May direct-to-consumer genetic testing have an impact on general practitioners' daily practice? a cross-sectional study of patients' intentions towards this approach.

BACKGROUND: Direct-to-consumer genetic testing (DTCGT) offers individuals access to information on their probable risks of suffering from a wide range of chronic diseases. General practitioners (GPs) will probably play a major role in supporting its use, but patients' perception of DTCGT remain unclear. This study aimed to describe those attitudes and expectations and how they might affect GPs' daily practices. METHODS: In 2018-2019, a study related to the use of DTCGT for preventive care in general medicine was conducted among patients in Switzerland's French-speaking areas. Data were collected in the waiting room using a self-administrated questionnaire about patients' interest in DTCGT and what their attitudes might be if testing revealed an elevated risk of diabetes, colorectal cancer, or Alzheimer's disease. RESULTS: About 40% of the 929 participating (participation rate about 80%) patients had heard about DTCGT and, once the test had been explained, 43% reported that they would be interested in being tested. If that testing suggested an elevated risk of disease, the majority of patients reported that they would change their lifestyle (65%-81%, depending on the disease), request more examinations (63%-77%), and expect changes in their GP's follow-up (48%-59%). Personal characteristics such as sex, age, urbanity, marital status, and perceived health were factors predictive of patients' attitudes. CONCLUSION: Findings indicated that the generalization of DTCGT might affect GPs' daily practices in terms of workload and knowledge about this approach. However, this result must be qualified by the fact that it is based on hypothetical situations.

Evolution of late-stage metastatic melanoma is dominated by aneuploidy and whole genome doubling.

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.

European Practical and Patient-Centred Guidelines for Adult Obesity Management in Primary Care.

The first contact for patients with obesity for any medical treatment or other issues is generally with General Practitioners (GPs). Therefore, given the complexity of the disease, continuing GPs' education on obesity management is essential. This article aims to provide obesity management guidelines specifically tailored to GPs, favouring a practical patient-centred approach. The focus is on GP communication and motivational interviewing as well as on therapeutic patient education. The new guidelines highlight the importance of avoiding stigmatization, something frequently seen in different health care settings. In addition, managing the psychological aspects of the disease, such as improving self-esteem, body image and quality of life must not be neglected. Finally, the report considers that achieving maximum weight loss in the shortest possible time is not the key to successful treatment. It suggests that 5-10% weight loss is sufficient to obtain substantial health benefits from decreasing comorbidities. Reducing waist circumference should be considered even more important than weight loss per se, as it is linked to a decrease in visceral fat and associated cardiometabolic risks. Finally, preventing weight regain is the cornerstone of lifelong treatment, for any weight loss techniques used (behavioural or pharmaceutical treatments or bariatric surgery).

Ethical, pedagogical, socio-political and anthropological implications of quaternary prevention / Implicações éticas, pedagógicas, sócio-políticas e antropológicas da prevenção quaternária / Implicaciones éticas, pedagógicas, sociopolíticas y antropológicas de la prevención cuaternaria

The concept of quaternary prevention, resulting from a reflection on the doctor-patient relationship, is presented as a renewal of the age-old ethical requirement: first, a doctor must do no harm; second, the doctor must control himself/herself. The origin of the concept, its endorsement by the World Organization of Family Doctors (WONCA) and the European Union of General Practitioners (UEMO), its dissemination, and the debates to which it has given rise, are presented by a panel of authors from 10 countries. This collective text deals more specifically with: the bioethics of prevention, the importance of teaching Quaternary prevention and factual medicine, the social and political implications of the concept of quaternary prevention, and its anthropological dimensions.

Multimorbidity and patterns of chronic conditions in a primary care population in Switzerland: a cross-sectional study.

OBJECTIVE: To characterise in details a random sample of multimorbid patients in Switzerland and to evaluate the clustering of chronic conditions in that sample. METHODS: 100 general practitioners (GPs) each enrolled 10 randomly selected multimorbid patients aged ≥18 years old and suffering from at least three chronic conditions. The prevalence of 75 separate chronic conditions from the International Classification of Primary Care-2 (ICPC-2) was evaluated in these patients. Clusters of chronic conditions were studied in parallel. RESULTS: The final database included 888 patients. Mean (SD) patient age was 73.0 (12.0) years old. They suffered from 5.5 (2.2) chronic conditions and were prescribed 7.7 (3.5) drugs; 25.7% suffered from depression. Psychological conditions were more prevalent among younger individuals (≤66 years old). Cluster analysis of chronic conditions with a prevalence ≥5% in the sample revealed four main groups of conditions: (1) cardiovascular risk factors and conditions, (2) general age-related and metabolic conditions, (3) tobacco and alcohol dependencies, and (4) pain, musculoskeletal and psychological conditions. CONCLUSION: Given the emerging epidemic of multimorbidity in industrialised countries, accurately depicting the multiple expressions of multimorbidity in family practices' patients is a high priority. Indeed, even in a setting where patients have direct access to medical specialists, GPs nevertheless retain a key role as coordinators and often as the sole medical reference for multimorbid patients.

