A high-throughput protocol for isolating cell-free circulating tumor DNA from peripheral blood

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Abstract

The analysis of cell-free circulating tumor DNA (ctDNA) is potentially a less invasive, more dynamic assessment of cancer progression and treatment response than characterizing solid tumor biopsies. Standard isolation methods require separation of plasma by centrifugation, a time-consuming step that complicates automation. To address these limitations, we present an automatable magnetic bead-based ctDNA isolation method that eliminates centrifugation to purify ctDNA directly from peripheral blood (PB). To develop and test our method, ctDNA from cancer patients was purified from PB and plasma. We found that allelic fractions of somatic single-nucleotide variants from target gene capture libraries were comparable, indicating that the PB ctDNA purification method may be a suitable replacement for the plasma-based protocols currently in use.

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A field guide for cancer diagnostics using cell-free DNA: From principles to practice and clinical applications.

Peter Schirmacher, Volker Endris, Albrecht Stenzinger … (2018)

Recently, many genome-wide profiling studies provided insights into the molecular make-up of major cancer types. The deeper understanding of these genetic alterations and their functional consequences led to the discovery of novel therapeutic opportunities improving clinical management of cancer patients. While tissue-based molecular patient stratification is the gold standard for precision medicine, it has certain limitations: Tissue biopsies are invasive sampling procedures carrying the risk of complications and may not represent the entire tumor due to underlying genetic heterogeneity. In this context, complementary characterization of genetic information in the blood of cancer patients can serve as minimal-invasive 'liquid biopsy'. Fragments of circulating cell-free DNA (cfDNA) are released from tissues of healthy individuals as well as cancer patients. The fraction of cfDNA that is released from primary tumors or metastases (i.e. circulating tumor DNA, ctDNA) represents genetic aberrations in cancer cells, which are a potential source for diagnostic, prognostic, and predictive biomarkers. Recent studies have demonstrated technical feasibility and clinical applications including detection of drug targets and resistance mutations as well as longitudinal monitoring of tumors under therapy. To this end, a variety of pre-analytical procedures for blood processing, isolation and quantification of cfDNA are being employed and several analytical methods and technologies ranging from PCR-based single locus assays to genome-wide approaches are available, which considerably differ in sensitivity, specificity, and throughput. However, broad implementation of ctDNA analysis in daily clinical practice requires a thorough understanding of theoretical, technical, and biological concepts and necessitates standardization and validation of pre-analytical and analytical procedures across different technologies. Here, we review the pertinent literature and discuss the advantages and limitations of available methodologies and their potential applications in molecular diagnostics.

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Personalized Circulating Tumor DNA Biomarkers Dynamically Predict Treatment Response and Survival In Gynecologic Cancers

Elena Pereira, Olga Camacho-Vanegas, Sanya Anand … (2015)

Background High-grade serous ovarian and endometrial cancers are the most lethal female reproductive tract malignancies worldwide. In part, failure to treat these two aggressive cancers successfully centers on the fact that while the majority of patients are diagnosed based on current surveillance strategies as having a complete clinical response to their primary therapy, nearly half will develop disease recurrence within 18 months and the majority will die from disease recurrence within 5 years. Moreover, no currently used biomarkers or imaging studies can predict outcome following initial treatment. Circulating tumor DNA (ctDNA) represents a theoretically powerful biomarker for detecting otherwise occult disease. We therefore explored the use of personalized ctDNA markers as both a surveillance and prognostic biomarker in gynecologic cancers and compared this to current FDA-approved surveillance tools. Methods and Findings Tumor and serum samples were collected at time of surgery and then throughout treatment course for 44 patients with gynecologic cancers, representing 22 ovarian cancer cases, 17 uterine cancer cases, one peritoneal, three fallopian tube, and one patient with synchronous fallopian tube and uterine cancer. Patient/tumor-specific mutations were identified using whole-exome and targeted gene sequencing and ctDNA levels quantified using droplet digital PCR. CtDNA was detected in 93.8% of patients for whom probes were designed and levels were highly correlated with CA-125 serum and computed tomography (CT) scanning results. In six patients, ctDNA detected the presence of cancer even when CT scanning was negative and, on average, had a predictive lead time of seven months over CT imaging. Most notably, undetectable levels of ctDNA at six months following initial treatment was associated with markedly improved progression free and overall survival. Conclusions Detection of residual disease in gynecologic, and indeed all cancers, represents a diagnostic dilemma and a potential critical inflection point in precision medicine. This study suggests that the use of personalized ctDNA biomarkers in gynecologic cancers can identify the presence of residual tumor while also more dynamically predicting response to treatment relative to currently used serum and imaging studies. Of particular interest, ctDNA was an independent predictor of survival in patients with ovarian and endometrial cancers. Earlier recognition of disease persistence and/or recurrence and the ability to stratify into better and worse outcome groups through ctDNA surveillance may open the window for improved survival and quality and life in these cancers.

