RESUMEN
Sequence-specific DNA extractions have the potential to improve the detection of low-abundance mutations from complex matrices, making them ideal for circulating tumor DNA analysis during the early stages of cancer. Ion-tagged oligonucleotides (ITOs) are oligonucleotides modified with an allylimidazolium salt via thiolene click chemistry. The allylimidazolium-based tag allows the ITO-DNA duplex to be selectively captured by a hydrophobic magnetic ionic liquid (MIL). In this study, the selectivity of the ITO-MIL method was examined by extracting low abundance of the BRAF V600E mutation-a common single-nucleotide polymorphism associated with several different cancers-from diluted human plasma, artificial urine, and diluted artificial sputum. Quantitative polymerase chain reaction (qPCR) was not able to distinguish a 9% BRAF V600E standard (50 fg·µL-1 BRAF V600E, 500 fg·µL-1 wild-type BRAF) from the 100% wild-type BRAF (50 fg·µL-1) standard. However, introducing the ITO-MIL extraction prior to qPCR allowed for samples consisting of 0.1% BRAF V600E (50 fg·µL-1 V600E BRAF, 50,000 fg·µL-1 wild-type BRAF) to be distinguished from the 100% wild-type BRAF standard. Ion-tagged oligonucleotides (ITOs) are combined with magnetic ionic liquids (MILs) to extract low-abundance BRAF V600E mutation from diluted human plasma, artificial urine, and diluted artificial sputum. The ITO-MIL extraction prior to qPCR allowed for samples consisting of 0.1% BRAF V600E to be distinguished from the 100% wild-type BRAF standard.
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Líquidos Iónicos/química , Neoplasias/genética , Oligonucleótidos/química , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas B-raf/genética , Humanos , Fenómenos Magnéticos , Mutación , Proteínas Proto-Oncogénicas B-raf/orina , Esputo/químicaRESUMEN
Importance: Thyroid cancer, predominantly papillary thyroid carcinoma (PTC), is common, but an estimated 30% of ultrasonography-guided fine-needle aspiration (FNA) biopsies of thyroid nodules are indeterminate. BRAF variation, associated with poor clinicopathological characteristics, is a useful molecular marker for diagnostics. Objective: To develop a sensitive molecular assay for BRAF V600E detection in remaining tissue of thyroid FNA biopsies to identify patients with cancer carrying a BRAF variation. Design, Setting, and Participants: This diagnostic study used tumor tissue from surgical formalin-fixed, paraffin-embedded (FFPE) specimens and residual tissue from thyroid FNA biopsies for genomic DNA extraction. FFPE specimens served as the validation set, and residual tissue from FNA biopsies served as the test set. A molecular assay was developed for accurate detection of BRAF V600E variation using locked nucleic acid (LNA) probe-based droplet digital polymerase chain reaction (dPCR), and the assay was validated by BRAF V600E immunohistochemical staining (IHC). The study was conducted between February 2019 and May 2021. Results: A total of 271 specimens, including 77 FFPE specimens (with a follow-up of 48 matched surgical specimens) and 146 residual FNA samples, were collected from 223 patients (mean [SD] age, 53.8 [15.3] years; 174 [78.0%] women; 49 [22.0%] men). The molecular assay by dPCR was first established to specifically and accurately detect and quantify wild-type BRAF and variant BRAF in DNA from human follicular thyroid carcinoma-derived FTC-133 and papillary thyroid carcinoma-derived BCPAP cells. The linearity of quantification of BRAF V600E was calculated (y = 0.7339x; R2 = 0.9996) with sensitivity at 0.02 copies/µL and reproducibility in detecting variant DNA at various dilutions(coefficient of variance in 0.3% DNA, 9.63%; coefficient of variance in 1.0% DNA, 7.41%). In validation testing, the dPCR assay and IHC staining exhibited 100% specificity in concordantly identifying BRAF V600E in PTCs (κ = 0.873; P < .001) and sensitivity of 32.0% (95% CI, 19.1% to 44.9%) in dPCR and 26.0% (95% CI, 13.1% to 38.9%) in IHC staining, with an improvement by 23.08% in dPCR compared with the IHC staining. The dPCR assay further detected BRAF V600E in 39 of 146 residual FNA specimens (26.7%). At short-term follow-up, 48 patients, including 14 of 39 patients with BRAF variation and 34 of 107 patients without BRAF variation on residual FNA specimens, underwent resection. The dPCR assay of BRAF status in the matched surgical specimens showed BRAF V600E variations in 12 patients and wild-type BRAF in 36 patients, with a high agreement to that in residual tissue of FNA specimens (κ = 0.789; P < .001). Among 14 patients with BRAF variations on residual FNA, 13 were diagnosed with PTC and 1 was diagnosed with anaplastic thyroid cancer at the thyroidectomy. Conclusions and Relevance: This diagnostic study developed a sensitive molecular assay for detection and quantification of BRAF V600E variation in residual tissue from thyroid FNA biopsies to identify patients with cancer harboring BRAF V600E in a cost-effective manner, highlighting the clinical value of molecular assay of the remaining FNA tissue in the management of thyroid nodules.
