Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.
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ABSTRACT:
SUBMITTER: Deveson IW
PROVIDER: S-EPMC8434938 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In China, circulating tumor DNA analysis is widely used and numerous assays are available. Systematic evaluation to help users make informed selections is needed. Nine circulating tumor DNA assays, including one benchmark assay, were evaluated using 23 contrived reference samples. There were two sample types (cell-free DNA and plasma samples), three circulating tumor DNA inputs (low, < 20 ng; medium, 20-50 ng; high, > 50 ng), two variant allele frequency ranges (low, 0.1-0.5%; intermediate, 0.5-2.5%), and four variant types (single nucleotide, insertion/deletion, structural, and copy number). Sensitivity, specificity, reproducibility, and all processes from cell-free DNA extraction to bioinformatics analysis were assessed. The test assays were generally comparable or superior to the benchmark assay, demonstrating high analytical sensitivity. Variations in circulating tumor DNA extraction and quantification efficiency, sensitivity, and reproducibility were observed, particularly at lower inputs. These findings will guide circulating tumor DNA assay choice for research and clinical studies, allowing consideration of multiple technical parameters.
| S-EPMC10904763 | biostudies-literature
Project description:Circulating tumor DNA (ctDNA) is a component of cell-free DNA (cfDNA) that is shed by malignant tumors into the bloodstream and other bodily fluids. ctDNA can comprise up to 10% of a patient's cfDNA depending on their tumor type and burden. The short half-life of ctDNA ensures that its detection captures tumor burden in real-time and offers a non-invasive method of repeatedly evaluating the genomic profile of a patient's tumor. A challenge in ctDNA detection includes clonal hematopoiesis of indeterminate potential (CHIP), which can be distinguished from tumor variants using a paired whole-blood control. Most assays for ctDNA quantification rely on measurements of somatic variant allele frequency (VAF), which is a mutation-dependent method. Patients with certain types of solid tumors, including colorectal cancer (CRC), can have levels of cfDNA 50 times higher than healthy patients. ctDNA undergoes a precipitous drop shortly after tumor resection and therapy, and rising levels can foreshadow radiologic recurrence on the order of months. The amount of tumor bulk required for ctDNA detection is lower than that for computed tomography (CT) scan detection, with ctDNA detection preceding radiologic recurrence in many cases. cfDNA/ctDNA can be used for tumor molecular profiling to identify resistance mutations when tumor biopsy is not available, to detect minimal residual disease (MRD), to monitor therapy response, and for the detection of tumor relapse. Although ctDNA is not yet implemented in clinical practice, studies are ongoing to define the appropriate way to use it as a tool in the clinic. In this review article, we examine the general aspects of ctDNA, its status as a biomarker, and its role in the management of early (II-III) and late (IV; mCRC) stage colorectal cancer (CRC).
| S-EPMC9024503 | biostudies-literature
Project description:BACKGROUND:With the accelerated development of next-generation sequencing (NGS), identified variants, and targeted therapies, clinicians who confront the complicated and multifarious genetic information may not effectively incorporate NGS-based circulating tumor DNA (ctDNA) analysis into routine patient care. Consequently, standardized ctDNA testing reports are of vital importance. In an effort to guarantee high-quality reporting performance, we conducted an investigation of the current detection and reporting practices for NGS-based ctDNA analysis. MATERIALS AND METHODS:A set of simulated ctDNA samples with known variants at known allelic frequencies and a corresponding case scenario were distributed to 66 genetic testing laboratories for ctDNA analysis. Written reports were collected to evaluate the detection accuracy, reporting integrity, and information sufficiency using 21 predefined criteria. RESULTS:Current reporting practices for NGS-based ctDNA analysis were found to be far from satisfactory, especially regarding testing interpretation and methodological details. Only 42.4% of laboratories reported the results in complete concordance with the expected results. Moreover, 74.2% of reports only listed aberrations with direct and well-known treatment consequences for the tumor type in question. Genetic aberrations for which experimental agents and/or drug access programs are available may thus be overlooked. Furthermore, methodological details for the interpretation of results were missing from the majority of reports (87.9%). CONCLUSION:This proof-of-principle study suggests that the capacity for accurate identification of variants, rational interpretation of genotypes, comprehensive recommendation of potential medications, and detailed description of methodologies need to be further improved before ctDNA analysis can be formally implemented in the clinic. IMPLICATIONS FOR PRACTICE:Accurate, comprehensive, and standardized clinical sequencing reports can help to translate complex genetic information into patient-centered clinical decisions, thereby shepherding precision oncology into daily practice. However, standards, guidelines, and quality requirements for clinical reports of next-generation sequencing (NGS)-based circulating tumor DNA (ctDNA) analysis are currently absent. By using a set of simulated clinical ctDNA samples and a corresponding case scenario, current practices were evaluated to identify deficiencies in clinical sequencing reports of ctDNA analysis. The recommendations provided here may serve as a roadmap for the improved implementation of NGS-based ctDNA analysis in the clinic.