Co-existence of BRAF and NRAS driver mutations in the same melanoma cells results in heterogeneity of targeted therapy resistance.

Acquired chemotherapeutic resistance of cancer cells can result from a Darwinistic evolution process in which heterogeneity plays an important role. In order to understand the impact of genetic heterogeneity on acquired resistance and second line therapy selection in metastatic melanoma, we sequenced the exomes of 27 lesions which were collected from 3 metastatic melanoma patients treated with targeted or non-targeted inhibitors. Furthermore, we tested the impact of a second NRAS mutation in 7 BRAF inhibitor resistant early passage cell cultures on the selection of second line therapies.We observed a rapid monophyletic evolution of melanoma subpopulations in response to targeted therapy that was not observed in non-targeted therapy. We observed the acquisition of NRAS mutations in the BRAF mutated patient treated with a BRAF inhibitor in 1 of 5 of his post-resistant samples. In an additional cohort of 5 BRAF-inhibitor treated patients we detected 7 NRAS mutations in 18 post-resistant samples. No NRAS mutations were detected in pre-resistant samples. By sequencing 65 single cell clones we prove that NRAS mutations co-occur with BRAF mutations in single cells. The double mutated cells revealed a heterogeneous response to MEK, ERK, PI3K, AKT and multi RTK - inhibitors.We conclude that BRAF and NRAS co-mutations are not mutually exclusive. However, the sole finding of double mutated cells in a resistant tumor is not sufficient to determine follow-up therapy. In order to target the large pool of heterogeneous cells in a patient, we think combinational therapy targeting different pathways will be necessary.

Tumour hypoxia promotes melanoma growth and metastasis via High Mobility Group Box-1 and M2-like macrophages.

Hypoxia is a hallmark of cancer that is strongly associated with invasion, metastasis, resistance to therapy and poor clinical outcome. Tumour hypoxia affects immune responses and promotes the accumulation of macrophages in the tumour microenvironment. However, the signals linking tumour hypoxia to tumour-associated macrophage recruitment and tumour promotion are incompletely understood. Here we show that the damage-associated molecular pattern High-Mobility Group Box 1 protein (HMGB1) is released by melanoma tumour cells as a consequence of hypoxia and promotes M2-like tumour-associated macrophage accumulation and an IL-10 rich milieu within the tumour. Furthermore, we demonstrate that HMGB1 drives IL-10 production in M2-like macrophages by selectively signalling through the Receptor for Advanced Glycation End products (RAGE). Finally, we show that HMGB1 has an important role in murine B16 melanoma growth and metastasis, whereas in humans its serum concentration is significantly increased in metastatic melanoma. Collectively, our findings identify a mechanism by which hypoxia affects tumour growth and metastasis in melanoma and depict HMGB1 as a potential therapeutic target.

Romidepsin and Azacitidine Synergize in their Epigenetic Modulatory Effects to Induce Apoptosis in CTCL.

PURPOSE: Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of malignancies that despite available therapies commonly relapse. The emergence of combination epigenetic therapies in other hematologic malignancies have made investigation of such combinations in CTCL a priority. Here, we explore the synergistic antiproliferative effects of romidepsin, an HDAC inhibitor, and azacitidine, a demethylating agent, combination in CTCL. EXPERIMENTAL DESIGN: The growth inhibition under combination treatment and single agent was explored by the MTT cell viability assay and the Annexin V/propidium iodide (PI) apoptosis assay in different CTCL cell lines and tumor cells derived from Sézary syndrome patients. Quantitative analysis of a dose-effect relationship of romidepsin and azacitidine was done by the CompuSyn software. Investigation of mechanism of action was performed by flow cytometry, immunoblotting, qRT-PCR arrays, and chromatin immunoprecipitation. Global CpG methylation sequencing was utilized to study genome methylation alteration under the treatment modalities. RESULTS: The combination of romidepsin and azacitidine exerts synergistic antiproliferative effects and induction of apoptosis involving activation of the caspase cascade in CTCL cell lines and tumor cells derived from Sézary syndrome patients. We identified genes that were selectively induced by the combination treatment, such as the tumor suppressor geneRhoBthat is linked to enhanced histone acetylation at its promoter region in parallel with pronounced expression of p21. Global CpG methylation sequencing in a CTCL cell line and tumor cells demonstrated a subset of genes with a unique change in methylation profile in the combination treatment. CONCLUSIONS: The synergistic antiproliferative effects of romidepsin and azacitidine combination treatment justify further exploration in clinical trials for advanced CTCL.

