparisons of the metastatic to intracranial tumors highlighted a high similarity in molecular profile but contrasting evidence regarding the origin of the metastasis. Subclonal reconstruction suggested a parallel evolution of the recurrent tumors, and that the metastatic tumor was largely derived from the first recurrence. We conclude that metastasis in glioma can be a late event in tumorigenesis.
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Transformación Celular Neoplásica/genética , Evolución Clonal/genética , Gliosarcoma/etiología , Gliosarcoma/patología , Adulto , Alelos , Biomarcadores de Tumor , Biopsia , Terapia Combinada , Variaciones en el Número de Copia de ADN , Femenino , Gliosarcoma/terapia , Humanos , Inmunohistoquímica , Imagen Multimodal/métodos , Mutación , Metástasis de la Neoplasia , Estadificación de Neoplasias , RecurrenciaRESUMEN
Brain, lung, and colon tissue experience deleterious immune-related adverse events when immune-oncological agents or radiation are administered. However, there is a paucity of information regarding whether the addition of radiation to immuno-oncological regimens exacerbates the tissue inflammatory response. We used a murine model to evaluate sub-acute tissue damage and the systemic immune response in C57Bl/6 mice when administered systemic anti-programmed cell death protein 1 (αPD-1) immunotherapy alone or in combination with stereotactic fractionated 10 gray/5 X-ray radiation to normal brain, lung or colon tissue. The model indicated that combinatorial αPD-1 immunotherapy and radiation may alter normal colon cell proliferation and cerebral blood vasculature, and induce systemic thrombocytopenia, lymphopenia, immune suppression, and altered immune repertoire (including interleukin-1ß). Therein our data supports close monitoring of hematological and immune-related adverse events in patients receiving combination therapy.
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Thioredoxins are a family of small proteins conserved through evolution, which are essential for the maintenance of cellular homeostasis. The "classic" thioredoxin, identified in most species, is a 12-kDa protein with a Cys-Pro-Gly-Cys (CPGC) active site. However, in nematodes a larger protein, 16 kDa, with a Cys-Pro-Pro-Cys (CPPC) active site was identified. We report that in the parasitic nematode Haemonchus contortus, both the 12-kDa (HcTrx1) and the 16-kDa (HcTrx3) species are expressed through the life cycle. However, the HcTrx3 is expressed at higher concentrations. Recombinant HcTrx1 and HcTrx3 were produced and both reduced insulin at a rate similar to that observed with ovine (host) and Escherichia coli thioredoxins and both were regenerated by a mammalian thioredoxin reductase, demonstrating that they have similar thioredoxin activity. Unlike mammalian thioredoxins, both proteins were able to reduce oxidised glutathione and hydrogen peroxide. This suggests essential roles for these proteins in response to oxidative stress and the host immune attack. Analysis of ivermectin-resistant H. contortus showed that expression of both genes were increased in a drug-resistant strain relative to a sensitive strain. Involvement in drug resistance identifies these thioredoxin proteins as potential drug targets for parasite control.
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Haemonchus/metabolismo , Tiorredoxinas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
While treatment with surgery, radiotherapy and/or chemotherapy may prolong life for patients with glioblastoma, recurrence is inevitable. What is still being discovered is how much these treatments and recurrence of disease affect the molecular profiles of these tumors and how these tumors adapt to withstand these treatment pressures. Understanding such changes will uncover pathways used by the tumor to evade destruction and will elucidate new targets for treatment development. Nineteen matched pre-treatment and post-treatment glioblastoma tumors were subjected to gene expression profiling (Fluidigm, TaqMan assays), MGMT promoter methylation analysis (pyrosequencing) and protein expression analysis of the DNA repair pathways, known to be involved in temozolomide resistance (immunohistochemistry). Gene expression profiling to molecularly subtype tumors revealed that 26% of recurrent post-treatment specimens did not match their primary diagnostic specimen subtype. Post-treatment specimens had molecular changes which correlated with known resistance mechanisms including increased expression of APEX1 (p < 0.05) and altered MGMT methylation status. In addition, genes associated with immune suppression, invasion and aggression (GPNMB, CCL5, and KLRC1) and polarization toward an M2 phenotype (CD163 and MSR1) were up-regulated in post-treatment tumors, demonstrating an overall change in the tumor microenvironment favoring aggressive tumor growth and disease recurrence. This was confirmed by in vitro studies that determined that glioma cell migration was enhanced in the presence of M2 polarized macrophage conditioned media. Further, M2 macrophage-modulated migration was markedly enhanced in post-treatment (temozolomide resistant) glioma cells. These findings highlight the ability of glioblastomas to evade not only the toxic onslaught of therapy but also to evade the immune system suggesting that immune-altering therapies may be of value in treating this terrible disease.
