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1.
Clin Chem ; 67(3): 554-563, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33439966

RESUMO

BACKGROUND: With the advent of precision oncology, liquid biopsies are quickly gaining acceptance in the clinical setting. However, in some cases, the amount of DNA isolated is insufficient for Next-Generation Sequencing (NGS) analysis. The nCounter platform could be an alternative, but it has never been explored for detection of clinically relevant alterations in fluids. METHODS: Circulating-free DNA (cfDNA) was purified from blood, cerebrospinal fluid, and ascites of patients with cancer and analyzed with the nCounter 3 D Single Nucleotide Variant (SNV) Solid Tumor Panel, which allows for detection of 97 driver mutations in 24 genes. RESULTS: Validation experiments revealed that the nCounter SNV panel could detect mutations at allelic fractions of 0.02-2% in samples with ≥5 pg mutant DNA/µL. In a retrospective analysis of 70 cfDNAs from patients with cancer, the panel successfully detected EGFR, KRAS, BRAF, PIK3CA, and NRAS mutations when compared with previous genotyping in the same liquid biopsies and paired tumor tissues [Cohen kappa of 0.96 (CI = 0.92-1.00) and 0.90 (CI = 0.74-1.00), respectively]. In a prospective study including 91 liquid biopsies from patients with different malignancies, 90 yielded valid results with the SNV panel and mutations in EGFR, KRAS, BRAF, PIK3CA, TP53, NFE2L2, CTNNB1, ALK, FBXW7, and PTEN were found. Finally, serial liquid biopsies from a patient with NSCLC revealed that the semiquantitative results of the mutation analysis by the SNV panel correlated with the evolution of the disease. CONCLUSIONS: The nCounter platform requires less DNA than NGS and can be employed for routine mutation testing in liquid biopsies of patients with cancer.


Assuntos
DNA Tumoral Circulante/genética , Análise Mutacional de DNA/métodos , Biópsia Líquida , Neoplasias/genética , Neoplasias/patologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes , Estudos Retrospectivos
2.
Br J Cancer ; 116(6): 802-810, 2017 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-28170370

RESUMO

BACKGROUND: When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA. METHODS: Our panel ('SiRe') covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice. RESULTS: SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression. CONCLUSIONS: SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Colorretais/genética , DNA de Neoplasias/genética , Tumores do Estroma Gastrointestinal/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Melanoma/genética , Mutação/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Colorretais/sangue , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , DNA de Neoplasias/sangue , Feminino , Seguimentos , Neoplasias Gastrointestinais/sangue , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/sangue , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/patologia , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Melanoma/sangue , Melanoma/tratamento farmacológico , Melanoma/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Estudos Prospectivos , Curva ROC
3.
Mol Oncol ; 13(12): 2633-2645, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31529604

RESUMO

Many advanced cases of cancer show central nervous system, pleural, or peritoneal involvement. In this study, we prospectively analyzed if cerebrospinal fluid (CSF), pleural effusion (PE), and/or ascites (ASC) can be used to detect driver mutations and guide treatment decisions. We collected 42 CSF, PE, and ASC samples from advanced non-small-cell lung cancer and melanoma patients. Cell-free DNA (cfDNA) was purified and driver mutations analyzed and quantified by PNA-Q-PCR or next-generation sequencing. All 42 fluid samples were evaluable; clinically relevant mutations were detected in 41 (97.6%). Twenty-three fluids had paired blood samples, 22 were mutation positive in fluid but only 14 in blood, and the abundance of the mutant alleles was significantly higher in fluids. Of the 34 fluids obtained at progression to different therapies, EGFR resistance mutations were detected in nine and ALK acquired mutations in two. The results of testing of CSF, PE, and ASC were used to guide treatment decisions, such as initiation of osimertinib treatment or selection of specific ALK tyrosine-kinase inhibitors. In conclusion, fluids close to metastatic sites are superior to blood for the detection of relevant mutations and can offer valuable clinical information, particularly in patients progressing to targeted therapies.


Assuntos
Acrilamidas/administração & dosagem , Quinase do Linfoma Anaplásico , Compostos de Anilina/administração & dosagem , Líquido Ascítico , Neoplasias Pulmonares , Mutação , Proteínas de Neoplasias , Derrame Pleural Maligno , Idoso , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Líquido Ascítico/metabolismo , Líquido Ascítico/patologia , Carcinoma Pulmonar de Células não Pequenas/líquido cefalorraquidiano , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/líquido cefalorraquidiano , Receptores ErbB/genética , Feminino , Células HT29 , Humanos , Neoplasias Pulmonares/líquido cefalorraquidiano , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/líquido cefalorraquidiano , Proteínas de Neoplasias/genética , Derrame Pleural Maligno/líquido cefalorraquidiano , Derrame Pleural Maligno/tratamento farmacológico , Derrame Pleural Maligno/genética , Estudos Prospectivos
4.
Transl Cancer Res ; 8(Suppl 1): S3-S15, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35117060

RESUMO

BACKGROUND: In a significant percentage of advanced non-small cell lung cancer (NSCLC) patients, tumor tissue is unavailable or insufficient for genetic analyses at time to progression. We prospectively analyzed the appearance of genetic alterations associated with resistance in liquid biopsies of advanced NSCLC patients progressing to targeted therapies using the NGS platform. METHODS: A total of 24 NSCLC patients were included in the study, 22 progressing to tyrosine kinase inhibitors and two to other treatments. Liquid biopsies samples were obtained and analyzed using the GeneReadTM QIAact Lung DNA UMI Panel, designed to enrich specific target regions and containing 550 variant positions in 19 selected genes frequently altered in lung cancer tumors. Previously, a retrospective validation of the panel was performed in clinical samples. RESULTS: Of the 21 patients progressing to tyrosine kinase inhibitors with valid results in liquid biopsy, NGS analysis identified a potential mechanism of resistance in 12 (57%). The most common were acquired mutations in ALK and EGFR, which appeared in 8/21 patients (38%), followed by amplifications in 5/21 patients (24%), and KRAS mutations in one patient (5%). Loss of the p.T790M was also identified in two patients progressing to osimertinib. Three of the 21 (14%) patients presented two or more concomitant alterations associated with resistance. Finally, an EGFR amplification was found in the only patient progressing to immunotherapy included in the study. CONCLUSIONS: NGS analysis in liquid biopsies of patients progressing to targeted therapies using the GeneReader platform is feasible and can help the oncologist to make treatment decisions.

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