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Biopsia con Aguja Fina/normas , Proteínas Proto-Oncogénicas B-raf/análisis , Nódulo Tiroideo/patología , Adulto , Anciano , Biopsia con Aguja Fina/métodos , Biopsia con Aguja Fina/estadística & datos numéricos , Análisis Mutacional de ADN/métodos , Técnicas y Procedimientos Diagnósticos/normas , Técnicas y Procedimientos Diagnósticos/estadística & datos numéricos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas B-raf/sangre , Reproducibilidad de los ResultadosRESUMEN
BRAF inhibitors are an effective treatment for BRAFV600E -mutated, risk-organ-positive Langerhans cell histiocytosis (RO+ LCH). However, cell-free BRAFV600E DNA often persists during therapy and recurrence frequently occurs after therapy discontinuation. To identify a pathological reservoir of BRAFV600E -mutated cells, we studied peripheral blood cells obtained from six infants with RO+ multisystem (MS) LCH that received targeted therapy. After cell sorting, the BRAFV600E mutation was detected in monocytes (n = 5), B lymphocytes (n = 3), T lymphocytes (n = 2), and myeloid and plasmacytoid dendritic cells (n = 2 each). This biomarker may offer an interesting tool for monitoring the effectiveness of new therapeutic approaches for weaning children with RO+ LCH from targeted therapy.
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Histiocitosis de Células de Langerhans/tratamiento farmacológico , Mutación Puntual , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Niño , Preescolar , Histiocitosis de Células de Langerhans/sangre , Histiocitosis de Células de Langerhans/genética , Humanos , Lactante , Mutación Puntual/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/sangreRESUMEN
BACKGROUND: Circulating tumor DNA (ctDNA) may complement radiography for interim assessment of patients with cancer. OBJECTIVE: Our objective was to explore the relationship between changes in plasma ctDNA versus radiographic imaging among patients with metastatic melanoma. METHODS: Using the Idylla system, we measured B-Raf proto-oncogene (BRAF) V600 ctDNA in plasma from 15 patients with BRAF V600E/K-positive primary tumors undergoing standard-of-care monitoring, including cross-sectional computed tomography (CT) imaging. BRAF V600 mutant allele frequency (%MAF) was calculated from the Idylla Cq values and directly measured using droplet digital polymerase chain reaction (ddPCR). RESULTS: The Idylla ctDNA assay demonstrated 91% sensitivity, 96% specificity, 91% positive predictive value, and 96% negative predictive value for the presence of > 93 mm metastatic disease. Qualitative ctDNA results corresponded to changes in RECIST (Response Evaluation Criteria in Solid Tumors) 1.1 status determined by CT imaging in 11 of 15 subjects (73%). Calculated %MAF results correlated with ddPCR (R2 = 0.94) and provided evidence of progressive disease 55 and 97 days in advance of CT imaging for two subjects with persistently positive qualitative results. CONCLUSIONS: Overall, interim ctDNA results provided evidence of partial response or progressive disease an average of 82 days before radiography. This pilot study supports the feasibility of using the Idylla plasma BRAF V600 ctDNA assay as a complement to CT scanning for routine monitoring of therapeutic response. Somatic mutation quantification based on Cq values shows promise for identifying disease progression and warrants further validation.
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Melanoma/diagnóstico por imagen , Melanoma/patología , Mutación , Proteínas Proto-Oncogénicas B-raf/sangre , ADN Tumoral Circulante/genética , Estudios Transversales , Estudios de Factibilidad , Humanos , Estudios Longitudinales , Masculino , Melanoma/sangre , Melanoma/genética , Metástasis de la Neoplasia , Proyectos Piloto , Proteínas Proto-Oncogénicas B-raf/genética , Sensibilidad y Especificidad , Nivel de Atención , Tomografía Computarizada por Rayos X , Carga TumoralRESUMEN
We investigated the current role of interferon-alpha (IFNα) in hairy cell leukaemia (HCL) in a retrospective analysis of patients with HCL. A cohort of 74 patients with HCL was divided in to three groups: (A) patients aged >65 years with first-line treatment; (B) patients with comorbidities with first-line treatment; (C) patients who were purine analogues resistant. In total, 94% achieved a response, with a complete response rate of 24%. After a median (range) follow-up of 60 (7-236) months, 55 patients (78%) are still responding. The 5-year progression-free survival was 95%, 68%, and 96% in groups A, B and C respectively. A proportion of patients were monitored through their B-Raf proto-oncogene, serine/threonine kinase (BRAF)-V600E status. IFNα remains a possible option in select patients with HCL, where minimal residual disease negativity is achievable.