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Malignant pleural mesothelioma is an aggressive cancer with poor prognosis. Here we have investigated in vitro efficacy of BAMLET and BLAGLET complexes (anti-cancer complexes consisting of oleic acid and bovine α-lactalbumin or ß-lactoglobulin respectively) in killing mesothelioma cells, determined BAMLET and BLAGLET structures, and investigated possible biological mechanisms. We performed cell viability assays on 16 mesothelioma cell lines. BAMLET and BLAGLET having increasing oleic acid content inhibited human and rat mesothelioma cell line proliferation at decreasing doses. Most of the non-cancer primary human fibroblasts were more resistant to BAMLET than were human mesothelioma cells. BAMLET showed similar cytotoxicity to cisplatin-resistant, pemetrexed-resistant, vinorelbine-resistant, and parental rat mesothelioma cells, indicating the BAMLET anti-cancer mechanism may be different to drugs currently used to treat mesothelioma. Cisplatin, pemetrexed, gemcitabine, vinorelbine, and BAMLET, did not demonstrate a therapeutic window for mesothelioma compared with immortalised non-cancer mesothelial cells. We demonstrated by quantitative PCR that ATP synthase is downregulated in mesothelioma cells in response to regular dosing with BAMLET. We sought structural insight for BAMLET and BLAGLET activity by performing small angle X-ray scattering, circular dichroism, and scanning electron microscopy. Our results indicate the structural mechanism by which BAMLET and BLAGLET achieve increased cytotoxicity by holding increasing amounts of oleic acid in an active cytotoxic state encapsulated in increasingly unfolded protein. Our structural studies revealed similarity in the molecular structure of the protein components of these two complexes and in their encapsulation of the fatty acid, and differences in the microscopic structure and structural stability. BAMLET forms rounded aggregates and BLAGLET forms long fibre-like aggregates whose aggregation is more stable than that of BAMLET due to intermolecular disulphide bonds. The results reported here indicate that BAMLET and BLAGLET may be effective second-line treatment options for mesothelioma.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Lactalbúmina/farmacología , Neoplasias Pulmonares/patología , Mesotelioma/patología , Ácido Oléico/farmacología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Lactalbúmina/química , Mesotelioma Maligno , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Modelos Moleculares , Conformación Molecular , Ácido Oléico/químicaRESUMEN
The SV40 viral oncogene has been used since the 1970s as a reliable and reproducible method to generate transgenic mouse models. This seminal discovery has taught us an immense amount about how tumorigenesis occurs, and its success has led to the evolution of many mouse models of cancer. Despite the development of more modern and targeted approaches for developing genetically engineered mouse models of cancer, SV40-induced mouse models still remain frequently used today. This review discusses a number of cancer types in which SV40 mouse models of cancer have been developed and highlights their relevance and importance to preclinical research.
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Ratones Transgénicos , Neoplasias/virología , Animales , Ratones , Virus 40 de los Simios/fisiologíaRESUMEN
Heterogeneity is a hallmark of glioblastoma with intratumoral heterogeneity contributing to variability in responses and resistance to standard treatments. Promoter methylation status of the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) is the most important clinical biomarker in glioblastoma, predicting for therapeutic response. However, it does not always correlate with response. This may be due to intratumoral heterogeneity, with a single biopsy unlikely to represent the entire lesion. Aberrations in other DNA repair mechanisms may also contribute. This study investigated intratumoral heterogeneity in multiple glioblastoma tumors with a particular focus on the DNA repair pathways. Transcriptional intratumoral heterogeneity was identified in 40% of cases with variability in MGMT methylation status found in 14% of cases. As well as identifying intratumoral heterogeneity at the transcriptional and epigenetic levels, targeted next generation sequencing identified between 1 and 37 unique sequence variants per specimen. In-silico tools were then able to identify deleterious variants in both the base excision repair and the mismatch repair pathways that may contribute to therapeutic response. As these pathways have roles in temozolomide response, these findings may confound patient management and highlight the importance of assessing multiple tumor biopsies.
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Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Glioblastoma/genética , Regiones Promotoras Genéticas/genética , Proteínas Supresoras de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos Alquilantes/uso terapéutico , Biomarcadores Farmacológicos , Biopsia , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/tratamiento farmacológico , Metilación de ADN , Dacarbazina/análogos & derivados , Dacarbazina/uso terapéutico , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Glioblastoma/diagnóstico , Glioblastoma/tratamiento farmacológico , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , TemozolomidaRESUMEN
The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 µl whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.
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Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Mesotelioma/inmunología , Mesotelioma/patología , Trasplante de Neoplasias/métodos , Animales , Linfocitos B/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo/métodos , Células Asesinas Naturales/inmunología , Mesotelioma Maligno , Monitorización Inmunológica/métodos , Monocitos/inmunología , Ratas , Ratas Endogámicas F344 , Subgrupos de Linfocitos T/inmunologíaRESUMEN
The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.