When should we order a next generation sequencing test in a patient with cancer?

Technical advances in genome sequencing and the implementation of next-generation sequencing (NGS) in clinical oncology have paved the way for individualizing cancer patient therapy based on molecular profiles. When and how to use NGS testing in the clinic is at present an unsolved issue, although new research results provide evidence favoring this approach in some types of advanced cancer. Clinical research is evolving rapidly, from basket and umbrella trials to adaptative design precision oncology clinical studies, and genomic and molecular data often displace the classical clinical validation procedures of biomarkers. In this context, physicians must be aware of the clinical evidence behind these new biomarkers and NGS tests available, in order to use them in the right moment, and with a critical point of view. This review will present the status of currently available targeted drugs that can be effective based on actionable molecular alterations, and the NGS tests that are currently available, offering a practical guide for the application of Clinical Precision Oncology in the real world routine practice.probability of identifying a targetable mutation is low[62], the canceris in early stages with recognized and effective forms of standardtreatment, or the patient has an irreversible disease with very shortlife-expectancy. As with any other laboratory test, doctors andpatients must be sure before ordering an NGS test that its result willhave an impact of the therapeutic plan. In any case, standard single-gene molecular testing must always be performed when indicated,since important therapeutic targets might be potentially missed if nomolecular analyses were performed.Clinical trials are showing that NGS testing can have an impact inthe response rate and progression-free survival of patients, and cantherefore be a very useful strategy leading to new molecularly-tar-geted treatment indications. Key factors responsible for improvedresults in precision-oriented clinical research, include refining themolecular pathways studied, developing molecular testing that inte-grates standarised genomic tests with transcriptomic analysis andimmunohistochemistry, selecting more active targeted agents,designing combinations of targeted agents -also with other forms oftherapy, and providing early treatment recommendations with avail-able Molecular Multidisciplinary Boards. Interdisciplinary discussionare very important to help with the interpretation of unclear molecu-lar results that are oftentimes seen with NGS testing.Important unsolved issues that will need to be addressed in thefuture include deciding which is the best tissue to perform NGS (pri-mary tumor vs metastasis, tumor DNA vs circulating tumor DNA),when is the right moment to test (atfirst diagnosis of advanced dis-ease or when the disease is refractory), and whether there are NGSclinical trial designs that allow for the use of control groups. Finally,using a complete informed consent before NGS testing and communi-cating NGS reports to patients are two very important aspects of theprocedure that have raised ethical concerns, and that must be alwaysaddressed by the practicing oncologists when ordering a NGS test.Search strategy and selection criteriaWe identified references through PubMed with the search terms“cancer AND NGS,”“cancer AND next generation sequencing,”“can-cer AND genomics,”for articles published to March 30, 2020. Thefinalreference list was generated on the basis of originality and relevanceto the broad scope of this Review.FundingThis Review was funded in part by research funds from projectsPIE15/00068andPI17/01865(Instituto de Salud Carlos III) awardedto RC, projectsJR17/00007andPI17/008(Instituto de Salud CarlosIII), awarded to NR-L,PI15/01491andPI19/00549(Instituto de SaludCarlos III) awarded to AA, projectsSAF2017 82886-R(Ministerio deEconomía y Competitividad), INDISNET-S2011/BMD-2332(Fundaci on Ram on Areces), andHR17-00016("La Caixa" Foundation)awarded to FS-M, and projectsPI16/00354(Instituto de Salud CarlosIII) andB2017/BMD-3733from the Consejería de Educaci on, Juventudy Deporte, Comunidad de Madrid, awarded to MQ-F. The manuscriptis part of the activities of the endowed Chair of Personalised PrecisionOncology, Universidad Aut onoma de Madrid (UAM-Fundaci on Insti-tuto Roche)S

