R-Score: A New Parameter to Assess the Quality of Variants’ Calls Assessed by NGS Using Liquid Biopsies

Next-generation sequencing (NGS) has enabled a deeper knowledge of the molecular landscape in non-small cell lung cancer (NSCLC), identifying a growing number of targetable molecular alterations in key genes. However, NGS profiling of liquid biopsies risk for false positive and false negative calls and parameters assessing the quality of NGS calls remains lacking. In this study, we have evaluated the positive percent agreement (PPA) between NGS and digital PCR calls when assessing EGFR mutation status using 85 plasma samples from 82 EGFR-positive NSCLC patients. According to our data, variant allele fraction (VAF) was significantly lower in discordant calls and the median of the absolute values of all pairwise differences (MAPD) was significantly higher in discordant calls (p < 0.001 in both cases). Based on these results, we propose a new parameter that integrates both variables, named R-score. Next, we sought to evaluate the PPA for EGFR mutation calls between two independent NGS platforms using a subset of 40 samples from the same cohort. Remarkably, there was a significant linear correlation between the PPA and the R-score (r = 0.97; p < 0.001). Specifically, the PPA of samples with an R-score ≤ −1.25 was 95.83%, whereas PPA falls to 81.63% in samples with R-score ≤ 0.25. In conclusion, R-score significantly correlates with PPA and can assist laboratory medicine specialists and data scientists to select reliable variants detected by NGS.

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Reduced sensitivity for EGFR T790M mutations using the Idylla EGFR Mutation Test

Journal of Clinical Pathology ◽

10.1136/jclinpath-2020-206527 ◽

2020 ◽

Vol 74 (1) ◽

pp. 43-47 ◽

Cited By ~ 1

Author(s):

Eric Lee ◽

Victoria Jones ◽

Eleni Topkas ◽

James Harraway

Keyword(s):

Egfr Mutation ◽

Kinase Inhibitor ◽

Limit Of Detection ◽

False Negative ◽

Growth Factor Receptor ◽

Digital Pcr ◽

T790m Mutation ◽

Amplification Curve ◽

Two Samples ◽

Next Generation Sequencing Ngs

AimsOsimertinib is a third-generation EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor that is effective in non-small cell lung cancer (NSCLC) harbouring the EGFR T790M mutation. The Idylla EGFR Mutation Test is a rapid cartridge-based method for detecting T790M and other EGFR mutations. However, false negative T790M results have been reported, and the sensitivity of the assay for this mutation is uncertain.MethodsEighty NSCLC samples were tested by both Idylla and a next-generation sequencing (NGS) assay; 46 were from patients at disease progression, and 24 of these had known T790M mutations. Droplet digital PCR (ddPCR) was used to confirm NGS findings in samples with the T790M mutation.ResultsOf 19 samples with T790M variant allele frequencies (VAF) higher than the stated 5% limit of detection, 14 were detected by Idylla (sensitivity 74%, 95% CI 49% to 90%). Where sufficient sample remained, ddPCR was consistent with NGS findings in all samples. False negative T790M results were associated with higher EGFR control Cq values (median 22.8 vs 19.8), presence of the EGFR Q787Q polymorphism in cis (80% vs 44%) and presence of an invalid T790M amplification curve. An EGFR exon 19 indel with VAF >5% was also not detected by the Idylla assay in two samples.ConclusionsThe Idylla EGFR Mutation Test has reduced sensitivity for the T790M mutation compared with NGS and ddPCR methods. The presence of an invalid T790M amplification curve may indicate a possible false negative result that warrants further testing by an orthogonal method.

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Comparison of the SuperARMS and Droplet Digital PCR for Detecting EGFR Mutation in ctDNA From NSCLC Patients

Translational Oncology ◽

10.1016/j.tranon.2018.02.007 ◽

2018 ◽

Vol 11 (2) ◽

pp. 542-545 ◽

Cited By ~ 16

Author(s):

Wei-neng Feng ◽

Wei-quan Gu ◽

Ning Zhao ◽

Ying-ming Pan ◽

Wei Luo ◽

...

