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OBJECTIVES: Pathologic subtyping of tissue biopsies is the gold standard for the diagnosis of lung cancer (LC), which could be complicated in cases of e.g. inconclusive tissue biopsies or unreachable tumors. The diagnosis of LC could be supported in a minimally invasive manner using protein tumor markers (TMs) and circulating tumor DNA (ctDNA) measured in liquid biopsies (LBx). This study evaluates the performance of LBx-based decision-support algorithms for the diagnosis of LC and subtyping into small- and non-small-cell lung cancer (SCLC and NSCLC) aiming to directly impact clinical practice. MATERIALS AND METHODS: In this multicenter prospective study (NL9146), eight protein TMs (CA125, CA15.3, CEA, CYFRA 21-1, HE4, NSE, proGRP and SCCA) and ctDNA mutations in EGFR, KRAS and BRAF were analyzed in blood of 1096 patients suspected of LC. The performance of individual and combined TMs to identify LC, NSCLC or SCLC was established by evaluating logistic regression models at pre-specified positive predictive values (PPV) of ≥95% or ≥98%. The most informative protein TMs included in the multi-parametric models were selected by recursive feature elimination. RESULTS: Single TMs could identify LC, NSCLC and SCLC patients with 46%, 25% and 40% sensitivity, respectively, at pre-specified PPVs. Multi-parametric models combining TMs and ctDNA significantly improved sensitivities to 65%, 67% and 50%, respectively. CONCLUSION: In patients suspected of LC, the LBx-based decision-support algorithms allowed identification of about two-thirds of all LC and NSCLC patients and half of SCLC patients. These models therefore show clinical value and may support LC diagnostics, especially in patients for whom pathologic subtyping is impossible or incomplete.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos Prospectivos , Biomarcadores Tumorais , Fosfopiruvato Hidratase , Biópsia LíquidaRESUMO
Identification of actionable mutations in advanced stage non-squamous non-small-cell lung cancer (NSCLC) patients is recommended by guidelines as it enables treatment with targeted therapies. In current practice, mutations are identified by next-generation sequencing of tumor DNA (tDNA-NGS), which requires tissue biopsies of sufficient quality. Alternatively, circulating tumor DNA (ctDNA) could be used for mutation analysis. This prospective, multicenter study establishes the diagnostic value of ctDNA analysis by droplet digital PCR (ctDNA-ddPCR) in patients with primary lung cancer. CtDNA from 458 primary lung cancer patients was analyzed using a panel of multiplex ddPCRs for EGFR (Ex19Del, G719S, L858R, L861Q and S768I), KRAS G12/G13 and BRAF V600 mutations. For 142 of 175 advanced stage non-squamous NSCLC patients tDNA-NGS results were available to compare to ctDNA-ddPCR. tDNA-NGS identified 98 mutations, of which ctDNA-ddPCR found 53 mutations (54%), including 32 of 45 (71%) targetable driver mutations. In 2 of these 142 patients, a mutation was found by ctDNA-ddPCR only. In 33 advanced stage patients lacking tDNA-NGS results, ctDNA-ddPCR detected 15 additional mutations, of which 7 targetable. Overall, ctDNA-ddPCR detected 70 mutations and tDNA-NGS 98 mutations in 175 advanced NSCLC patients. Using an up-front ctDNA-ddPCR strategy, followed by tDNA-NGS only if ctDNA-ddPCR analysis is negative, increases the number of mutations found from 98 to 115 (17%). At the same time, up-front ctDNA-ddPCR reduces tDNA-NGS analyses by 40%, decreasing the need to perform (additional) biopsies.
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Supernatant pleural effusions (PE) have shown to be a valuable source for the detection of driver mutations in circulating tumor DNA (ctDNA). In this prospective study, the clinical value of ctDNA analysis in supernatant PE to support therapy selection and disease monitoring in lung cancer patients is assessed. Paired PE and plasma samples were collected from lung cancer patients before initiation of therapy (N = 2) and from EGFR positive patients during therapy (N = 3). Supernatant PE and plasma were tested for mutations in EGFR, KRAS and BRAF by droplet digital PCR. In PE of two patients with suspected lung cancer, a KRAS mutation was detected with a 5- and 8-fold higher fractional abundance (FA) compared to plasma. For three patients with progressive disease during therapy, both the EGFR L858R and T790M mutations were detected in PE. However, in plasma only for two of these patients the L858R mutation was detected with a 46- and 14- fold lower FA, and only for one patient the T790M mutation was detected with a 8-fold lower FA. For one patient, longitudinal ctDNA analysis in PE revealed the T790M and L858R mutations already two months prior to detection of progressive disease by CT-scan. In this study, a higher ctDNA concentration and FA was obtained from PE compared to the corresponding blood samples, which enables more sensitive mutation analysis. Thus, PE is a valuable liquid biopsy, complementing plasma, for ctDNA analysis to support therapy selection and disease monitoring in lung cancer patients.