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Antineoplásicos/uso terapéutico , Interferón-alfa/uso terapéutico , Leucemia de Células Pilosas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Alopecia/inducido químicamente , Antineoplásicos/efectos adversos , Biomarcadores de Tumor/sangre , Comorbilidad , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos , Sustitución de Medicamentos , Femenino , Estudios de Seguimiento , Humanos , Interferón-alfa/efectos adversos , Estimación de Kaplan-Meier , Leucemia de Células Pilosas/sangre , Leucemia de Células Pilosas/mortalidad , Masculino , Persona de Mediana Edad , Mutación Missense , Mutación Puntual , Supervivencia sin Progresión , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas B-raf/genética , Estudios Retrospectivos , Terapia RecuperativaRESUMEN
Liquid biopsy and cell-free DNA (cfDNA) show great promise in cancer diagnostics. In this study, we designed a custom droplet digital PCR (ddPCR) assay for the quantification and quality control of cfDNA isolated from serum. The assay was validated on a group of locally advanced colorectal cancer (CRC) patients and two control groups-patients with hemorrhoids and healthy individuals. The assay shows a high correlation with Qubit measurement (r = 0.976) but offers a higher dynamic range. Mean concentrations of cfDNA were 12.36 ng/µL, 5.17 ng/µL, and 0.29 ng/µL for CRC, hemorrhoid patients, and healthy controls, respectively. The quality of cfDNA was assessed with the measurement of B-cell DNA contamination. On a subset of CRC patients, we compared the mutation status on KRAS (G12A, G12D, G12V, G13D) and BRAF (V600E) genes in the primary tumor and cfDNA isolated from the serum. A total of 70.6% of primary tumor samples were mutated, and the mean fractional abundance of mutations was 9.50%. The matching serum samples were mutated in 38% cases with an average fractional abundance of 0.23%. We conclude that any decisions based solely on the amount of cfDNA present in patient serum must be interpreted carefully and in the context of co-morbidities. This study explores the potential of ddPCR somatic mutations detection from liquid biopsy as a supplement to tissue biopsy in targeted personalized CRC patient management.
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Ácidos Nucleicos Libres de Células/sangre , Neoplasias Colorrectales/sangre , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Ácidos Nucleicos Libres de Células/genética , ADN Tumoral Circulante , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Humanos , Biopsia Líquida , Masculino , Persona de Mediana Edad , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas p21(ras)/sangreRESUMEN
Tumor-derived cell-free DNA (cfDNA) is an emerging biomarker for guiding the personalized treatment of patients with metastatic colorectal cancer (CRC). While patients with CRC liver metastases (CRC-LM) have relatively high levels of plasma cfDNA, little is known about patients with CRC peritoneal metastases (CRC-PM). This study evaluated the presence of tumor-derived cfDNA in plasma and peritoneal fluid (i.e. ascites or peritoneal washing) in 20 patients with isolated CRC-PM and in the plasma of 100 patients with isolated CRC-LM. Among tumor tissue KRAS/BRAF mutation carriers, tumor-derived cfDNA was detected by droplet digital polymerase chain reaction (ddPCR) in plasma of 93% of CRC-LM and 20% of CRC-PM patients and in peritoneal fluid in all CRC-PM patients. Mutant allele fraction (MAF) and mutant copies per ml (MTc/ml) were lower in CRC-PM plasma than in CRC-LM plasma (median MAF = 0.28 versus 18.9%, p < 0.0001; median MTc/ml = 21 versus 1,758, p < 0.0001). Within patients with CRC-PM, higher cfDNA levels were observed in peritoneal fluid than in plasma (median MAF = 16.4 versus 0.28%, p = 0.0019; median MTc/ml = 305 versus 21, p = 0.0034). These data imply that tumor-derived cfDNA in plasma is a poor biomarker to monitor CRC-PM. Instead, cfDNA detection in peritoneal fluid may offer an alternative to guide CRC-PM treatment decisions.
