Technological considerations for genome-guided diagnosis and management of cancer

Lennon, Niall J.; Adalsteinsson, Viktor A.; Gabriel, Stacey

doi:10.1186/s13073-016-0370-4

Cited by 15 publications

(15 citation statements)

References 72 publications

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“…To perform ND-NaME-PrO-HRM-Miseq on circulating DNA from plasma obtained from two metastatic breast cancer patients (#301 and #284), we selected two samples where exome sequencing had been performed on matched tumor biopsies (36, 37). We selected ten mutations that were confidently detected by WES of the tumor biopsies.…”

Section: Resultsmentioning

confidence: 99%

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies

et al. 2017

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BACKGROUND The use of clinical samples and circulating cell free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of wild-type DNA (WT-DNA). Mutation enrichment is combined with high-resolution-melting (HRM) performed in multiplexed closed-tube reactions, as a rapid, cost-effective screening tool prior to targeted re-sequencing. METHODS We developed a homogeneous, closed-tube approach to utilize a double-strand DNA-specific nuclease (DSN) for degradation of WT-DNA at multiple targets simultaneously. The No-Denaturation Nuclease-assisted Minor Allele Enrichment with Probe-Overlap (ND-NaME-PrO) employs WT-oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA-breathing), the oligonucleotide-probes enhance double-stranded-DNA-specific nuclease digestion at the selected targets, with high preference towards WT over mutant DNA. To validate ND-NaME-PrO we employed multiplexed-HRM, digital PCR and Miseq targeted re-sequencing of mutated genomic DNA and cfDNA. RESULTS Serial dilution of KRAS mutation-containing DNA demonstrates mutation enrichment by 10–120-fold and detection of allelic-fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM demonstrated mutation scanning of 10–20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM-scanning over 10 DNA targets. cfDNA mutations were enriched up to ~100-fold, average ~25-fold, and identified via targeted re-sequencing. CONCLUSIONS Closed-tube homogeneous ND-NaME-PrO combined with multiplexed-HRM is a convenient approach to efficiently enrich for mutations on multiple DNA-targets and enabling pre-screening prior to targeted re-sequencing.

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Section: Resultsmentioning

confidence: 99%

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies

et al. 2017

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“…Both plasma and serum are derivatives of whole blood without the cells, and are acquired through centrifugation of blood[16], [52], [53]. Plasma and serum cannot be used for capture and analysis of CTCs because they are the remnants after removal of blood cells, which also removes CTCs.…”

Section: Sample Fluid Types For Liquid Biopsymentioning

confidence: 99%

“…The major reason for this is that nucleic acid screening technologies are quite developed, with an increasing understanding of the relationship between individual mutations and cancer prognosis[52], [119]. Another reason is that, compared to CTCs or exosomes, circulating nucleic acids require relatively little downstream analysis, as their sequence, mutation status, and copy number can be known based upon capture without further processing.…”

Section: Technologies For Capture Detection and Analysis Of Circulamentioning

confidence: 99%

Advances in liquid biopsy on-chip for cancer management: Technologies, biomarkers, and clinical analysis

Tadimety

Closson

et al. 2018

Critical Reviews in Clinical Laboratory Sciences

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Liquid biopsy, as a minimally invasive method of gleaning insight into the dynamics of diseases through a patient fluid sample, has been growing in popularity for cancer diagnosis, prognosis, and monitoring. While many technologies have been developed and validated in research laboratories, there has also been a push to expand these technologies into other clinical settings and as point of-care devices. In this review we discuss and evaluate microchip-based technologies for circulating tumor cell (CTC), exosome, and circulating tumor nucleic acid (ctNA) capture, detection, and analysis. Such integrated systems streamline otherwise multiple-step, manual operations to get a sample-to-answer quantitation. In addition, analysis of disease biomarkers is suited to point of care settings because of ease of use, low consumption of sample and reagents, and high throughput. We also cover the basics of biomarkers and their detection in biological fluid samples suitable for liquid biopsy on-chip. We focus on emerging technologies that process a small patient sample with high spatial-temporal resolution and derive clinically meaningful results through on-chip biomarker sensing and downstream molecular analysis in a simple workflow. This critical review is meant as a resource for those interested in developing technologies for capture, detection, and analysis platforms for liquid biopsy in a variety of settings.

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“…These trials may employ biopsy of multiple regions to assess spatial heterogeneity and serial liquid biopsies to assess temporal heterogeneity [104, 105]. Unfortunately, lab tests and their interpretation can take a long time and there may be no FDA-approved therapies targeting phenotypes or pathways identified by these tests.…”

Section: Targeting Tumor Heterogeneity In the Clinicmentioning

confidence: 99%

Mechanisms and clinical implications of tumor heterogeneity and convergence on recurrent phenotypes

et al. 2017

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Tumor heterogeneity has been identified at various -omic levels. The tumor genome, transcriptome, proteome, and phenome can vary widely across cells in patient tumors and are influenced by tumor cell interactions with heterogeneous physical conditions and cellular components of the tumor microenvironment. Here, we explore the concept that while variation exists at multiple -omic levels, changes at each of these levels converge on the same pathways and lead to convergent phenotypes in tumors that can provide common drug targets. These phenotypes include cellular growth and proliferation, sustained oncogenic signaling, and immune avoidance, among others. Tumor heterogeneity complicates treatment of patient cancers as it leads to varied response to therapies. Identification of convergent cellular phenotypes arising in patient cancers and targeted therapies that reverse them has the potential to transform the way clinicians treat these cancers and to improve patient outcome.

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Technological considerations for genome-guided diagnosis and management of cancer

Cited by 15 publications

References 72 publications

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies

Advances in liquid biopsy on-chip for cancer management: Technologies, biomarkers, and clinical analysis

Mechanisms and clinical implications of tumor heterogeneity and convergence on recurrent phenotypes

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