Microenvironmental Snail1-induced immunosuppression promotes melanoma growth

Melanoma is an aggressive form of skin cancer due to its high metastatic abilities and resistance to therapies. Melanoma cells reside in a heterogeneous tumour icroenvironment that acts as a crucial regulator of its progression. Snail1 is an epithelial-tomesenchymal transition transcription factor expressed during development and reactivated in pathological situations including fibrosis and cancer. In this work, we show that Snail1 is activated in the melanoma microenvironment, particularly in fibroblasts. Analysis of mouse models that allow stromal Snail1 depletion and therapeutic Snail1 blockade indicate that targeting Snail1 in the tumour microenvironment decreases melanoma growth and lung metastatic burden, extending mice survival. Transcriptomic analysis of melanoma-associated fibroblasts and analysis of the tumours indicate that stromal Snail1 induces melanoma growth by promoting an immunosuppressive microenvironment and a decrease in anti-tumour immunity. This study unveils a novel role of Snail1 in melanoma biology and supports its potential as a therapeutic targe

α-MSH regulates intergenic splicing of MC1R and TUBB3 in human melanocytes

Alternative splicing enables higher eukaryotes to increase their repertoire of proteins derived from a restricted number of genes. However, the possibility that functional diversity may also be augmented by splicing between adjacent genes has been largely neglected. Here, we show that the human melanocortin 1 receptor (MC1R) gene, a critical component of the facultative skin pigmentation system, has a highly complex and inefficient poly(A) site which is instrumental in allowing intergenic splicing between this locus and its immediate downstream neighbour tubulin-β-III (TUBB3). These transcripts, which produce two distinct protein isoforms localizing to the plasma membrane and the endoplasmic reticulum, seem to be restricted to humans as no detectable chimeric mRNA could be found in MC1R expressing mouse melanocytes. Significantly, treatment with the MC1R agonist α-MSH or activation of the stress response kinase p38-MAPK, both key molecules associated with ultraviolet radiation dermal insult and subsequent skin tanning, result in a shift in expression from MC1R in favour of chimeric MC1R-TUBB3 isoforms in cultured melanocytes. We propose that these chimeric proteins serve to equip melanocytes with novel cellular phenotypes required as part of the pigmentation response

Effect of excess weight and immune-related adverse events on the efficacy of cancer immunotherapy with anti-PD-1 antibodies

Immunotherapy is an effective treatment in advanced cancer, although predictors of response are limited. We studied whether excess weight influences the efficacy outcomes of immunotherapy. We have also evaluated the combined prognostic effect of excess weight and immune-related adverse events (irAEs). Efficacy of anti-PD-1 treatment was evaluated with both objective radiological response (ORR) rate and progression-free survival (PFS), and toxicity with irAEs. We studied the association between excess weight and ORR, PFS or irAEs. 132 patients diagnosed with advanced cancer were included. Median body mass index (BMI) was 24.9 kg/m2. 64 patients had normal weight (BMI<25 kg/m2), and 64 patients had excess weight (BMI≥25 kg/m2). Four patients had underweight and were excluded from further analysis. ORR was achieved in 50 patients (38.0%), median PFS was 6 months. 44 patients developed irAEs (33.3%). ORR was higher in excess weight patients than in patients with normal weight (51.6% vs 25.0%; OR 3.45, p = .0009). PFS was improved in patients with excess weight (7.25 months vs 4 months, HR 1.72, p = .01). The incidence of IrAEs was not different in patients with excess weight (54.5% vs 43.2%, p = .21). When high BMI and irAEs were combined, we observed a marked prognostic trend in ORR rate (87.5% vs 6.2%; OR 161.0, p < .00001), and in PFS (14 months vs 3 months; HR 5.89, p < .0001). Excess weight patients with advanced cancer that receive single-agent anti-PD-1 antibody therapy exhibit a significantly improved clinical outcome compared with normal BMI patients. This association was especially marked when BMI and irAEs were considered combined.This study was funded in part by the project Discovery, Validation and Implementation of Biomarkers for Precision Oncology [PIE15/00068], and the project FIS PI17/01865 from the Instituto de Salud Carlos III, Ministerio de Economia y Competitividad, awarded to RC, and the projects [JR 17/00007 and PI17/008], awarded to NRL

Metabolic rewiring induced by ranolazine improves melanoma responses to targeted therapy and immunotherapy

