Biology subjects

Wan, J. C.

Publications and source records attributed to Wan, J. C..

3 recordsLinked to original sources

bioRxiv2019

Comprehensive characterisation of cell-free tumour DNA in plasma and urine of patients with renal tumours

Cell-free tumour-derived DNA (ctDNA) allows non-invasive monitoring of cancers but its utility in renal cell cancer (RCC) has not been established. Here, untargeted and targeted sequencing methods, applied to two independent cohorts of renal tumour patients (n=90), were used to determine ctDNA content in plasma and urine. Our data revealed lower plasma ctDNA levels in RCC relative to other cancers, with untargeted detection of [~]33%. A sensitive personalised approach, applied to plasma and urine from select patients improved detection to [~]50%, including in patients with early-stage and even benign lesions.\n\nA machine-learning based model predicted detection, potentially offering a means of triaging samples for personalised analysis. In addition, with limited data we observed that plasma, and for the first time, urine ctDNA may better represent tumour heterogeneity than tissue biopsy. Furthermore, longitudinal sampling of >200 plasma samples revealed that ctDNA can track disease course. Additional datasets will be required to validate these findings.\n\nOverall, our data highlight RCC as a ctDNA-low malignancy, but indicate potential clinical utility provided improvement in detection approaches.\n\nOne sentence summaryComplementary sequencing methods show that cell-free tumour DNA levels are low in renal cancer though, via various strategies, may still be informative.

cancer biology↗

bioRxiv2019

High-sensitivity monitoring of ctDNA by patient-specific sequencing panels and integration of variant reads

Circulating tumor-derived DNA (ctDNA) can be used to monitor cancer dynamics noninvasively. Patients with small tumors have few copies of ctDNA in plasma, resulting in limited sensitivity to detect low-volume or residual disease. We show that sampling limitations can be overcome and sensitivity for ctDNA detection can be improved by massively parallel sequencing when hundreds to thousands of mutations are identified by tumor genotyping. We describe the INtegration of VAriant Reads (INVAR) analysis pipeline, which combines patient-specific mutation lists with both custom error-suppression methods and signal enrichment based on biological features of ctDNA. In this framework, the sensitivity can be estimated independently for each sample based on the number of informative reads, which is the product of the number of mutations analyzed and the average depth of unique sequencing reads. We applied INVAR to deep sequencing data generated by custom hybrid-capture panels, and showed that when ~106 informative reads were obtained INVAR allowed detection of tumor-derived DNA fractions to parts per million (ppm). In serial samples from patients with advanced melanoma on treatment, we detected ctDNA when imaging confirmed tumor volume of ~1cm3. In patients with resected early-stage melanoma, ctDNA was detected in 40% of patients who later relapsed, with higher rates of detection when more informative reads were obtained. We further demonstrated that INVAR can be generalized and allows improved detection of ctDNA from whole-exome and low-depth whole-genome sequencing data.

cancer biology↗

bioRxiv2019

Detection of ctDNA from dried blood spots after DNA size selection

Recent advances in the research and clinical applications of circulating tumour DNA (ctDNA) is limited by practical considerations of sample collection. Whole genome sequencing (WGS) is increasingly used for analysis of ctDNA, identifying copy-number alterations, fragment size patterns, and other genomic features. We hypothesised that low-depth WGS data may be generated from minute amounts of cell-free DNA, and that fragment-size selection may be effective to remove contaminating genomic DNA (gDNA) from small volumes of blood. There are practical advantages to using dried blood spots as these are easier to collect, facilitate serial sampling, and support novel study designs in prospective human studies, animal models and expand the utilisation of archival samples by the removal of gDNA in small volumes. We therefore developed a protocol for the isolation and analysis of cell-free DNA from dried blood spots. Analysing a dried blood spot of 50L frozen whole blood from a patient with melanoma, we identified ctDNA based on tumour-specific somatic copy-number alterations, and found a fragment size profile similar to that observed in plasma DNA processed by traditional methods. We extended this approach to detect tumour-derived cell-free DNA in a dried blood spot from a mouse xenograft model and were able to identify ctDNA from the originally grafted ascites. Together, our data suggests that ctDNA can be detected and monitored in dried blood spots. This will enable new approaches for sample collection from patients and in vivo models.

cancer biology↗