63 research outputs found
Investigating the diagnostic potential of circulating tumour DNA (ctDNA) as a non-invasive liquid biopsy: from research to clinic.
Recent advances in oncology have led to the development of targeted therapies, enabling patients to be treated based on their tumour molecular profile. Whilst tumour biopsies are routinely used for profiling, they can be highly invasive, may not fully reflect the heterogeneity present within the tumour mass, or accurately represent the genomic profile as the tumour evolves over time. Recent interest has focussed on the use of circulating tumour DNA (ctDNA) as a non-invasive ‘liquid biopsy’. Cell-free ctDNA, released from cancer cells, is highly fragmented, and carries the same genetic modifications present in the originating tumour, so has potential to be an exquisitely specific biomarker.
This thesis will focus on research I have performed over the last decade to investigate the diagnostic potential of ctDNA. I assessed the hypothesis that ctDNA is a clinically useful biomarker, able to correlate with disease burden, monitor tumour dynamics, and be used to guide treatment. I developed novel digital PCR and next generation sequencing (NGS) assays for the highly sensitive detection of ctDNA, and led the development and analytical validation of a clinical diagnostic ctDNA test to ISO15189:2012 regulatory standards, which is now being used in the clinic to stratify advanced non-small cell lung cancer patients to treatment. This thesis involves critical analysis of 14 publications that I have co-authored investigating the use of ctDNA in high-grade serous ovarian, breast and lung cancer, and the development of novel methods to improve sensitivity of detection.
When I started this work in 2009, very little was known about the clinical relevance of ctDNA. Since this time, work by myself and others has led to an explosion of interest in this area, leading to significant advances in the use of ctDNA for cancer diagnosis, treatment selection, patient monitoring and detection of minimal residual disease
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Detection of ctDNA from dried blood spots after DNA size selection
Background: Recent advances in the study and clinical applications of circulating tumor DNA (ctDNA) are limited by practical considerations of sample collection. Whole genome sequencing (WGS) is increasingly used for analysis of ctDNA, identifying copy-number alterations and fragmentation patterns. We hypothesized that low-depth/shallow WGS (sWGS) data may be generated from minute amounts of cell-free DNA, and that fragment-size selection may remove contaminating genomic DNA from small blood volumes. Dried blood spots have practical advantages for sample collection, may facilitate serial sampling, and could support novel study designs in humans and animal models.
Methods: We developed a protocol for the isolation and analysis of cell-free DNA from dried blood spots using filter paper cards and bead-based size selection. DNA extracted and size-selected from dried spots was analyzed using sWGS and PCR.
Results: Analyzing a 50L dried blood spot from frozen whole blood of a patient with melanoma, we identified ctDNA based on the presence of tumor-specific somatic copy-number alterations, and found a fragment size profile similar to that observed in plasma DNA. We found alterations in different chromosomes in blood-spots from two patients with high-grade serous ovarian carcinoma. Extending this approach to serial dried blood spots from mouse xenograft models, we detect tumor-derived cell-free DNA and identified ctDNA from the originally grafted ascites.
Conclusion: Our data suggest that ctDNA can be detected and monitored in dried blood spots from archived and fresh blood samples, enabling new approaches for sample collection and novel study/trial designs for both patients and in vivo models.The University of Cambridge and Cancer Research UK (grant numbers A20240 and A29580). The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement n.337905. Healthy volunteer samples were provided by the Cambridge Blood and Stem Cell Biobank, which is supported by the Cambridge NIHR Biomedical Research Centre, Wellcome Trust - MRC Stem Cell Institute and the Cambridge Experimental Cancer Medicine Centre, UK
Dynamics of multiple resistance mechanisms in plasma DNA during EGFR-targeted therapies in non-small cell lung cancer.
Tumour heterogeneity leads to the development of multiple resistance mechanisms during targeted therapies. Identifying the dominant driver(s) is critical for treatment decision. We studied the relative dynamics of multiple oncogenic drivers in longitudinal plasma of 50 EGFR-mutant non-small-cell lung cancer patients receiving gefitinib and hydroxychloroquine. We performed digital PCR and targeted sequencing on samples from all patients and shallow whole-genome sequencing on samples from three patients who underwent histological transformation to small-cell lung cancer. In 43 patients with known EGFR mutations from tumour, we identified them accurately in plasma of 41 patients (95%, 41/43). We also found additional mutations, including EGFR T790M (31/50, 62%), TP53 (23/50, 46%), PIK3CA (7/50, 14%) and PTEN (4/50, 8%). Patients with both TP53 and EGFR mutations before treatment had worse overall survival than those with only EGFR Patients who progressed without T790M had worse PFS during TKI continuation and developed alternative alterations, including small-cell lung cancer-associated copy number changes and TP53 mutations, that tracked subsequent treatment responses. Longitudinal plasma analysis can help identify dominant resistance mechanisms, including non-druggable genetic information that may guide clinical management.We would like to acknowledge the support of The University of Cambridge, Cancer Research UK (grant numbers A11906, A20240) (to N.R.), the European Research Council under the European Union's Seventh Framework Programme (FP/2007- 2013) / ERC Grant Agreement n. 337905 (to N.R.), and Hutchison Whampoa Limited (to N.R.
