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.
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.
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.
Assessment of actionable gene mutations and oncogene fusions have made a paradigm shift in treatment strategies of non-small cell lung cancer (NSCLC).
mutations involved around 0.2-0.8% of NSCLC ...patients, mostly on codon 61. For these patients, few data are available regarding clinical characteristics and response to therapies.
Next-Generation Sequencing (NGS) done routinely at Nantes University Hospital was used to identify
molecular alterations in NSCLC patients. We identified and described four
p.GlnQ61Leu mutated patients. Literature of previously
-mutant NSCLC cases was reviewed, and available data in solid tumour with the most advanced H-Ras specific inhibitor, tipifarnib, were presented.
Of 1614 patients diagnosed with advanced NSCLC from January 2018 to December 2020, four (0.25%) had
p.Gln61Leu mutation. Three of them died during the first-line systemic therapy. Furthermore, three additional cases were identified in literature. All cases were current or former smokers, most of them had pleural or pericardial effusion at diagnosis.
The clinical course of patients with
-mutant NSCLC remains unclear. Furthers cases should be identified in order to clarify prognosis and response to therapies. Tipifarnib, a farnesyl transferase inhibitor, is a promising candidate to target
-mutant tumours and should be explored in NSCLC patients.
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%;
= 12/102). Patients without initial bP had significantly better PFS and OS (4-month PFS = 78%; 1-year OS = 73%;
= 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%;
= 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.
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 < 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).
Purpose: MEM is an NGS algorithm that uses Expectation-Maximisation to detect the presence of unstable alleles from the NGS sequences of five microsatellites (BAT-25, BAT-26, NR-21, NR-24 and NR-27). ...The purpose of this study was to compare the MEM algorithm with a reference PCR method (MSI-PCR) and MisMatch Repair protein immunohistochemistry (MMR-IHC). Methods: FFPE colorectal cancer samples from 146 patients were analysed in parallel by MSI-PCR and NGS using the MEM algorithm. MMR-IHC results were available for 133 samples. Serial dilutions of an MSI positive control were performed to estimate the limit of detection. Results: the MEM algorithm was able to detect unstable alleles of each microsatellite with up to a 5% allelic fraction. Of the 146 samples, 28 (19.2%) were MSI in MSI-PCR. MEM algorithm results were in perfect agreement with those of MSI-PCR, at both MSI status and individual microsatellite level (Cohen’s kappa = 1). A high level of agreement was noted between MSI-PCR/MEM algorithm results and MMR-IHC results (Cohen’s kappa = 0.931). Conclusion: the MEM algorithm can determine the MSI status of colorectal cancer samples on a small NGS panel, using only five microsatellites approved by international guidelines, and can be combined with screening for targetable mutations.
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<.01) and S100 protein concentration (P<.01). Finally, seven patients presented discordant BRAF status in different tumor sites. In all these patients, the test performed on ctDNA was positive, suggesting that ctDNA analysis might be less sensitive to tumor heterogeneity. Altogether, these results suggest that plasmatic mutant BRAF DNA is a prognostic factor of OS, correlated with tumor burden. In addition, it represents an interesting alternative source of DNA to detect BRAF mutations before treatment.
Multiple myeloma (MM) is still considered incurable and new therapeutic approaches are therefore needed. Deep‐sequencing analysis revealed the presence of BRAF mutations in up to 15% of patients. The ...clinical experience of BRAF‐targeted therapy in myeloma patients harboring BRAF mutation is still limited. We here report the case of a patient with penta‐refractory (bortezomib, lenalidomide, carfilzomib, pomalidomide, and daratumumab) MM with extramedullary BRAF‐mutated disease that achieved clinical response to dual BRAF and MEK inhibition. At the time of disease progression, gene sequencing analysis of the tumor at the time of progression demonstrated a clonal evolution with emergence of a NRAS mutation and persistence of BRAF and TP53 mutations. Backtracking of the NRAS mutation was performed by digital polymerase chain reaction on the baseline biopsy and identified the pre‐existence of the NRAS at a subclonal level. This observation is the first report of acquired NRAS mutation leading to resistance to dual BRAF/MEK inhibitors in MM. These data suggest that a systematic search for RAS mutations using highly sensitive techniques should be performed before considering targeted therapy in relapsed myeloma with BRAF mutation.
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.
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.
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.
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