Philosophical roots of Quaternary Prevention / Raízes filosóficas da Prevenção Quaternária / Raíces filosóficas de la Prevención Cuaternaria

This article explores two philosophical dimensions of quaternary prevention since it represents the family doctors' response to overmedicalization. The first dimension refers to the theory of knowledge and the second to the theory of action. Despite their interconnectedness, they are addressed separately. Firstly, in the theories of knowledge (Epistemology) we argue that the positivism of Evidence-Based Medicine (EBM), so useful to select good practices, should be balanced against critical vision of the use of EBM coupled with a constructivist view through the narrative-based medicine. Secondly, in the theory of action (Ethics) we argue that the non-maleficence principle (primum non nocere) needs to be balanced by the beneficence principle. The latter is the primary medical obligation and doctors should cultivate this practical wisdom. Finally, some aspects of P4's future challenges are discussed such as health inequalities, interprofessional collaboration, responsibility, managerialism, and the integrative medicine, where a philosophical position should be considered.

Este artigo explora duas dimensões filosóficas da prevenção quaternária, uma vez que esta representa a resposta dos médicos de família para a sobremedicalização. A primeira dimensão se refere à teoria do conhecimento e a segunda à teoria da ação. Apesar de suas interconexões, elas são abordadas separadamente. Em primeiro lugar, com relação à teoria do conhecimento (Epistemologia) argumenta-se que o positivismo da Medicina Baseada em Evidências (MBE), tão útil para selecionar boas práticas, deve ser equilibrado com uma visão crítica do uso da EBM, juntamente com uma visão construtivista através da medicina baseada em narrativas. Em segundo lugar, com relação à teoria da ação (Ética) argumenta-se que o princípio da não-maleficência (primum non nocere) precisa ser equilibrado pelo princípio da beneficência. Este último constitui-se obrigação médica primária e os médicos deveriam cultivar essa sabedoria prática. Finalmente, alguns aspectos dos futuros desafios da P4 são discutidos, tais como as desigualdades na saúde, a colaboração interprofissional, responsabilidade, gerencialismo, e a medicina integrativa, onde suas posições filosóficas deveriam ser consideradas

Este artículo explora dos dimensiones filosóficas de la prevención cuaternaria, ya que esta representa la respuesta de los médicos de familia a la sobremedicalización. La primera dimensión se refiere a la teoría del conocimiento y la segunda a la teoría de la acción. A pesar de sus interconexiones, ellas serán discutidas en separado. En primer lugar, con respecto a las teorías del conocimiento (Epistemología) argumentase que el positivismo de la Medicina Basada en la Evidencia (MBE), tan útil para seleccionar las buenas prácticas, debería ser equilibrada con una visión crítica del uso de la MBE, junto con una visión constructivista por medio de la medicina basada en la narrativa. En segundo lugar, con respecto a la teoría de la acción (Ética) argumentase que el principio de no maleficencia (primum non nocere) debe ser equilibrado por el principio de beneficencia. Este último es la obligación médica primaria y los médicos deberían cultivar esta sabiduría en la práctica. Finalmente, se discuten algunos aspectos de los retos futuros de la P4, como las desigualdades en salud, la colaboración interprofesional, la responsabilidad, el gerencialismo, y la medicina integral, donde sus posiciones filosóficas deberían ser consideradas.

Methylation-dependent SOX9 expression mediates invasion in human melanoma cells and is a negative prognostic factor in advanced melanoma.