| S-EPMC7066684 | biostudies-literature
Project description:The importance of circulating free DNA (cfDNA) in cancer clinical research was recognized in 1994 when a mutated RAS gene fragment was detected in a patient's blood sample. Up to 1% of the total circulating DNA in patients with cancer is circulating tumor DNA (ctDNA) that originates from tumor cells. As ctDNA is rapidly cleared from the blood stream and can be obtained by minimally invasive methods, it can be used as a dynamic cancer biomarker for cancer early detection, diagnosis, and treatment monitoring. Despite the potential for clinical use, few ctDNA assays have been cleared or approved by the US Food and Drug Administration. As tools for clinical and translational research, current ctDNA assays face some challenges, and more research is needed to advance use of these assays. On September 29-30, 2016, the Division of Cancer Treatment and Diagnosis at the National Cancer Institute convened a workshop entitled "Circulating Tumor DNA Assays in Clinical Cancer Research" to garner input from industry experts, academia, and government research and regulatory agencies to understand and promote the translation of ctDNA assays to clinical research, with potential to advance to use in clinical practice. This Commentary presents the topics of the workshop covered in the presentations and points made in the discussions that followed: 1) background on ctDNA, 2) potential clinical utility of ctDNA assays, 3) assay technology, 4) assay clinical and analytical validation, and 5) industry perspectives. Additional relevant information that has come to light since the workshop has been included.
| S-EPMC6136923 | biostudies-literature
Project description:BackgroundIncreasing knowledge of cancer biology and an expanding spectrum of molecularly targeted therapies provide the basis for precision oncology. Despite extensive gene diagnostics, previous reports indicate that less than 10% of patients benefit from this concept.MethodsWe retrospectively analyzed all patients referred to our center's Molecular Tumor Board (MTB) from 2018 to 2021. Molecular testing by next-generation sequencing (NGS) included a 67-gene panel for the detection of short-sequence variants and copy-number alterations, a 53- or 137-gene fusion panel and an ultra-low-coverage whole-genome sequencing for the detection of additional copy-number alterations outside the panel's target regions. Immunohistochemistry for microsatellite instability and PD-L1 expression complemented NGS.ResultsA total of 109 patients were referred to the MTB. In all, 78 patients received therapeutic proposals (70 based on NGS) and 33 were treated accordingly. Evaluable patients treated with MTB-recommended therapy (n = 30) had significantly longer progression-free survival than patients treated with other therapies (n = 17) (4.3 vs. 1.9 months, p = 0.0094). Seven patients treated with off-label regimens experienced major clinical benefits.ConclusionThe combined focused sequencing assays detected targetable alterations in the majority of patients. Patient benefits appeared to lie in the same range as with large-scale sequencing approaches.
| S-EPMC9496918 | biostudies-literature
Project description:Background This retrospective single center study aimed to describe circulating tumor DNA (ctDNA) comprehensive genomic profiling (CGP) utilization in a community practice for patients with advanced solid tumors. Methods All patients were included who were seen at the Scripps Hillcrest Oncology Clinic (San Diego, CA, USA) between September 2016 to March 2018 who had ctDNA assay testing performed. In this cohort, all ctDNA testing was performed to aid therapeutic decision making with wide variety in both the type of advanced solid tumor, as well as the line of therapy. Results Of the assays performed in the 41 patients included in this review, 42% of therapeutic actions following ctDNA assay results were influenced by the ctDNA result, including initiation of the corresponding Federal Drug Administration (FDA) approved therapy, placement on clinical trial, and initiation of off label-targeted options. In addition, CGP results guided clinicians away from futile or harmful treatments, such as EGFR inhibition in colorectal cancer patients with discovered KRAS mutations. No additional prognostic or therapeutic information was gathered in one quarter of patients for which ctDNA was drawn. Furthermore, discovered genomic alterations by ctDNA testing did not influence therapeutic action in 58% of cases. Conclusions These results highlight the conundrum that having additional information regarding an individual’s tumor biology does not yet translate into meaningful targeted therapy in the majority of cases. Further studies are needed regarding ctDNA utilization to help guide community oncologists who will continue to face the choice between targeted therapy, immunotherapy, and cytotoxic chemotherapy as science advances.