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Lessons learned from the application of whole-genome analysis to the treatment of patients with advanced cancers

Janessa Laskin, Steven Jones, Samuel Aparicio … (2015)

Given the success of targeted agents in specific populations it is expected that some degree of molecular biomarker testing will become standard of care for many, if not all, cancers. To facilitate this, cancer centers worldwide are experimenting with targeted “panel” sequencing of selected mutations. Recent advances in genomic technology enable the generation of genome-scale data sets for individual patients. Recognizing the risk, inherent in panel sequencing, of failing to detect meaningful somatic alterations, we sought to establish processes to integrate data from whole-genome analysis (WGA) into routine cancer care. Between June 2012 and August 2014, 100 adult patients with incurable cancers consented to participate in the Personalized OncoGenomics (POG) study. Fresh tumor and blood samples were obtained and used for whole-genome and RNA sequencing. Computational approaches were used to identify candidate driver mutations, genes, and pathways. Diagnostic and drug information were then sought based on these candidate “drivers.” Reports were generated and discussed weekly in a multidisciplinary team setting. Other multidisciplinary working groups were assembled to establish guidelines on the interpretation, communication, and integration of individual genomic findings into patient care. Of 78 patients for whom WGA was possible, results were considered actionable in 55 cases. In 23 of these 55 cases, the patients received treatments motivated by WGA. Our experience indicates that a multidisciplinary team of clinicians and scientists can implement a paradigm in which WGA is integrated into the care of late stage cancer patients to inform systemic therapy decisions.

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Author and article information

Journal

Journal ID (publisher-id): BTN

Title: BioTechniques

Abbreviated Title: BioTechniques

Publisher: Future Science Ltd (London, UK )

ISSN (Print): 0736-6205

ISSN (Electronic): 1940-9818

Publication date (Print): February 2019

Publication date (Electronic): 29 November 2018

Volume: 66

Issue: 2

Pages: 85-92

Affiliations

[1] ¹Canada's Michael Smith Genome Sciences Centre, BC Cancer, 675 West 10th Avenue, Vancouver, BC, Canada

[2] ²Department of Medical Genetics, Faculty of Medicine, University of British Columbia, Vancouver, Canada

[3] ³Department of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby, BC, Canada

[4] ⁴Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, Canada

Author notes

*Author for correspondence: mmarra@ 123456bcgsc.ca

Article

DOI: 10.2144/btn-2018-0148

SO-VID: 8f6cc3c0-1de8-4d75-b3f5-86289521b9a4

License:

This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License

History

Date received : 01 October 2018

Date accepted : 31 October 2018

Date: 29 November 2018

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A high-throughput protocol for isolating cell-free circulating tumor DNA from peripheral blood

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Abstract

Most cited references 13

A field guide for cancer diagnostics using cell-free DNA: From principles to practice and clinical applications.

Personalized Circulating Tumor DNA Biomarkers Dynamically Predict Treatment Response and Survival In Gynecologic Cancers

Lessons learned from the application of whole-genome analysis to the treatment of patients with advanced cancers

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