<div>Abstract<p>The factors responsible for the low detection rate of cell-free tumor DNA (ctDNA) in the plasma of patients with glioblastoma (GBM) are currently unknown. In this study, we measured circulating nucleic acids in patient-derived orthotopically implanted xenograft (PDOX) models of GBM (<i>n</i> = 64) and show that tumor size and cell proliferation, but not the integrity of the blood–brain barrier or cell death, affect the release of ctDNA in treatment-naïve GBM PDOX. Analysis of fragment length profiles by shallow genome-wide sequencing (<0.2× coverage) of host (rat) and tumor (human) circulating DNA identified a peak at 145 bp in the human DNA fragments, indicating a difference in the origin or processing of the ctDNA. The concentration of ctDNA correlated with cell death only after treatment with temozolomide and radiotherapy. Digital PCR detection of plasma tumor mitochondrial DNA (tmtDNA), an alternative to detection of nuclear ctDNA, improved plasma DNA detection rate (82% vs. 24%) and allowed detection in cerebrospinal fluid and urine. Mitochondrial mutations are prevalent across all cancers and can be detected with high sensitivity, at low cost, and without prior knowledge of tumor mutations via capture-panel sequencing. Coupled with the observation that mitochondrial copy number increases in glioma, these data suggest analyzing tmtDNA as a more sensitive method to detect and monitor tumor burden in cancer, specifically in GBM, where current methods have largely failed.</p>Significance:<p>These findings show that detection of tumor mitochondrial DNA is more sensitive than circulating tumor DNA analysis to detect and monitor tumor burden in patient-derived orthotopic xenografts of glioblastoma.</p></div>
Abstract Novel biomarkers are required to assess tumor burden and response in cancer as conventional biopsies are invasive, costly and only provide a snapshot of the mutational profile at a given time and location. A promising biomarker is the detection of genomic material released from tumors into the blood plasma of patients, known as circulating tumor DNA (ctDNA). ctDNA has been detected in plasma for a wide range of solid tumors and can be distinguished from other (germline) cell-free DNA by the presence of tumor-specific DNA alterations or known hotspot mutations. However, the potential of ctDNA as a biomarker has not yet been fully realized due to technical challenges associated with its detection and analysis, including the short fragment sizes (140-170 bp), small number of amplifiable copies and low/variable allele fractions of ctDNA. We have developed an enhanced platform for tagged-amplicon deep sequencing (TAm-Seq™). Using a combination of improved library preparation and bespoke data analysis methods, this platform can be used to sequence established cancer hotspots and the entire coding regions of selected genes, while preserving high levels of specificity and sensitivity. Using this approach, we have developed an assay that analyzes ∼20 kb of the genome (including regions of interest in more than 30 genes) with sensitivity down to a few mutant copies. Performance of this assay has been demonstrated using spike-in experiments, dilution series and clinical sample cohorts. Proof of concept studies have shown the potential of ctDNA to be used to assess tumor mutation status, monitor tumor dynamics, assess response to treatment and identify mutations associated with acquired drug resistance and disease progression. This non-invasive approach - a “liquid biopsy” - offers a revolution in how cancer can be detected, monitored and treated. Citation Format: Andrew RJ Lawson, Vincent Plagnol, Abdelaziz Fahem, Tim Forshew, James D. Brenton, Davina Gale, Nitzan Rosenfeld. Assessment of clinical applications of circulating tumor DNA using an enhanced TAm-Seq platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2412. doi:10.1158/1538-7445.AM2015-2412
Abstract Introduction Identification of minimal residual disease (MRD) following curative intervention of localized non-small cell lung cancer (NSCLC) holds promise for identifying patients who are at higher risk of relapse and who would benefit from adjuvant treatment. Current routine clinical practice involves serial radiographic imaging following surgery to detect macroscopic disease. Liquid biopsy can identify patients who have MRD without macroscopic disease. Currently available assays have only identified circulating tumor DNA (ctDNA) in a limited number of cases with early stage NSCLC. More sensitive methods are needed to accurately identify the majority of patients who will relapse. Here we evaluate the performance of InVision®MRD, a personalized sequencing assay for plasma cell-free DNA, for detection of ctDNA in patients with early-stage NSCLC undergoing treatment with curative intent. Methods InVision®MRD is a highly sensitive in vitro diagnostic assay, currently available for research use only (RUO), that can detect the presence of tumor DNA traces in cell-free DNA from plasma samples of cancer patients. InVision®MRD identifies tumor-specific variants from exome sequencing of tumor tissue and tracks them in plasma specimens by multiplex PCR and high-depth next-generation sequencing. We evaluated