| S-EPMC8798503 | biostudies-literature
Project description:Circulating tumor DNA (ctDNA) has emerged as a promising tool for various clinical applications, including early diagnosis, therapeutic target identification, treatment response monitoring, prognosis evaluation, and minimal residual disease detection. Consequently, ctDNA assays have been incorporated into clinical practice. In this review, we offer an in-depth exploration of the clinical implementation of ctDNA assays. Notably, we examined existing evidence related to pre-analytical procedures, analytical components in current technologies, and result interpretation and reporting processes. The primary objective of this guidelines is to provide recommendations for the clinical utilization of ctDNA assays.
| S-EPMC10813828 | biostudies-literature
Project description:Circulating tumor (ct) DNA is a powerful tool that can be used to track cancer beyond a single snapshot in space and time. It has potential applications in detecting minimal residual disease and predicting relapse, in selecting patients for tailored treatments, and in revealing mechanisms of response or resistance. Here, we discuss the incorporation of ctDNA into clinical trials.
| S-EPMC6474042 | biostudies-literature
Project description:Next-generation sequencing of circulating tumor DNA presents a promising approach to cancer diagnostics, complementing conventional tissue-based diagnostic testing by enabling minimally invasive serial testing and broad genomic coverage through a simple blood draw to maximize therapeutic benefit to patients. LiquidHALLMARK® is an amplicon-based next-generation sequencing assay developed for the genomic profiling of plasma-derived cell-free DNA (cfDNA). The comprehensive 80-gene panel profiles point mutations, insertions/deletions, copy number alterations, and gene fusions, and further detects oncogenic viruses (Epstein-Barr virus (EBV) and hepatitis B virus (HBV)) and microsatellite instability (MSI). Here, the analytical and clinical validation of the assay is reported. Analytical validation using reference genetic materials demonstrated a sensitivity of 99.38% for point mutations and 95.83% for insertions/deletions at 0.1% variant allele frequency (VAF), and a sensitivity of 91.67% for gene fusions at 0.5% VAF. In non-cancer samples, a high specificity (≥99.9999% per-base) was observed. The limit of detection for copy number alterations, EBV, HBV, and MSI were also empirically determined. Orthogonal comparison of epidermal growth factor receptor (EGFR) variant calls made by LiquidHALLMARK and a reference allele-specific polymerase chain reaction (AS-PCR) method for 355 lung cancer specimens revealed an overall concordance of 93.80%, while external validation with cobas® EGFR Mutation Test v2 for 50 lung cancer specimens demonstrated an overall concordance of 84.00%, with a 100% concordance rate for EGFR variants above 0.4% VAF. Clinical application of LiquidHALLMARK in 1,592 consecutive patients demonstrated a high detection rate (74.8% circulating tumor DNA (ctDNA)-positive in cancer samples) and broad actionability (50.0% of cancer samples harboring alterations with biological evidence for actionability). Among ctDNA-positive lung cancers, 72.5% harbored at least one biomarker with a guideline-approved drug indication. These results establish the high sensitivity, specificity, accuracy, and precision of the LiquidHALLMARK assay and supports its clinical application for blood-based genomic testing.
| S-EPMC9053827 | biostudies-literature
Project description:BackgroundLung cancer (LC) is a leading cause of cancer-deaths globally. Its lethality is due in large part to the paucity of accurate screening markers. Precision Medicine includes the use of omics technology and novel analytic approaches for biomarker development. We combined Artificial Intelligence (AI) and DNA methylation analysis of circulating cell-free tumor DNA (ctDNA), to identify putative biomarkers for and to elucidate the pathogenesis of LC.MethodsIllumina Infinium MethylationEPIC BeadChip array analysis was used to measure cytosine (CpG) methylation changes across the genome in LC. Six different AI platforms including support vector machine (SVM) and Deep Learning (DL) were used to identify CpG biomarkers and for LC detection. Training set and validation sets were generated, and 10-fold cross validation performed. Gene enrichment analysis using g:profiler and GREAT enrichment was used to elucidate the LC pathogenesis.ResultsUsing a stringent GWAS significance threshold, p-value <5x10-8, we identified 4389 CpGs (cytosine methylation loci) in coding genes and 1812 CpGs in non-protein coding DNA regions that were differentially methylated in LC. SVM and three other AI platforms achieved an AUC=1.00; 95% CI (0.90-1.00) for LC detection. DL achieved an AUC=1.00; 95% CI (0.95-1.00) and 100% sensitivity and specificity. High diagnostic accuracies were achieved with only intragenic or only intergenic CpG loci. Gene enrichment analysis found dysregulation of molecular pathways involved in the development of small cell and non-small cell LC.ConclusionUsing AI and DNA methylation analysis of ctDNA, high LC detection rates were achieved. Further, many of the genes that were epigenetically altered are known to be involved in the biology of neoplasms in general and lung cancer in particular.
| S-EPMC9114890 | biostudies-literature