RESUMO
Liquid biopsy provides a minimally invasive platform for the detection of tumor-derived information, including hotspot mutations, such as BRAF V600E. In this study, we provide evidence of the technical development of a ddPCR assay for the detection of BRAF V600E mutations in the plasma of patients with glioma or brain metastasis. In a small patient cohort (n = 9, n = 5 BRAF V600E, n = 4 BRAF WT, n = 4 healthy control), we were able to detect the BRAF V600E mutation in the plasma of 4/5 patients with BRAF V600E-tissue confirmed mutant tumors, and none of the BRAF WT tumors. We also provide evidence in two metastatic patients with longitudinal monitoring, where the plasma-based BRAF V600E mutation correlated with clinical disease status. This proof of principle study demonstrates the potential of this assay to serve as an adjunctive tool for the detection, monitoring, and molecular characterization of BRAF mutant gliomas and brain metastasis.
RESUMO
PURPOSE: Liquid biopsy offers a minimally invasive tool to diagnose and monitor the heterogeneous molecular landscape of tumors over time and therapy. Detection of TERT promoter mutations (C228T, C250T) in cfDNA has been successful for some systemic cancers but has yet to be demonstrated in gliomas, despite the high prevalence of these mutations in glioma tissue (>60% of all tumors). EXPERIMENTAL DESIGN: Here, we developed a novel digital droplet PCR (ddPCR) assay that incorporates features to improve sensitivity and allows for the simultaneous detection and longitudinal monitoring of two TERT promoter mutations (C228T and C250T) in cfDNA from the plasma of patients with glioma. RESULTS: In baseline performance in tumor tissue, the assay had perfect concordance with an independently performed clinical pathology laboratory assessment of TERT promoter mutations in the same tumor samples [95% confidence interval (CI), 94%-100%]. Extending to matched plasma samples, we detected TERT mutations in both discovery and blinded multi-institution validation cohorts with an overall sensitivity of 62.5% (95% CI, 52%-73%) and a specificity of 90% (95% CI, 80%-96%) compared with the gold-standard tumor tissue-based detection of TERT mutations. Upon longitudinal monitoring in 5 patients, we report that peripheral TERT-mutant allele frequency reflects the clinical course of the disease, with levels decreasing after surgical intervention and therapy and increasing with tumor progression. CONCLUSIONS: Our results demonstrate the feasibility of detecting circulating cfDNA TERT promoter mutations in patients with glioma with clinically relevant sensitivity and specificity.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/diagnóstico , Glioma/diagnóstico , Telomerase/genética , Adulto , Idoso , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Estudos de Coortes , Análise Mutacional de DNA/métodos , Estudos de Viabilidade , Feminino , Glioma/sangue , Glioma/terapia , Humanos , Biópsia Líquida/métodos , Masculino , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Sensibilidade e EspecificidadeRESUMO
Liquid biopsy for the detection and monitoring of central nervous system tumors is of significant clinical interest. At initial diagnosis, the majority of patients with central nervous system tumors undergo magnetic resonance imaging (MRI), followed by invasive brain biopsy to determine the molecular diagnosis of the WHO 2016 classification paradigm. Despite the importance of MRI for long-term treatment monitoring, in the majority of patients who receive chemoradiation therapy for glioblastoma, it can be challenging to distinguish between radiation treatment effects including pseudoprogression, radiation necrosis, and recurrent/progressive disease based on imaging alone. Tissue biopsy-based monitoring is high risk and not always feasible. However, distinguishing these entities is of critical importance for the management of patients and can significantly affect survival. Liquid biopsy strategies including circulating tumor cells, circulating free DNA, and extracellular vesicles have the potential to afford significant useful molecular information at both the stage of diagnosis and monitoring for these tumors. Here, current liquid biopsy-based approaches in the context of tumor monitoring to differentiate progressive disease from pseudoprogression and radiation necrosis are reviewed.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Biópsia Líquida/métodos , Lesões por Radiação , Biomarcadores Tumorais/sangue , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , DNA Tumoral Circulante/sangue , Progressão da Doença , Vesículas Extracelulares/patologia , Glioblastoma/diagnóstico , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Necrose , Células Neoplásicas Circulantes/patologia , Lesões por Radiação/diagnóstico , Lesões por Radiação/patologia , Radioterapia/efeitos adversosRESUMO
Sequencing studies have provided novel insights into the heterogeneous molecular landscape of glioblastoma (GBM), unveiling a subset of patients with gene fusions. Tissue biopsy is highly invasive, limited by sampling frequency and incompletely representative of intra-tumor heterogeneity. Extracellular vesicle-based liquid biopsy provides a minimally invasive alternative to diagnose and monitor tumor-specific molecular aberrations in patient biofluids. Here, we used targeted RNA sequencing to screen GBM tissue and the matched plasma of patients (n = 9) for RNA fusion transcripts. We identified two novel fusion transcripts in GBM tissue and five novel fusions in the matched plasma of GBM patients. The fusion transcripts FGFR3-TACC3 and VTI1A-TCF7L2 were detected in both tissue and matched plasma. A longitudinal follow-up of a GBM patient with a FGFR3-TACC3 positive glioma revealed the potential of monitoring RNA fusions in plasma. In summary, we report a sensitive RNA-seq-based liquid biopsy strategy to detect RNA level fusion status in the plasma of GBM patients.