Keyword(s):

Egfr Mutation ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Nsclc Patients

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Lungful: An observational prospective study to assess the molecular epidemiology of EGFR mutations upon progression on or after first line EGFR-TKI therapy, in real-life clinical settings in Greece.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e21641 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e21641-e21641

Author(s):

Giannis Socrates Mountzios ◽

Dimitrios Mavroudis ◽

Epaminondas Samantas ◽

Anna Koumarianou ◽

Evangelos Georgios Konstantinos Fergadis ◽

...

Keyword(s):

Molecular Epidemiology ◽

Second Generation ◽

Egfr Mutation ◽

Locally Advanced ◽

Real Life ◽

Egfr Mutations ◽

Liquid Biopsies ◽

T790m Mutation ◽

Nsclc Patients ◽

Egfr Tki

e21641 Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the gold standard 1st line strategy for non-small-cell lung cancer (NSCLC) patients with activating EGFR mutations (EGFRm), associated with improved survival outcomes and quality of life compared to chemotherapy. Despite the high response rate with first- and second- generation TKIs, most patients develop resistance to treatment and progress. The acquisition of T790M mutation in exon 20 is considered the most common resistance mechanism. This study aims to investigate the molecular epidemiology of EGFR resistance mutations, focusing on T790M in EGFRm NSCLC patients treated with TKIs. Methods: The study included patients with locally advanced/metastatic EGFRm NSCLC who have progressed on or after 1st line treatment with first- or second- generation TKI. Samples either from plasma-based liquid biopsy and/or tissue re-biopsy were analysed using the Cobas EGFR Mutation Test v2. All patients signed informed consent and were enrolled between July 2017 and September 2019. Statistical analyses were performed using SAS software, Version 9.4. Results: Ninety-six eligible patients were enrolled. At the time of progression, T790M mutation was detected in 16.7%of the patients using plasma-based liquid biopsies. Among patients with negative T790M result, in plasma, tissue re-biopsy was performed in 22,7% with evaluable/valid results in 72.2% of them. T790M mutation was identified in 38.5% of re-biopsy samples. According to Cobas EGFR Mutation test results (combined plasma and tissue), T790M mutation was identified in 21.9% of the patients. Of T790M-positive patients 42.9% had previously received first and 57.1% second generation EGFR-TKI. Conclusions: Results from this study in real world clinical setting in Greece, show that EGFR-T790M acquired resistance positivity rate in plasma is lower compared to previous reports. Moreover, these data underline the challenges of implementing precision medicine using tissue re-biopsy in advanced/metastatic NSCLC. Clinical trial information: D133FR00126. [Table: see text]

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EGFR status evaluation by liquid biopsy during first-line therapy in patients with advanced NSCLC.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e20524 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e20524-e20524

Author(s):

Francesca Zanelli ◽

Maria Pagano ◽

Candida Bonelli ◽

Bruno Casali ◽

Alberto Cavazza ◽

...

Keyword(s):