Fragmentation patterns and personalized sequencing of cell-free DNA in urine and plasma of glioma patients.
Glioma-derived cell-free DNA (cfDNA) is challenging to detect using liquid biopsy because quantities in body fluids are low. We determined the glioma-derived DNA fraction in cerebrospinal fluid (CSF), plasma, and urine samples from patients using sequencing of personalized capture panels guided by analysis of matched tumor biopsies. By sequencing cfDNA across thousands of mutations, identified individually in each patient's tumor, we detected tumor-derived DNA in the majority of CSF (7/8), plasma (10/12), and urine samples (10/16), with a median tumor fraction of 6.4 × 10-3 , 3.1 × 10-5 , and 4.7 × 10-5 , respectively. We identified a shift in the size distribution of tumor-derived cfDNA fragments in these body fluids. We further analyzed cfDNA fragment sizes using whole-genome sequencing, in urine samples from 35 glioma patients, 27 individuals with non-malignant brain disorders, and 26 healthy individuals. cfDNA in urine of glioma patients was significantly more fragmented compared to urine from patients with non-malignant brain disorders (P = 1.7 × 10-2 ) and healthy individuals (P = 5.2 × 10-9 ). Machine learning models integrating fragment length could differentiate urine samples from glioma patients (AUC = 0.80-0.91) suggesting possibilities for truly non-invasive cancer detection
Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer.
Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution.We thank the Human Research Tissue Bank at Addenbrooke’s Hospital which is supported by the NIHR Cambridge Biomedical Research Centre. We acknowledge the support of Cancer Research UK, the University of Cambridge, National Institute for Health Research Cambridge Biomedical Research Centre and Cambridge Experimental Cancer Medicine Centre. Dr. Dawson was supported by an Australian National Breast Cancer Foundation and Victorian Cancer Agency Early Career Fellowship. Dr. Murtaza was supported by Science Foundation Arizona’s Bisgrove Scholars Early Tenure Track award.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms976
Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.
Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications
Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.
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
CXCR4 inhibition in human pancreatic and colorectal cancers induces an integrated immune response.
Inhibition of the chemokine receptor CXCR4 in combination with blockade of the PD-1/PD-L1 T cell checkpoint induces T cell infiltration and anticancer responses in murine and human pancreatic cancer. Here we elucidate the mechanism by which CXCR4 inhibition affects the tumor immune microenvironment. In human immune cell-based chemotaxis assays, we find that CXCL12-stimulated CXCR4 inhibits the directed migration mediated by CXCR1, CXCR3, CXCR5, CXCR6, and CCR2, respectively, chemokine receptors expressed by all of the immune cell types that participate in an integrated immune response. Inhibiting CXCR4 in an experimental cancer medicine study by 1-wk continuous infusion of the small-molecule inhibitor AMD3100 (plerixafor) induces an integrated immune response that is detected by transcriptional analysis of paired biopsies of metastases from patients with microsatellite stable colorectal and pancreatic cancer. This integrated immune response occurs in three other examples of immune-mediated damage to noninfected tissues: Rejecting renal allografts, melanomas clinically responding to anti-PD1 antibody therapy, and microsatellite instable colorectal cancers. Thus, signaling by CXCR4 causes immune suppression in human pancreatic ductal adenocarcinoma and colorectal cancer by impairing the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells.Stand Up 2 Cancer, Lustgarten Foundation, NIH
Correlating Radiomic Features of Heterogeneity on CT with Circulating Tumor DNA in Metastatic Melanoma
Clinical imaging methods, such as computed tomography (CT), are used for routine tumor response monitoring. Imaging can also reveal intratumoral, intermetastatic, and interpatient heterogeneity, which can be quantified using radiomics. Circulating tumor DNA (ctDNA) in the plasma is a sensitive and specific biomarker for response monitoring. Here we evaluated the interrelationship between circulating tumor DNA mutant allele fraction (ctDNAmaf), obtained by targeted amplicon sequencing and shallow whole genome sequencing, and radiomic measurements of CT heterogeneity in patients with stage IV melanoma. ctDNAmaf and radiomic observations were obtained from 15 patients with a total of 70 CT examinations acquired as part of a prospective trial. 26 of 39 radiomic features showed a significant relationship with log(ctDNAmaf). Principal component analysis was used to define a radiomics signature that predicted ctDNAmaf independent of lesion volume. This radiomics signature and serum lactate dehydrogenase were independent predictors of ctDNAmaf. Together, these results suggest that radiomic features and ctDNAmaf may serve as complementary clinical tools for treatment monitoring
Comprehensive characterization of cell-free tumor DNA in plasma and urine of patients with renal tumors.
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
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