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Líquido Ascítico/química , Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Neoplasias Colorrectales/genética , Neoplasias Peritoneales/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Anciano , Biomarcadores de Tumor/sangre , ADN Tumoral Circulante/sangre , Toma de Decisiones Clínicas , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Análisis Mutacional de ADN , Femenino , Humanos , Biopsia Líquida , Masculino , Persona de Mediana Edad , Mutación , Países Bajos , Neoplasias Peritoneales/sangre , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/terapia , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Pronóstico , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas p21(ras)/sangreRESUMEN
BACKGROUND: The absence of high-quality next-generation sequencing (NGS) reference material (RM) has impeded the clinical use of liquid biopsies with plasma cell-free DNA (cfDNA) in China. OBJECTIVE: This study aimed to develop a national RM panel for external quality assessment and performance evaluation during kit registration of non-small-cell lung cancer (NSCLC)-related Kirsten rat sarcoma viral oncogene (KRAS)/neuroblastoma ras oncogene (NRAS)/epidermal growth factor receptor (EGFR)/B-type Raf kinase (BRAF)/mesenchymal-epithelial transition factor (MET) genetic assays using plasma circulating tumor DNA (ctDNA). METHODS: Mutation cell lines detected by NGS and validated by Sanger sequencing were selected to establish the RM. Cell line genomic DNA was sheared and used to spike basal plasma cfDNA at 10% concentration. Then, the calibration accuracy was determined by four sequencing platforms. Average values were adopted and diluted to 0.1%, 0.3%, 1% and 3% concentrations with basal plasma as the RM panel. Then, five manufacturers were invited to evaluate the performance of the RM panel. RESULTS: 20 cell lines with 23 clinically important mutations were selected, including six mutations in KRAS, two mutations in NRAS, three in BRAF, four in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), six in EGFR, one EGFR Gain (4-5 copy) and one MET Gain (2-5 copy). The RM panel consisted of 87 samples, including these 21 mutations at four concentrations (0.1%, 0.3%, 1% and 3%), one MET gain, one EGFR gain and one wild type. The detection rate was 100% for the 3%, 1% and 0.3% samples at all five companies. For the 0.1% concentration, 15 samples had inconsistent results, but at least three companies had correct results for each mutation. CONCLUSION: RM for a KRAS/NRAS/EGFR/BRAF/MET mutation panel for plasma ctDNA was developed, which will be essential for quality control of the performance of independent laboratories.
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Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , ADN Tumoral Circulante/genética , Análisis Mutacional de ADN/normas , GTP Fosfohidrolasas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Neoplasias Pulmonares/genética , Proteínas de la Membrana/genética , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Adulto , Beijing , Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Línea Celular Tumoral , ADN Tumoral Circulante/sangre , Receptores ErbB/sangre , Receptores ErbB/genética , Femenino , GTP Fosfohidrolasas/sangre , Humanos , Biopsia Líquida/normas , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/diagnóstico , Masculino , Proteínas de la Membrana/sangre , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas c-met/sangre , Proteínas Proto-Oncogénicas p21(ras)/sangre , Estándares de Referencia , Adulto JovenRESUMEN
PURPOSE: We evaluated the predictive value of pretreatment ctDNA to inform therapeutic outcomes in patients with metastatic melanoma relative to type and line of treatment. EXPERIMENTAL DESIGN: Plasma circulating tumor DNA (ctDNA) was quantified in 125 samples collected from 110 patients prior to commencing treatment with immune checkpoint inhibitors (ICIs), as first- (n = 32) or second-line (n = 27) regimens, or prior to commencing first-line BRAF/MEK inhibitor therapy (n = 66). An external validation cohort included 128 patients commencing ICI therapies in the first- (N = 77) or second-line (N = 51) settings. RESULTS: In the discovery cohort, low ctDNA (≤20 copies/mL) prior to commencing therapy predicted longer progression-free survival (PFS) in patients treated with first-line ICIs [HR, 0.20; 95% confidence interval (CI) 0.07-0.53; P < 0.0001], but not in the second-line setting. An independent cohort validated that ctDNA is predictive of PFS in the first-line setting (HR, 0.42; 95% CI, 0.22-0.83; P = 0.006), but not in the second-line ICI setting. Moreover, ctDNA prior to commencing ICI treatment was not predictive of PFS for patients pretreated with BRAF/MEK inhibitors in either the discovery or validation cohorts. Reduced PFS and overall survival were observed in patients with high ctDNA receiving anti-PD-1 monotherapy, relative to those treated with combination anti-CTLA-4/anti-PD-1 inhibitors. CONCLUSIONS: Pretreatment ctDNA is a reliable indicator of patient outcome in the first-line ICI treatment setting, but not in the second-line ICI setting, especially in patients pretreated with BRAF/MEK inhibitors. Preliminary evidence indicated that treatment-naïve patients with high ctDNA may preferentially benefit from combined ICIs.