Disease Progression ◽

Liquid Biopsy ◽

Egfr Mutation ◽

Advanced Nsclc ◽

First Line ◽

Liquid Biopsies ◽

T790m Mutation ◽

First Line Therapy ◽

Line Therapy ◽

Nsclc Patients

e20524 Background: over the past decade, personalized management based on the molecular features of tumours in patients with advanced non small-cell lung cancer (NSCLC) has entered routine clinical practice. The poor performance of many advanced NSCLC patients may limit invasive biopsies. The liquid biopsy is a diagnostic procedure performed on cancer-derived material obtained in blood samples. In this abstract, we will describe our experience with liquid biopsies. Methods: In the Reggio Emilia Clinical Cancer Centrefrom March 2016 to December 2016, 42 patients with advanced NSCLC were analyzed that had had or had already started first line therapy. The liquid biopsy was repeated at each imaging response evaluation by thoracic-abdominal compound tomography (CT) scan performed every 3 months. In the liquid biopsy, the mutational status of EGFR was analyzed with real time PCR (KIT cobas EGFR mutation test v2 CE-IVD Roche); in tissue, it was evaluated by pyrosequencing. Results: 21/42 liquid biopsies were EGFR-mutated (12/21 eson 19 and 9/21 eson 21). In 3/21 (14.3%) cases, the tissue biopsies showed wild type (WT) EGFR. 6 liquid biopsies were also performed at time 0 (diagnosis). All liquid biopsies of EGFR WT remained WT during treatment and imaging evaluation. The median number of liquid biopsy tests for patients was 2 (range 1-3). In 4/21 cases, T790M was performed: 3 cases in both liquid biopsies and tissue, and 1 case in tissue but not in liquid biopsy. TKi therapy was ineffective in this patient with T790M mutation detected in tissue, but not in liquid biopsy. In all patients, the disappearance of the T790M mutation during TKi therapy was related to disease progression. In 11 cases, modification of EGFR mutation status during treatment anticipated CT scan evidence of disease progression (median = three months). Conclusions: the liquid biopsy is an excellent resource. In our experience the liquid biopsy is the sensitive method of choice during treatment of advanced NSCLC patients. EGFR modification status during TKi therapy showed advanced disease progression.

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Future Perspectives in Detecting EGFR and ALK Gene Alterations in Liquid Biopsies of Patients with NSCLC

International Journal of Molecular Sciences ◽

10.3390/ijms22083815 ◽

2021 ◽

Vol 22 (8) ◽

pp. 3815

Author(s):

Daniela Ferreira ◽

Juliana Miranda ◽

Paula Martins-Lopes ◽

Filomena Adega ◽

Raquel Chaves

Keyword(s):

Kinase Inhibitors ◽

Liquid Biopsies ◽

Assay Sensitivity ◽

Non Invasive ◽

Tki Treatment ◽

Nsclc Patients ◽

Next Generation Sequencing Ngs ◽

Routine Procedures ◽

The Impact

Non-small-cell lung cancer (NSCLC) is a major cause of death worldwide. Alterations in such genes as EGFR and ALK are considered important biomarkers in NSCLC due to the existence of targeted therapies with specific tyrosine kinase inhibitors (TKIs). However, specific resistance-related mutations can occur during TKI treatment, which often result in therapy inefficacy. Liquid biopsies arise as a reliable tool for the early detection of these types of alterations, allowing a non-invasive follow-up of the patients. Furthermore, they can be essential for cancer screening, initial diagnosis and to check surgery success. Despite the great advantages of liquid biopsies in NSCLC and the high input that next-generation sequencing (NGS) approaches can provide in this field, its use in oncology is still limited. With improvement of assay sensitivity and the establishment of clinical guidelines for liquid biopsy analysis, it is expected that they will be used in routine procedures. This review focuses on the usefulness of liquid biopsies of NSCLC patients as a means to detect alterations in EGFR and ALK genes and in disease management, highlighting the impact of NGS methods.

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A Targeted Gene Panel for Circulating Tumor DNA Sequencing in Neuroblastoma

Frontiers in Oncology ◽

10.3389/fonc.2020.596191 ◽

2020 ◽

Vol 10 ◽

Author(s):

Flora Cimmino ◽

Vito Alessandro Lasorsa ◽

Simona Vetrella ◽

Achille Iolascon ◽

Mario Capasso

Keyword(s):