BACKGROUND: Melanoma is the most fatal skin cancer displaying a high degree of molecular heterogeneity. Phenotype switching is a mechanism that contributes to melanoma heterogeneity by altering transcription profiles for the transition between states of proliferation/differentiation and invasion/stemness. As phenotype switching is reversible, epigenetic mechanisms, like DNA methylation, could contribute to the changes in gene expression. RESULTS: Integrative analysis of methylation and gene expression datasets of five proliferative and five invasion melanoma cell cultures reveal two distinct clusters. SOX9 is methylated and lowly expressed in the highly proliferative group. SOX9 overexpression results in decreased proliferation but increased invasion in vitro. In a B16 mouse model, sox9 overexpression increases the number of lung metastases. Transcriptional analysis of SOX9-overexpressing melanoma cells reveals enrichment in epithelial to mesenchymal transition (EMT) pathways. Survival analysis of The Cancer Genome Atlas melanoma dataset shows that metastatic patients with high expression levels of SOX9 have significantly worse survival rates. Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients. CONCLUSIONS: Our genome-wide DNA methylation and gene expression study of 10 early passage melanoma cell cultures reveals two phenotypically distinct groups. One of the genes regulated by DNA methylation between the two groups is SOX9. SOX9 induces melanoma cell invasion and metastasis and decreases patient survival. A number of genes downregulated by SOX9 have a negative impact on patient survival. In conclusion, SOX9 is an important gene involved in melanoma invasion and negatively impacts melanoma patient survival.

A new live-cell biobank workflow efficiently recovers heterogeneous melanoma cells from native biopsies.

Fibroblast contamination can make establishing primary melanoma cell cultures from native biopsies a major challenge, due to fibroblasts overgrowing the melanoma cells. Standard protocols therefore enrich for highly proliferative melanoma cells that grow well in vitro but may not represent the full range of in vivo tumor heterogeneity. Here we apply conditional methods that more effectively retrieve melanoma cells by differential trypsinization or by inducing fibroblast senescence through contact inhibition, serum starvation or deprivation of adhesion. Simple mixing experiments of melanoma and fibroblast cells demonstrated the efficacy of the new protocols in retrieving slow-growing melanoma cells. Applying our protocols to 20 cultures that had failed to grow by conventional methods, we could retrieve 12 (60%) validated melanoma cell cultures. Further application of the protocols in the live-cell biobank of 124 early passage cultures significantly improved recovery rates from 13% using standard protocols to 70% overall for the new workflow.

PI3K signalling is required for a TGFß-induced epithelial-mesenchymal-like transition (EMT-like) in human melanoma cells.

Epithelial to mesenchymal transition (EMT) is a programme defined in epithelial cells and recognized as playing a critical role in cancer progression. Although melanoma is not a cancer of epithelial cells, hallmarks of EMT have been described to play a critical role in melanoma progression. Here, we demonstrate that long-term TGFß exposure can induce a dedifferentiated EMT-like state resembling a previously described invasive phenotype (EMT-like). TGFß-induced EMT-like is marked by the downregulation of melanocyte differentiation markers, such as MITF, and the upregulation of mesenchymal markers, such as N-cadherin, and an increase in melanoma cell migration and cell invasion. Pharmacological interference shows the dependency of TGFß-induced EMT-like on the activation of the PDGF signalling pathway and the subsequent activation of PI3K in human melanoma cells. Together, the data provide novel insights into the transcriptional plasticity of melanoma cells that might contribute to tumor progression in patients and propose avenues to therapeutic interventions.

Characterization of pigmented dermo-epidermal skin substitutes in a long-term in vivo assay.

In our laboratory, we have been using human pigmented dermo-epidermal skin substitutes for short-term experiments since several years. Little is known, however, about the long-term biology of such constructs after transplantation. We constructed human, melanocyte-containing dermo-epidermal skin substitutes of different (light and dark) pigmentation types and studied them in a long-term animal experiment. Developmental and maturational stages of the epidermal and dermal compartment as well as signs of homoeostasis were analysed 15 weeks after transplantation. Keratinocytes, melanocytes and fibroblasts from human skin biopsies were isolated and assembled into dermo-epidermal skin substitutes. These were transplanted onto immuno-incompetent rats and investigated 15 weeks after transplantation. Chromameter evaluation showed a consistent skin colour between 3 and 4 months after transplantation. Melanocytes resided in the epidermal basal layer in physiological numbers and melanin accumulated in keratinocytes in a supranuclear position. Skin substitutes showed a mature epidermis in a homoeostatic state and the presence of dermal components such as Fibrillin and Tropoelastin suggested advanced maturation. Overall, pigmented dermo-epidermal skin substitutes show a promising development towards achieving near-normal skin characteristics and epidermal and dermal tissue homoeostasis. In particular, melanocytes function correctly over several months whilst remaining in a physiological, epidermal position and yield a pigmentation resembling original donor skin colour.