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Antígeno CTLA-4/sangre , ADN Tumoral Circulante/sangre , Melanoma/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/genética , Proteínas Proto-Oncogénicas B-raf/sangre , Anciano , Antígeno CTLA-4/antagonistas & inhibidores , Terapia Combinada/efectos adversos , Quimioterapia Combinada/métodos , Femenino , Humanos , Inmunoterapia/efectos adversos , Quinasas Quinasa Quinasa PAM/genética , Masculino , Melanoma/sangre , Melanoma/genética , Melanoma/inmunología , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Supervivencia sin Progresión , Inhibidores de Proteínas Quinasas/administración & dosificaciónRESUMEN
A tunable plasmon-assisted electrochemiluminescence (ECL) strategy is reported using concave Au nanocubes (Au CBs) for rapidly accelerated fibrosarcoma B-type (BRAF) detection. Concave Au CBs exhibit a strong surface plasmon coupling (SPC) effect between its sharp apexes and edges. The high spectral overlap with graphite phase carbon nitride quantum dots (g-C3N4 QDs) is achieved by tuning surface plasmon absorption peak of the concave Au CBs. It maximizes the SPC effect and enhances the ECL signal of g-C3N4 QDs 3-fold. The SPC effect of Au CBs is twice as high as with Au NPs. We also employed a toehold-mediated strand displacement (TMSD) strategy for sensitive target recycling amplification. Under optimal conditions, this sensor can determine BRAF gene from 1 pM to 1 nM with a detection limit of 3.06 × 10-5 nM (S/N = 3) and RSD 3.67%. With the aid of the TMSD strategy and tunable plasmon-assisted ECL sensing mode, this sensor also exhibits good analytical performance in human serum with satisfactory recovery of 90.2~109%. The proposed strategy provides a promising method to effectively enhance spectral overlap and detect BRAF gene.
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Técnicas Electroquímicas/instrumentación , Oro/química , Mediciones Luminiscentes/instrumentación , Nanoestructuras/química , Proteínas Proto-Oncogénicas B-raf/genética , Resonancia por Plasmón de Superficie/métodos , Técnicas Electroquímicas/métodos , Humanos , Mediciones Luminiscentes/métodos , Microscopía Electrónica de Transmisión , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas B-raf/químicaRESUMEN
PURPOSE: Brain involvement occurs in the majority of patients with metastatic melanoma. The potential of circulating tumor DNA (ctDNA) for surveillance and monitoring systemic therapy response in patients with melanoma brain metastases merits investigation. EXPERIMENTAL DESIGN: This study examined circulating BRAF, NRAS, and c-KIT mutations in patients with melanoma with active brain metastases receiving PD-1 inhibitor-based therapy. Intracranial and extracranial disease volumes were measured using the sum of product of diameters, and response assessment performed using RECIST. Longitudinal plasma samples were analyzed for ctDNA over the first 12 weeks of treatment (threshold 2.5 copies/mL plasma). RESULTS: Of a total of 72 patients, 13 patients had intracranial metastases only and 59 patients had concurrent intracranial and extracranial metastases. ctDNA detectability was 0% and 64%, respectively, and detectability was associated with extracranial disease volume (P < 0.01). Undetectable ctDNA on-therapy was associated with extracranial response (P < 0.01) but not intracranial response. The median overall survival in patients with undetectable (n = 34) versus detectable (n = 38) ctDNA at baseline was 39.2 versus 10.6 months [HR, 0.51; 95% confidence interval (CI), 0.28-0.94; P = 0.03] and on-therapy was 39.2 versus 9.2 months (HR, 0.32; 95% CI, 0.16-0.63; P < 0.01). CONCLUSIONS: ctDNA remains a strong prognostic biomarker in patients with melanoma with brain metastases, especially in patients with concurrent extracranial disease. However, ctDNA was not able to detect or monitor intracranial disease activity, and we recommend against using ctDNA as a sole test during surveillance and therapeutic monitoring in patients with melanoma.