Ad Hoc ◽

Circulating Tumor Dna ◽

Gene Panel ◽

Liquid Biopsies ◽

Targeted Next Generation Sequencing ◽

Pathogenic Variation ◽

Targeted Ngs ◽

Molecular Landscape ◽

Next Generation Sequencing Ngs ◽

Tumor Biopsies

BackgroundLiquid biopsies do not reflect the complete mutation profile of the tumor but have the potential to identify actionable mutations when tumor biopsies are not available as well as variants with low allele frequency. Most retrospective studies conducted in small cohorts of pediatric cancers have illustrated that the technology yield substantial potential in neuroblastoma.AimThe molecular landscape of neuroblastoma harbors potentially actionable genomic alterations. We aimed to study the utility of liquid biopsy to characterize the mutational landscape of primary neuroblastoma using a custom gene panel for ctDNA targeted sequencing.MethodsTargeted next-generation sequencing (NGS) was performed on ctDNA of 11 patients with primary neuroblastoma stage 4. To avoid the detection of false variants, we used UMIs (unique molecular identifiers) for the library construction, increased the sequencing depth and developed ad hoc bioinformatic analyses including the hard filtering of the variant calls.ResultsWe identified 9/11 (81.8%) patients who carry at least one pathogenic variation. The most frequently mutated genes were KMT2C (five cases), NOTCH1/2 (four cases), CREBBP (three cases), ARID1A/B (three cases), ALK (two cases), FGFR1 (two cases), FAT4 (two cases) and CARD11 (two cases).ConclusionsWe developed a targeted NGS approach to identify tumor-specific alterations in ctDNA of neuroblastoma patients. Our results show the reliability of our approach to generate genomic information which can be integrated with clinical and pathological data at diagnosis.

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Metastatic Site Location Influences the Diagnostic Accuracy of ctDNA EGFR- Mutation Testing in NSCLC Patients: a Pooled Analysis

Current Cancer Drug Targets ◽

10.2174/1568009618666180308125110 ◽

2018 ◽

Vol 18 (7) ◽

pp. 697-705 ◽

Cited By ~ 12

Author(s):

Francesco Passiglia ◽

Sergio Rizzo ◽

Christian Rolfo ◽

Antonio Galvano ◽

Enrico Bronte ◽

...

Keyword(s):

Diagnostic Accuracy ◽

Egfr Mutation ◽

Digital Pcr ◽

Pooled Analysis ◽

Egfr Mutations ◽

Site Location ◽

Metastatic Site ◽

Ctdna Analysis ◽

Nsclc Patients ◽

Egfr Mutation Testing

Background: Recent studies evaluated the diagnostic accuracy of circulating tumor DNA (ctDNA) analysis in the detection of epidermal growth factor receptor (EGFR) mutations from plasma of NSCLC patients, overall showing a high concordance as compared to standard tissue genotyping. However it is less clear if the location of metastatic site may influence the ability to identify EGFR mutations. Objective: This pooled analysis aims to evaluate the association between the metastatic site location and the sensitivity of ctDNA analysis in detecting EGFR mutations in NSCLC patients. Methods: Data from all published studies, evaluating the sensitivity of plasma-based EGFRmutation testing, stratified by metastatic site location (extrathoracic (M1b) vs intrathoracic (M1a)) were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, and World Conference of Lung Cancer, meeting proceedings. Pooled Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated for the ctDNA analysis sensitivity, according to metastatic site location. Results: A total of ten studies, with 1425 patients, were eligible. Pooled analysis showed that the sensitivity of ctDNA-based EGFR-mutation testing is significantly higher in patients with M1b vs M1a disease (OR: 5.09; 95% CIs: 2.93 – 8.84). A significant association was observed for both EGFR-activating (OR: 4.30, 95% CI: 2.35-7.88) and resistant T790M mutations (OR: 11.89, 95% CI: 1.45-97.22), regardless of the use of digital-PCR (OR: 5.85, 95% CI: 3.56-9.60) or non-digital PCR technologies (OR: 2.96, 95% CI: 2.24-3.91). Conclusions: These data suggest that the location of metastatic sites significantly influences the diagnostic accuracy of ctDNA analysis in detecting EGFR mutations in NSCLC patients.

