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The management of metastatic cutaneous melanoma is conditioned by the identification of BRAF-activating mutations in tumor DNA. Tumor genotyping is usually performed on DNA extracted from tissue samples. However, these invasive samples are rarely repeated during follow-up, and their analysis requires a sample pre-treatment which may take several weeks. Circulating tumor DNA (ctDNA), released into blood by cancer cells, is a good alternative to tissue sampling. ctDNA is not subject to tumor heterogeneity, and can be analyzed rapidly, making possible the detection of mutations in emergency or in patients whose tumor cannot be sampled. ctDNA can also be analyzed repeatedly during follow-up, for postresection minimal residual disease assessment, for therapeutic response monitoring and for early relapse detection.

The ability of early (first weeks of treatment) ctDNA kinetics to identify primary resistance to anti-PD1 immunotherapies was evaluated with a validation cohort of 49 patients treated with anti‑PD1 for metastatic BRAF or NRAS-mutated melanoma, alone and pooled with the 53 patients from a previously described derivation cohort. BRAF or NRAS mutations were quantified on plasma DNA by digital PCR at baseline and after two or four weeks of treatment. ctDNA kinetics were interpreted according to pre-established biological response criteria. A biological progression (bP, i.e., a significant increase in ctDNA levels) at week two or week four was associated with a lack of benefit from anti-PD1 (4-month PFS = 0%, 1‑year OS = 13%, n = 12/102). Patients without initial bP had significantly better PFS and OS (4-month PFS = 78%, 1‑year OS = 73%, n = 26/102), as did patients whose ctDNA kinetics were not evaluable, due to low/undetectable baseline ctDNA (4-month PFS = 80%, 1‑year OS = 81%, n = 64/102). ctDNA detection at first-line anti-PD1 initiation was an independent prognostic factor for OS and PFS in multivariate analysis. Overall, early ctDNA quantitative monitoring may allow the detection of primary resistances of metastatic melanoma to anti-PD1 immunotherapies.

Systemic fungal infections are associated with high mortality rates despite adequate treatment. Moreover, acquired resistance to antifungals is increasing, which further complicates the therapeutic management. One strategy to overcome antifungal resistance is to use antifungal combinations. In vitro, several techniques are used to assess drug interactions, such as the broth microdilution checkerboard, agar-diffusion methods, and time-kill curves. Currently, the most widely used technique is the checkerboard method. The aim of all these techniques is to determine if the interaction between antifungal agents is synergistic, indifferent, or antagonistic. However, the interpretation of the results remains difficult. Several methods of analysis can be used, based on different theories. The most commonly used method is the calculation of the fractional inhibitory concentration index. Determination of the usefulness of combination treatments in patients needs well-conducted clinical trials, which are difficult. It is therefore important to study antifungal combinations in vivo, in experimental animal models of fungal infections. Although mammalian models have mostly been used, new alternative animal models in invertebrates look promising. To evaluate the antifungal efficacy, the most commonly used criteria are the mortality rate and the fungal load in the target organs.

Circulating tumour DNA (ctDNA) can be used to identify gene alterations. The purpose of this study was to determine whether the detection of ctDNA, based on the identification of BRAF and NRAS mutations before systemic treatment initiation, was associated with the prognosis of metastatic melanoma. In total, 68 BRAF or NRAS-mutated stage IV or unresectable stage III metastatic cutaneous melanoma patients were included and tested for the presence of BRAF and NRAS mutations in circulating DNA before treatment initiation, using the Cobas BRAF/NRAS Mutation Test (Roche). The expected mutation was detected in the plasma of 34/68 patients (50% sensitivity). ctDNA detection was associated with AJCC stage, along with the number and nature of metastases. ctDNA was less frequently detected in NRAS-mutated than in BRAF-mutated melanoma (36% and 66%, respectively). At initiation of first-line treatment, ctDNA detection was associated with poor prognosis in Progression Free Survival (PFS) and Overall Survival (OS) in univariate analysis (log-rank: p = 0.002 and p &lt, 0.0001, respectively). In multivariate analysis, ctDNA detection was an independent factor of poor prognosis in OS, after adjustment for AJCC stage, number and nature of metastases and gender (HR = 4.384, 95% CI: (1.308, 14.699), p = 0.017).