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Biomarcadores de Tumor/sangre , Neoplasias Encefálicas/tratamiento farmacológico , ADN Tumoral Circulante/sangre , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Anciano , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/sangre , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/secundario , ADN Tumoral Circulante/genética , Femenino , Estudios de Seguimiento , GTP Fosfohidrolasas/sangre , GTP Fosfohidrolasas/genética , Humanos , Estimación de Kaplan-Meier , Estudios Longitudinales , Masculino , Melanoma/sangre , Melanoma/mortalidad , Melanoma/secundario , Proteínas de la Membrana/sangre , Proteínas de la Membrana/genética , Persona de Mediana Edad , Mutación , Pronóstico , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-kit/genética , Medición de Riesgo/métodos , Medición de Riesgo/estadística & datos numéricos , Neoplasias Cutáneas/sangre , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Resultado del TratamientoRESUMEN
Circulating tumor DNA (ctDNA), which carries tumor-specific mutations, is an emerging candidate biomarker for malignancies and for monitoring disease status in various human tumors. Recently, BRAF V595E mutation has been reported in 80% of dogs with urothelial carcinoma. This study investigates the BRAF V595E allele concentration in circulating cell-free DNA (cfDNA) and assesses the clinical significance of BRAF-mutated ctDNA levels in canines with urothelial carcinoma. A total of 15 dogs with urothelial carcinoma were included. cfDNA concentration was measured using a real-time polymerase chain reaction (PCR) of the LINE-1 gene. To measure the concentration of the mutated BRAF gene in cfDNA, allele-specific real-time PCR with a locked nucleic acid probe was performed. BRAF mutations were detected in 11 (73%) of the 15 tested tumor samples. BRAF-mutated ctDNA concentrations were significantly higher in dogs with the BRAF mutation (14.05 ± 13.51 ng/ml) than in wild-type dogs (0.21 ± 0.41 ng/ml) (p = 0.031). The amount of BRAF-mutated ctDNA in plasma increased with disease progression and responded to treatment. Our results show that BRAF-mutated ctDNA can be detected using allele-specific real-time PCR in plasma samples of canines with urothelial carcinoma with the BRAF V595E mutation. This ctDNA analysis may be a potentially useful tool for monitoring the progression of urothelial carcinoma and its response to treatment.
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Carcinoma de Células Transicionales/veterinaria , Ácidos Nucleicos Libres de Células/sangre , Enfermedades de los Perros/genética , Mutación Missense , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Urológicas/veterinaria , Alelos , Animales , Biomarcadores de Tumor/sangre , Carcinoma de Células Transicionales/sangre , Carcinoma de Células Transicionales/genética , ADN de Neoplasias/sangre , Enfermedades de los Perros/sangre , Perros , Femenino , Masculino , Proteínas Proto-Oncogénicas B-raf/sangre , Neoplasias Urológicas/sangre , Neoplasias Urológicas/genéticaRESUMEN
Over the past years, the emergence of liquid biopsy technologies has dramatically expanded our ability to assess multiple myeloma without the need for invasive sampling. Interrogation of cell-free DNA from the peripheral blood recapitulates the mutational landscape at excellent concordance with matching bone marrow aspirates. It can quantify disease burden and identify previously undetected resistance mechanisms which may inform clinical management in real-time. The convenience of sample acquisition and storage provides strong procedural benefits over currently available testing. Further investigations will have to define the role of cell-free DNA as a diagnostic measure by determining clinically relevant tumor thresholds in comparison to existing routine parameters. This review presents an overview of currently available assays and discusses the clinical value, potential and limitations of cell-free DNA technologies for the assessment of this challenging disease.
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Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Genoma Humano , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/genética , Mutación , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/sangre , ADN Tumoral Circulante/sangre , GTP Fosfohidrolasas/sangre , GTP Fosfohidrolasas/genética , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Biopsia Líquida/métodos , Proteínas de la Membrana/sangre , Proteínas de la Membrana/genética , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Neoplasia Residual , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/sangre , Proteínas Proto-Oncogénicas p21(ras)/genética , Recurrencia , Proteína p53 Supresora de Tumor/sangre , Proteína p53 Supresora de Tumor/genéticaAsunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Mutación , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Proteínas Proto-Oncogénicas B-raf/genética , Anciano , Biomarcadores de Tumor/sangre , Carboplatino/administración & dosificación , ADN Tumoral Circulante/sangre , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Neoplasias de la Próstata Resistentes a la Castración/sangre , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Proteínas Proto-Oncogénicas B-raf/sangre , Taxoides/administración & dosificaciónRESUMEN
Background In metastatic melanoma, 40%-50% of patients harbor a BRAF V600E mutation and are thereby eligible to receive a combined BRAF/MEK inhibitor therapy. Compared to standard-of-care tissue-based genetic testing, analysis of circulating tumor DNA (ctDNA) from blood enables a comprehensive assessment of tumor mutational status in real-time and can be used for monitoring response to therapy. The aim of our study was to directly compare the performance of two highly sensitive methodologies, droplet digital PCR (ddPCR) and a combination of ARMS/asymmetric-rapid PCR/melting curve analysis, for the detection of BRAF V600E in plasma from melanoma patients. Methods Cell-free DNA (cfDNA) was isolated from 120 plasma samples of stage I to IV melanoma patients. Identical plasma-cfDNA samples were subjected to BRAF V600E mutational analysis using in parallel, ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis. Results BRAF V600E mutation was detected in 9/117 (7.7%) ctDNA samples by ddPCR and in 22/117 (18.8%) ctDNA samples by the combination of ARMS/asymmetric- rapid PCR/melting curve analysis. The concordance between these two methodologies was 85.5% (100/117). The comparison of plasma-ctDNA analysis using ddPCR and tissue testing revealed an overall agreement of 79.4% (27/34), while the corresponding agreement using the combination of ARMS/asymmetric-rapid PCR/melting curve analysis was 73.5% (25/34). Moreover, comparing the detection of BRAF-mutant ctDNA with the clinics, overall agreement of 87.2% (48/55) for ddPCR and 79.2% (42/53) was demonstrated. Remarkably, the duration of sample storage was negatively correlated with correctness of genotyping results highlighting the importance of pre-analytical factors. Conclusions Our direct comparison study has shown a high level of concordance between ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis for the detection of BRAF V600E mutations in plasma.