the detection of ctDNA in plasma samples collected from the LUng cancer - CIrculating tumor DNA (LUCID) study, which collected plasma samples from 100 patients with NSCLC stages I-III who underwent radical treatment with curative intent, either surgery or radiotherapy ± chemotherapy. Of patients in the LUCID study, 60% had stage I NSCLC and 40% patients had stage II/III disease, according to TNM 7th edition. Results To evaluate the InVision®MRD assay, a subset of samples from the LUCID study were analyzed. Samples were collected before and after surgery and chemo-radiotherapy from patients with early-stage NSCLC. Using multiplexed analysis of 48 patient-specific variants and high-depth sequencing, ctDNA was detected in 50% of pre-treatment samples analyzed from the first set of 18 patients, at ctDNA fractions ranging from 20 ppm (equivalent to 0.002%) to 19576 ppm (equivalent to 1.958%). Conclusions These findings highlight an opportunity to improve ctDNA detection for early stage NSCLC using a patient-specific plasma sequencing assay. Initial detection rates have reached 50% for patients with early-stage disease prior to treatment, including detection of ctDNA to levels as low as a few parts per million. Together with further data to be presented, this suggests a possible route to improving treatment for early stage NSCLC by detection of residual disease post treatment and for monitoring for early detection of relapse. Citation Format: Katrin Heider, Davina Gale, Andrea Ruiz-Valdepenas, Giovanni Marsico, Garima Sharma, Malcolm Perry, Robert Osborne, Karen Howarth, Tadd Lazarus, Viona Rundell, Jelena Belic, Jerome Wulff, Susan Harden, Doris M. Rassl, Robert C. Rintoul, Nitzan Rosenfeld. Sensitive detection of ctDNA in early stage non-small cell lung cancer patients with a personalized sequencing assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 735.
Current evidence suggests that plasma cell-free DNA (cfDNA) is fragmented around a mode of 166 bp. Data supporting this view has been mainly acquired through the analysis of double-stranded cfDNA. The characteristics and diagnostic potential of single-stranded and damaged double-stranded cfDNA in healthy individuals and cancer patients remain unclear. Here, through a combination of high-affinity magnetic bead-based DNA extraction and single-stranded DNA sequencing library preparation (MB-ssDNA), we report the discovery of a large proportion of cfDNA fragments centered at ∼50 bp. We show that these "ultrashort" cfDNA fragments have a greater relative abundance in plasma of healthy individuals (median = 19.1% of all sequenced cfDNA fragments,
Abstract Background Cell-free tumor-derived DNA (ctDNA) allows non-invasive monitoring of cancers, but its utility in renal cell cancer (RCC) has not been established. Methods Here, a combination of untargeted and targeted sequencing methods, applied to two independent cohorts of patients ( n = 91) with various renal tumor subtypes, were used to determine ctDNA content in plasma and urine. Results Our data revealed lower plasma ctDNA levels in RCC relative to other cancers of similar size and stage, with untargeted detection in 27.5% of patients from both cohorts. A sensitive personalized approach, applied to plasma and urine from select patients ( n = 22) improved detection to ~ 50%, including in patients with early-stage disease and even benign lesions. Detection in plasma, but not urine, was more frequent amongst patients with larger tumors and in those patients with venous tumor thrombus. With data from one extensively characterized patient, we observed that plasma and, for the first time, urine ctDNA may better represent tumor heterogeneity than a single tissue biopsy. Furthermore, in a subset of patients ( n = 16), longitudinal sampling revealed that ctDNA can track disease course and may pre-empt radiological identification of minimal residual disease or disease progression on systemic therapy. Additional datasets will be required to validate these findings. Conclusions These data highlight RCC as a ctDNA-low malignancy. The biological reasons for this are yet to be determined. Nonetheless, our findings indicate potential clinical utility in the management of patients with renal tumors, provided improvement in isolation and detection approaches.
Circulating tumor DNA (ctDNA) offers new opportunities for noninvasive cancer management. Detecting ctDNA in plasma is challenging because it constitutes only a minor fraction of the total cell-free DNA (cfDNA). Pre-analytical factors affect cfDNA levels contributed from leukocyte lysis, hence the ability to detect low-frequency mutant alleles. This study investigates the effects of the delay in processing, storage temperatures, different blood collection tubes, centrifugation protocols, and sample shipment on cfDNA levels. Peripheral blood (n = 231) from cancer patients (n = 62) were collected into K
Introduction Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. Materials and methods We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. Results The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. Conclusions These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.
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