International audience; BACKGROUND:Fixed tissues are the standard samples used in routine practice for molecular testing. But sometimes tissues are lacking or difficult to obtain. In these cases, circulating tumor DNA released from tumor cells can be used as an alternative source of tumor DNA.CASE PRESENTATION:We present the case of a 63-year-old Caucasian woman with a metastatic melanoma and a very poor performance status. A plasma sample was tested and the BRAF p.V600E mutation was detected. Based on this result, a treatment combining a BRAF inhibitor and a MEK inhibitor was immediately started. This patient achieved a complete response. In addition, by repeating the plasma test, we could obtain a precise kinetic of release of mutated BRAF DNA in plasma.CONCLUSIONS:We report here for the first time the efficient treatment of a metastatic melanoma patient on the basis of circulating tumor DNA analysis. This urgent treatment provided a dramatic response in a patient with a very poor initial condition. The kinetic data most likely reflect treatment efficacy.

Screening for theranostic biomarkers is mandatory for the therapeutic management of cutaneous melanoma. BRAF and NRAS genes must be tested in routine clinical practice. The methods used to identify these alterations must be sensitive to detect mutant alleles in a background of wild type alleles, and specific to identify the correct mutation. They should not require too much material, since in some cases the available samples are small biopsies. Finally, they should also be quick enough to allow a rapid therapeutic management of patients. Sixty five consecutive formalin-fixed paraffin-embedded (FFPE) melanoma samples were prospectively tested for BRAF mutations with the VE1 (anti-BRAF V600E) antibody and for both BRAF and NRAS mutations with the Idylla NRAS-BRAF-EGFR S492R Mutation Assay cartridges. Results were compared to our routine laboratory practice, allele specific amplification and/or Sanger sequencing and discordant cases confirmed by digital PCR. Excluding discordant by-design-mutations, system failures and DNA quantity or quality failures, BRAF IHC demonstrated an overall concordance of 89% for BRAF V600E mutation detection, the Idylla system gave a concordance of 100% for BRAF mutation detection and of 92.1% for NRAS mutation detection when compared to our reference. When discrepancies were observed, all routine results were confirmed by digital PCR. Finally, BRAF IHC positive predictive value (PPV) was of 82% and negative predictive value (NPV) of 92%. The Idylla cartridges showed a PPV and NPV of both 100% for BRAF mutation detection and a PPV and NPV of 100% and 87% respectively, for NRAS mutation detection. In conclusion, BRAF V600E immunohistochemistry is efficient for detecting the V600E mutation, but negative cases should be further evaluated by molecular approaches for other BRAF mutations. Since 3 NRAS mutations have not been detected by the Idylla NRAS-BRAF-EGFR S492R Mutation Assay, these cartridges should not be used as a substitute for traditional molecular methods in the conventional patient therapeutic care process without the expertise needed to have a critical view of the produced results.

Major advances in the treatment of non-small cell lung cancer (NSCLC) patients have been obtained during the last decade. Molecular testing of tumor samples is therefore mandatory in routine clinical practice. Tumor DNA is also present as cell-free molecules in blood, which is therefore a very useful and convenient source of tumor DNA. In this review, we discuss pre-analytical and analytical aspects of circulating tumor DNA (ctDNA) analysis. We also describe the use of ctDNA analysis in routine clinical practice, and discuss the potential use of ctDNA monitoring both to identify minimal residual disease and as a potential tool to early identify patients’ response to treatment.