To B-(RAF) or not to be.

The identification of targetable mutations has revolutionized the therapy of metastatic melanoma. In particular, BRAF and MEK inhibitors have a well-documented impact on overall survival in metastatic disease. However, therapeutic success is highly dependent on the correct identification of these mutations. We discuss the impact of molecular heterogeneity in this context.

An Aggressive Hypoxia Related Subpopulation of Melanoma Cells is TRP-2 Negative.

Despite existing vaccination strategies targeting TRP-2, its function is not yet fully understood. TRP-2 is an enzyme involved in melanin biosynthesis and therefore discussed as a differentiation antigen. However, in mice Trp-2 was shown to be expressed in melanocyte stem cells of the hair follicle and therefore also considered as an indicator of stemness. A proper understanding of the TRP-2 function is crucial, considering a vaccination targeting cells with stemness properties would be highly effective in contrast to a therapy targeting differentiated melanoma cells. Analysing over 200 melanomas including primaries, partly matched metastases and patients' cell cultures we show that TRP-2 is correlated with Melan A expression and decreases with tumor progression. In mice it is expressed in differentiated melanocytes as well as in stem cells. Furthermore, we identify a TRP-2 negative, proliferative, hypoxia related cell subpopulation which is significantly associated with tumor thickness and diseases progression. Patients with a higher percentage of those cells have a less favourable tumor specific survival. Our findings underline that TRP-2 is a differentiation antigen, highlighting the importance to combine TRP-2 vaccination with other strategies targeting the aggressive undifferentiated hypoxia related subpopulation.

[Interprofessional pill box management in an ambulatory care setting]. / Gérer un semainier a plusieurs chez un patient bénéficiaire de soins a domicile.

Complex multimorbid patients are now more common in ambulatory care and the management of their medication more frequently needs interprofessional collaboration. This qualitative study explored health professional's main challenges when introducing, preparing and sharing the use of a pill box for a patient. Another objective of this study was to explore options for improving care in these situations.

Hypoxia contributes to melanoma heterogeneity by triggering HIF1α-dependent phenotype switching.

We have previously reported a model for melanoma progression in which oscillation between melanoma cell phenotypes characterized by invasion or proliferation is fundamental to tumor heterogeneity and disease progression. In this study we examine the possible role of hypoxia as one of the microenvironmental influences driving metastatic progression by promoting a switch from a proliferative to an invasive phenotype. Immunohistochemistry on primary human cutaneous melanoma biopsies showed intratumoral heterogeneity for cells expressing melanocytic markers, and a loss of these markers correlated with hypoxic regions. Furthermore, we show that the downregulation of melanocytic markers is dependent on hypoxia inducible factor 1α (HIF1α), a known regulator of the hypoxic response. In vitro invasion assays showed that a hypoxic environment increases the invasiveness of proliferative melanoma cell cultures in a HIF1α-dependent manner. In contrast, invasive phenotype melanoma cells showed no increase in invasive potential upon exposure to hypoxia. Thus, exposure of proliferative melanoma cells to hypoxic microenvironments is sufficient, in a HIF1α-dependent manner, to downregulate melanocytic marker expression and increase their invasive potential.

Melanoma immunotherapy: historical precedents, recent successes and future prospects.

The idea of cancer immunotherapy has been around for more than a century; however, the first immunotherapeutic ipilimumab, an anti-CTLA-4 antibody, has only recently been approved by the US FDA for melanoma. With an increasing understanding of the immune response, it is expected that more therapies will follow. This review aims to provide a general overview of immunotherapy in melanoma. We first explain the development of cancer immunotherapy more than a century ago and the general opinions about it over time. This is followed by a general overview of the immune reaction in order to give insight into the possible targets for therapy. Finally, we will discuss the current therapies for melanoma, their shortcomings and why it is important to develop patient stratification criteria. We conclude with an overview of recent discoveries and possible future therapies.

Systematic classification of melanoma cells by phenotype-specific gene expression mapping.

There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression-based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype-specific signatures with measurements of characteristics of melanoma phenotype-specific biology in different melanoma cell lines and short-term cultures. We further show that 86% of 536 tested melanoma lines and short-term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two-state system, as described by the phenotype switching model, underlies melanoma progression.