Resistance of melanoma to targeted therapy and immunotherapy is linked to metabolic rewiring. Here, we show that increased fatty acid oxidation (FAO) during prolonged BRAF inhibitor (BRAFi) treatment contributes to acquired therapy resistance in mice. Targeting FAO using the US Food and Drug Administration-approved and European Medicines Agency-approved anti-anginal drug ranolazine (RANO) delays tumour recurrence with acquired BRAFi resistance. Single-cell RNA-sequencing analysis reveals that RANO diminishes the abundance of the therapy-resistant NGFRhi neural crest stem cell subpopulation. Moreover, by rewiring the methionine salvage pathway, RANO enhances melanoma immunogenicity through increased antigen presentation and interferon signalling. Combination of RANO with anti-PD-L1 antibodies strongly improves survival by increasing antitumour immune responses. Altogether, we show that RANO increases the efficacy of targeted melanoma therapy through its effects on FAO and the methionine salvage pathway. Importantly, our study suggests that RANO could sensitize BRAFi-resistant tumours to immunotherapy. Since RANO has very mild side-effects, it might constitute a therapeutic option to improve the two main strategies currently used to treat metastatic melanoma

An adaptive signaling network in melanoma inflammatory niches confers tolerance to MAPK signaling inhibition

Mitogen-activated protein kinase (MAPK) pathway antagonists induce profound clinical responses in advanced cutaneous melanoma, but complete remissions are frustrated by the development of acquired resistance. Before resistance emerges, adaptive responses establish a mutation-independent drug tolerance. Antagonizing these adaptive responses could improve drug effects, thereby thwarting the emergence of acquired resistance. In this study, we reveal that inflammatory niches consisting of tumor-associated macrophages and fibroblasts contribute to treatment tolerance through a cytokine-signaling network that involves macrophage-derived IL-1β and fibroblast-derived CXCR2 ligands. Fibroblasts require IL-1β to produce CXCR2 ligands, and loss of host IL-1R signaling in vivo reduces melanoma growth. In tumors from patients on treatment, signaling from inflammatory niches is amplified in the presence of MAPK inhibitors. Signaling from inflammatory niches counteracts combined BRAF/MEK (MAPK/extracellular signal–regulated kinase kinase) inhibitor treatment, and consequently, inhibiting IL-1R or CXCR2 signaling in vivo enhanced the efficacy of MAPK inhibitors. We conclude that melanoma inflammatory niches adapt to and confer drug tolerance toward BRAF and MEK inhibitors early during treatmen

Distinct Pigmentary and Melanocortin 1 Receptor–Dependent Components of Cutaneous Defense against Ultraviolet Radiation

Genetic variation at the melanocortin 1 receptor (MC1R) is an important risk factor for developing ultraviolet (UV) radiation–induced skin cancer, the most common form of cancer in humans. The underlying mechanisms by which the MC1R defends against UV-induced skin cancer are not known. We used neonatal mouse skin (which, like human skin, contains a mixture of melanocytes and keratinocytes) to study how pigment cells and Mc1r genotype affect the genome-level response to UV radiation. Animals without viable melanocytes (Kit(W-v)/Kit(W-v)) or animals lacking a functional Mc1r (Mc1r(e)/Mc1r(e)) were exposed to sunburn-level doses of UVB radiation, and the patterns of large-scale gene expression in the basal epidermis were compared to each other and to nonmutant animals. Our analysis revealed discrete Kit- and Mc1r-dependent UVB transcriptional responses in the basal epidermis. The Kit-dependent UVB response was characterized largely by an enrichment of oxidative and endoplasmic reticulum stress genes, highlighting a distinctive role for pigmented melanocytes in mediating antioxidant defenses against genotoxic stresses within the basal epidermal environment. By contrast, the Mc1r-dependent UVB response contained an abundance of genes associated with regulating the cell cycle and oncogenesis. To test the clinical relevance of these observations, we analyzed publicly available data sets for primary melanoma and melanoma metastases and found that the set of genes specific for the Mc1r-dependent UVB response was able to differentiate between different clinical subtypes. Our analysis also revealed that the classes of genes induced by UVB differ from those repressed by UVB with regard to their biological functions, their overall number, and their size. The findings described here offer new insights into the transcriptional nature of the UV response in the skin and provide a molecular framework for the underlying mechanisms by which melanocytes and the Mc1r independently mediate and afford protection against UV radiation