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ADN Tumoral Circulante/sangre , Melanoma/sangre , Proteínas Proto-Oncogénicas B-raf/sangre , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Humanos , Biopsia Líquida , Masculino , Melanoma/genética , Persona de Mediana Edad , Mutación , Reacción en Cadena de la Polimerasa/métodos , Proteínas Proto-Oncogénicas B-raf/genética , Manejo de Especímenes/métodos , Factores de TiempoRESUMEN
BACKGROUND: Circulating tumour DNA (ctDNA) has emerged as a promising blood-based biomarker for monitoring disease status of patients with advanced cancers. The presence of ctDNA in the blood is a result of biological processes, namely tumour cell apoptosis and/or necrosis, and can be used to monitor different cancers by targeting cancer-specific mutation. CASE PRESENTATION: We present the case of a 67 year old Caucasian male that was initially treated with BRAF inhibitors followed by anti-CTLA4 and then anti-PD1 immunotherapy for metastatic melanoma but later developed colorectal cancer. The kinetics of ctDNA derived from each cancer type were monitored targeting BRAF V600R (melanoma) and KRAS G13D (colon cancer), specifically reflected the status of the patient's tumours. In fact, the discordant pattern of BRAF and KRAS ctDNA was significantly correlated with the clinical response of melanoma to pembrolizumab treatment and progression of colorectal cancer noted by PET and/or CT scan. Based on these results, ctDNA can be used to specifically clarify disease status of patients with metachronous cancers. CONCLUSIONS: Using cancer-specific mutational targets, we report here for the first time the efficacy of ctDNA to accurately provide a comprehensive outlook of the tumour status of two different cancers within one patient. Thus, ctDNA analysis has a potential clinical utility to delineate clinical information in patients with multiple cancer types.
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ADN Tumoral Circulante/sangre , Neoplasias Colorrectales/sangre , Melanoma/sangre , Neoplasias Primarias Secundarias/tratamiento farmacológico , Anciano , Biomarcadores de Tumor/sangre , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/genética , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Humanos , Masculino , Melanoma/patología , Melanoma/secundario , Mutación , Neoplasias Primarias Secundarias/sangre , Neoplasias Primarias Secundarias/genética , Neoplasias Primarias Secundarias/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas p21(ras)/sangreAsunto(s)
Histiocitosis de Células de Langerhans/tratamiento farmacológico , Histiocitosis Sinusal/tratamiento farmacológico , Proteínas Proto-Oncogénicas B-raf/metabolismo , Adulto , Niño , Preescolar , Femenino , Fluorodesoxiglucosa F18/metabolismo , Histiocitosis de Células de Langerhans/complicaciones , Histiocitosis de Células de Langerhans/diagnóstico por imagen , Histiocitosis de Células de Langerhans/genética , Histiocitosis Sinusal/complicaciones , Histiocitosis Sinusal/diagnóstico por imagen , Histiocitosis Sinusal/genética , Humanos , Imidazoles/administración & dosificación , Imidazoles/uso terapéutico , Lactante , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Mutación , Oximas/administración & dosificación , Oximas/uso terapéutico , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas B-raf/efectos de los fármacos , Resultado del TratamientoRESUMEN
PURPOSE: Nivolumab improves survival in patients with metastatic melanoma. Unfortunately, most patients do not respond to this treatment. Preclinical data indicate that radiation therapy could work synergistically with nivolumab and improve response rates. METHODS AND MATERIALS: We conducted a phase 2 trial in 20 patients with inoperable or metastatic melanoma with ≥2 measurable lesions (Response Evaluation Criteria in Solid Tumors v1.1). Stereotactic body radiation therapy (SBRT) of 3 × 8 Gy to the largest lesion was delivered before the second nivolumab cycle. The primary endpoint was overall response rate (ORR) in the nonirradiated lesions (Response Evaluation Criteria in Solid Tumors v1.1). Secondary endpoints included toxicity. An exploratory endpoint was mutant BRAF and NRAS circulating tumor DNA (ctDNA) on serial blood samples. RESULTS: An ORR of 45% was noted with 3 complete and 6 partial responses. Three patients experienced stable disease and 7 had progressive disease as best response. All patients with a complete response in the nonirradiated lesions exhibited a local complete response in the irradiated lesion. Grade 1 to 2 treatment-related adverse events (AEs) occurred in 17 patients; 3 patients experienced grade 3 AEs (lymphopenia, gastroenteritis, and bullous pemphigoid). No grade 4 to 5 AEs occurred. ctDNA was detected in 8 patients, and changes corresponded to clinical response and suggested that a subset of patients with a low programmed death ligand-1 score only started responding after SBRT. CONCLUSIONS: We conclude that the combination treatment was well tolerated and led to an ORR of 45% in patients with metastatic or inoperable melanoma, similar to historical response rates of nivolumab monotherapy. Although underpowered, our data therefore do not indicate a substantial abscopal response. Nonetheless, serial ctDNA analyses suggest that a subset of patients responded only after the addition of SBRT.