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DNA Tumoral Circulante/sangue , Técnicas de Genotipagem/métodos , Neoplasias Pulmonares/patologia , Derrame Pleural/patologia , Feminino , Humanos , MasculinoRESUMO
Liquid biopsies have become of interest as minimally invasive ways to monitor treatment response in lung cancer patients. Circulating tumor DNA (ctDNA) and protein biomarkers are evaluated for their added value in monitoring therapy response and early detection of disease progression. Plasma and serum samples of non-small cell or small cell lung cancer patients were analyzed for driver mutations in ctDNA (EGFR, KRAS or BRAF) using droplet digital PCR and protein biomarkers (CA125, CEA, CA15.3, Cyfra 21-1, HE4, NSE, proGRP and SCCA) using electrochemiluminescence immunoassays. Biomarker concentration changes were compared with the outcome of CT-scans during therapy. The median difference of the concentration of ctDNA, CA125 and Cyfra21-1 was significantly lower in patients with partial response (PR) compared to patients with progressive disease (PD) on the first evaluation CT-scan (P<0.001, P=0.042 and P=0.020, respectively). A substantial agreement between ctDNA or CA125 response and radiographic response was observed (k=0.692 and k=0.792, respectively). The median difference of the concentration of ctDNA and Cyfra21-1 was also significantly lower in PR patients compared to PD patients at the last CT-scan during therapy (P<0.001 and P=0.026, respectively). An almost perfect agreement between ctDNA and radiographic response (k=0.827) and a moderate agreement between Cyfra21-1 response and radiographic response was observed (k=0.553). Serial testing of the concentration of ctDNA, Cyfra21-1, and possibly CA125 could be a useful added tool for monitoring therapy response and early detection of disease progression in lung cancer patients.
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Biomarcadores Tumorais/metabolismo , Biópsia Líquida/métodos , Neoplasias Pulmonares/terapia , Idoso , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
The detection of EGFR-sensitizing and EGFR-resistance mutations in advanced non-small-cell lung cancer patients is important for the selection and monitoring of EGFR tyrosine-kinase inhibitor therapy. Droplet digital PCR (ddPCR) multiplex assays allow for sensitive and simultaneous detection of multiple mutations in cell-free DNA (cfDNA) with a minimum of extract needed and at lower cost. Patients were screened for the EGFR tyrosine-kinase inhibitor-sensitizing mutations Ex19Del, L858R, L861Q, G719S, and S768I using a novel ddPCR pentaplex assay. Patients who tested positive subsequently were monitored during treatment for the EGFR-sensitizing mutation and two EGFR-resistance mutations, T790M and C797S, using a ddPCR monitor triplex assay. The ddPCR multiplex assays enabled reliable detection of each mutation with a fractional abundance of at least 0.1%. For six patients, longitudinal data were analyzed and the ddPCR results provided a good reflection of the course of the disease and radiologic response. This study confirms that ddPCR on cfDNA supports the diagnosis and therapy selection, and shows that ddPCR multiplex assays on cfDNA could be a valuable additional diagnostic tool for therapy monitoring of non-small-cell lung cancer patients.
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Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Terapia de Alvo Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/epidemiologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Seguimentos , Humanos , Biópsia Líquida/métodos , Estudos Longitudinais , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/patologia , Países Baixos/epidemiologia , Inibidores de Proteínas Quinases/farmacologia , Reprodutibilidade dos Testes , Resultado do TratamentoRESUMO
The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 patients. A one-step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19-negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality and to enable quantification of SARS-CoV-2 RNA. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. It was found that assay sensitivity of the RT-ddPCR was not affected by the total NA background while assay sensitivity of the gold standard RT-PCR assay is drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared with water. The present study describes a robust and sensitive one-step ddRT-PCR multiplex assay for reliable quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.
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Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , DNA/análise , Reação em Cadeia da Polimerase Multiplex/métodos , RNA Mensageiro/análise , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Autoantígenos/genética , Proteínas do Envelope de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/genética , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Genes Essenciais , Glucuronidase/genética , Humanos , Fosfoproteínas/genética , Reação em Cadeia da Polimerase em Tempo Real , Ribonuclease P/genética , SARS-CoV-2 , Sensibilidade e EspecificidadeRESUMO
For patients with suspected lung carcinoma, the analysis of circulating tumor DNA, obtained by liquid biopsy, has the potential to support cancer diagnosis and guide targeted therapy. To ensure sensitive and reproducible detection of circulating tumor DNA in routine clinical practice, a standardized (pre) analytical workflow is required. Plasma was obtained from patients and healthy volunteers. Using the QIAmp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany), six different procedures for the isolation of cell-free DNA (cfDNA) were compared. cfDNA was analyzed by droplet digital PCR (ddPCR) for KRAS G12/13 mutations and for EGFR Ex19Del, L858R, and L861Q mutations using an in-house EGFR multiplex assay. A new isolation procedure that yields extracts with significantly higher cfDNA concentrations than described previously was selected (P < 0.001). EGFR and KRAS assay sensitivity of at least 0.2% fractional abundance was guaranteed for approximately 76% of patient samples in one run. A flowchart that includes validity criteria for a standardized analytical workflow of ddPCR analysis was designed. An improved protocol for cfDNA isolation enables a higher cfDNA input for ddPCR. The use of sensitive KRAS and EGFR multiplex assays and accompanying validity criteria allows for controlled and efficient testing of patient samples at lower costs. Using the suggested workflow, a guaranteed, reliable, and sensitive analysis of cfDNA can be performed using ddPCR in routine clinical practice.