International audience; Objective: Mucosal associated invariant T cells (MAIT) and innate lymphoid cells (ILCs) have immuno-regulatory functions at mucosal sites and have been involved in various inflammatory and autoimmune diseases. The aim of this study was to assess their frequencies in blood in ANCA-associated vasculitis (AAV).Methods: The frequencies and function of MAIT cells, ILCs, gdT, iNKT, NK cells were analyzed by flow cytometry on PBMC of patients with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) without any treatment, in acute (AP) and remission phase (RP) and compared with healthy controls (HC).Results: The frequencies of MAIT cells were strongly decreased in GPA and MPA in AP compared to HC, both in never treated and in relapsing patients and independently of patient age. This was associated with an activated phenotype of patient MAIT cells, as shown by increased expression of CD69 and IFNg. MAIT cells remained decreased during RP in AAV patients. The frequencies of iNKT and gdT cells were unaffected compared to HC, whereas those of NK cells were slightly reduced during AP in MPA. We also observed a significant decrease in frequencies of total ILCs with decreased ILC2 and ILC3 and increased ILC1 during AP in both GPA and MPA compared to HC. These frequencies normalized during RP. Interestingly , we observed a significant correlation between the frequency of total ILCs and BVAS.Conclusion: We show for the first time that AAV are associated with a major decrease and an activated phenotype of blood MAIT cell. These features persisted during remission suggesting a role for MAIT cells in the pathogenesis of AAV.

International audience; Immunotherapies have changed the medical management of metastatic melanoma. However, the early detection of patients who do not respond to these treatments is a key issue. We evaluated the quantitative monitoring of circulating tumor DNA (ctDNA) as an early predictor of response to anti-PD1. Patients treated with anti-PD1 for metastatic mutated melanoma were selected. The somatic alteration detected on the tumor tissue was quantified on plasma DNA by digital PCR (dPCR) at treatment initiation, after 2 and 4 weeks of treatment, and then every 4 weeks until progression. The absence of biological response (defined as a significant decrease in the amount of ctDNA relative to the baseline level) after 2 weeks of treatment was associated with a lack of clinical benefit under anti-PD1. In the presence of a biological response at week 2, detection of subsequent biological progression (significant increase in the amount of ctDNA relative to its nadir) was 100% predictive of progressive disease, on average 75 days prior to radiological detection. Patients with a persistent biological response beyond week 16 did not experience any progressive disease and exhibited sustained responses. In conclusion, we show that quantitative monitoring of ctDNA, using criteria accounting for dPCR measurement imprecision, allows the early and specific detection of patients who do not respond to anti-PD1 therapy.

The management of metastatic melanoma has evolved since the onset of treatments with BRAF inhibitors. In order to predict which patients are likely to respond to these treatments, the therapeutic strategy is now conditioned by the search for the activating mutations of the BRAF gene. Tumor genotyping is routinely performed from DNA extracted from tissue or cellular specimens from the primary tumor, metastases, or neoplastic effusions. Due to their invasiveness, these specimens are rarely repeated during the management. In addition, the analysis of the tumor material requires a pretreatment of the sample (formalin fixation, paraffin inclusion, preparation of tissue sections) and may take up to several weeks, making emergency treatment with BRAF inhibitors impossible. Circulating tumor DNA (ctDNA), released by cancer cells in the blood stream, appears as an alternative to tissue sampling. The pre-analytical conditions are now well defined, and several technological approaches can be used to demonstrate the desired molecular alterations. ctDNA is less affected by tumor heterogeneity, can be collected in a minimally invasive manner and analyzed rapidly. Furthermore, ctDNA can be repeatedly analyzed during follow-up, which makes it possible to envisage its use as a specific tumor marker, in order to monitor the response to the treatment and to detect treatment failure.

International audience; Circulating tumor DNA is a promising non-invasive tool for cancer monitoring. The main objective of our work was to investigate the relationship between mutant BRAF DNA in plasma and clinical response. Thirty-eight stage IV patients with a V600 mutated BRAF melanoma were included prior to any treatment. DNA was extracted from plasma and mutant DNA was detected using the amplification-refractory mutation system method. Before the beginning of any treatment, the corresponding BRAF mutation was detected in 29 of the 38 tested plasma samples (76.3% positive per cent agreement). We observed a strong correlation between the presence of circulating mutated DNA and overall survival (OS; P=.02), and with the number of metastatic sites (P=.01). The presence of circulating mutated DNA was also strongly correlated with serum LDH activity (P