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Antineoplásicos Inmunológicos/uso terapéutico , Quimioradioterapia/métodos , Melanoma/terapia , Nivolumab/uso terapéutico , Radiocirugia/métodos , Neoplasias Cutáneas/terapia , Adulto , Anciano , Antineoplásicos Inmunológicos/efectos adversos , Biomarcadores de Tumor/sangre , Quimioradioterapia/efectos adversos , ADN de Neoplasias/sangre , Esquema de Medicación , Femenino , GTP Fosfohidrolasas/sangre , Humanos , Estimación de Kaplan-Meier , Masculino , Melanoma/sangre , Melanoma/patología , Melanoma/secundario , Proteínas de la Membrana/sangre , Persona de Mediana Edad , Nivolumab/efectos adversos , Receptor de Muerte Celular Programada 1/sangre , Supervivencia sin Progresión , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas B-raf/genética , Radiocirugia/efectos adversos , Criterios de Evaluación de Respuesta en Tumores Sólidos , Neoplasias Cutáneas/sangre , Neoplasias Cutáneas/patologíaRESUMEN
Because boron nitride quantum dots (BN QDs) have a wider gap (5.0-6.0 eV) than other QDs, the edge configurations, chemical functionalities, and heteroatom dopants can decrease and regulate the band gap of BN QDs, thereby ameliorating the QDs' properties. Now, the precise control and regulation of BN QDs are still at an early stage and is a challenging task. Therefore, we used thiourea and l-cysteine as different sulfur precursors to regulate the BN QDs' optoelectronic properties in this study. It is interesting that two kinds of S-regulated BN QDs present significantly different electrochemiluminescence (ECL) properties and electro-optical activity. Furthermore, a ratiometric and enzyme-free ECL sensing mode is constructed with the amplified surface plasmon coupled-ECL (SPC-ECL) strategy. The proposed DNA sensor can quantify the BRAF gene from 1 pmol/L to 1.5 nmol/L with a limit of detection (LOD) of 0.3 pmol/L. The change of BN QDs' ECL signal was easily observed with a smartphone camera. This work for the first time provides insight into the role of sulfur regulation in enhancing ECL efficiency and the electro-optical activity of BN QDs.
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Técnicas Biosensibles , Técnicas Electroquímicas , Mediciones Luminiscentes , Proteínas Proto-Oncogénicas B-raf/genética , Resonancia por Plasmón de Superficie , Compuestos de Boro/química , Cisteína/química , ADN/sangre , ADN/química , Oro/química , Humanos , Nanopartículas del Metal/química , Proteínas Proto-Oncogénicas B-raf/sangre , Puntos Cuánticos/química , Tiourea/químicaRESUMEN
BACKGROUND: Langerhans cell histiocytosis (LCH) is a histiocytic disorder driven by a constitutive activation of the MAPK signaling pathway in myeloid cells. In 50-60% of cases, it is caused by the BRAFV600E mutation. There is evidence that levels of BRAFV600E in the peripheral blood of patients with LCH correlate with disease burden and could be used as marker for disease extent and response to therapy. However, there is currently no consensus on how testing for minimal disseminated disease should be performed. METHODS: Different approaches to determine the mutation load in patients with LCH were assessed and longitudinal evaluation of patient DNA during treatment with chemotherapy and/or the RAF inhibitor vemurafenib was performed. DNA was isolated from whole blood, different leukocyte subsets, and circulating cell-free DNA (ccf-DNA). RESULTS: We show that determining BRAF levels from whole blood is superior to using ccfDNA. Furthermore, it is important to identify the clinically relevant BRAF-mutated cellular subpopulations such as CD14+ monocytes or CD1c+ DCs, since other blood cells can also harbor the mutation and therefore confound whole blood or ccfDNA measurements. CONCLUSION: Our data support the view that single-agent treatment with an RAF inhibitor reduces disease activity but does not cure LCH.