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Current progress and quality of radiomic studies for predicting EGFR mutation in patients with non-small cell lung cancer using PET/CT images: a systematic review

British Journal of Radiology ◽

10.1259/bjr.20201272 ◽

2021 ◽

pp. 20201272

Author(s):

Meilinuer Abdurixiti ◽

Mayila Nijiati ◽

Rongfang Shen ◽

Qiu Ya ◽

Naibijiang Abuduxiku ◽

...

Keyword(s):

Lung Cancer ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Egfr Mutation ◽

Small Cell ◽

Small Cell Lung ◽

Mutation Status ◽

Pet Ct ◽

Nsclc Patients

Objectives: To assess the methodological quality of radiomic studies based on positron emission tomography/computed tomography (PET/CT) images predicting epidermal growth factor receptor (EGFR) mutation status in patients with non-small cell lung cancer (NSCLC). Methods: We systematically searched for eligible studies in the PubMed and Web of Science datasets using the terms “radiomics”, “PET/CT”, “NSCLC”, and “EGFR”. The included studies were screened by two reviewers independently. The quality of the radiomic workflow of studies was assessed using the Radiomics Quality Score (RQS). Interclass correlation coefficient (ICC) was used to determine inter rater agreement for the RQS. An overview of the methodologies used in steps of the radiomics workflow and current results are presented. Results: Six studies were included with sample sizes of 973 ranging from 115 to 248 patients. Methodologies in the radiomic workflow varied greatly. The first-order statistics were the most reproducible features. The RQS scores varied from 13.9 to 47.2%. All studies were scored below 50% due to defects on multiple segmentations, phantom study on all scanners, imaging at multiple time points, cut-off analyses, calibration statistics, prospective study, potential clinical utility, and cost-effectiveness analysis. The ICC results for majority of RQS items were excellent. The ICC for summed RQS was 0.986 [95% confidence interval (CI): 0.898–0.998]. Conclusions: : The PET/CT based radiomics signature could serve as a diagnostic indicator of EGFR mutation status in NSCLC patients. However, the current conclusions should be interpreted with care due to the suboptimal quality of the studies. Consensus for standardization of PET/CT based radiomic workflow for EGFR mutation status in NSCLC patients is warranted to further improve research. Advances in knowledge: Radiomics can offer clinicians better insight into the prediction of EGFR mutation status in NSCLC patients, whereas the quality of relative studies should be improved before application to the clinical setting.

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Plasma Circulating Tumor DNA Levels for the Monitoring of Melanoma Patients: Landscape of Available Technologies and Clinical Applications

BioMed Research International ◽

10.1155/2017/5986129 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 23

Author(s):

Benoit Busser ◽

Julien Lupo ◽

Lucie Sancey ◽

Stéphane Mouret ◽

Patrice Faure ◽

...

Keyword(s):

Acquired Resistance ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Clinical Applications ◽

Molecular Heterogeneity ◽

Liquid Biopsies ◽

Melanoma Patients ◽

Ctdna Analysis ◽

Next Generation Sequencing Ngs ◽

Tumor Dna

Melanoma is a cutaneous cancer with an increasing worldwide prevalence and high mortality due to unresectable or metastatic stages. Mutations inBRAF,NRAS, orKITare present in more than 60% of melanoma cases, but a useful blood-based biomarker for the clinical monitoring of melanoma patients is still lacking. Thus, the analysis of circulating tumor cells (CTCs) and/or cell-free circulating tumor DNA (ctDNA) analysis from blood (liquid biopsies) appears to be a promising noninvasive, repeatable, and systemic sampling tool for detecting and monitoring melanoma. Here, we review the molecular biology-based strategies used for ctDNA quantification in melanoma patients, as well as their main clinical applications. Droplet digital PCR (ddPCR) and next generation sequencing (NGS) technologies appear to be two versatile and complementary strategies to study rare variant mutations for the detection and monitoring of melanoma progression. Among the different clinical uses of ctDNA, we highlight the assessment of molecular heterogeneity and the identification of genetic determinants for targeted therapy as well as the analysis of acquired resistance. Importantly, ctDNA quantification might also be a novel biomarker with a prognostic value for melanoma patients.

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