Factors that influence quality and yield of circulating-free DNA: A systematic review of the methodology literature.

BACKGROUND: Circulating-free DNA (cfDNA) is under investigation as a liquid biopsy of cancer for early detection, monitoring disease progression and therapeutic response. This systematic review of the primary cfDNA literature aims to identify and evaluate factors that influence recovery of cfDNA, and to outline evidence-based recommendations for standardization of methods. METHODS: A search of the Ovid and Cochrane databases was undertaken in May 2018 to obtain relevant literature on cfDNA isolation and quantification. Retrieved titles and abstracts were reviewed by two authors. The factors evaluated include choice of specimen type (plasma or serum); time-to-processing of whole blood; blood specimen tube; centrifugation protocol (speed, time, temperature and number of spins); and methods of cfDNA isolation and quantification. FINDINGS: Of 4,172 articles identified through the database search, 52 proceeded to full-text review and 37 met the criteria for inclusion. A quantitative analysis was not possible, due to significant heterogeneity in methodological approaches between studies. Therefore, included data was tabulated and a textual qualitative synthesis approach was taken. INTERPRETATION: This is the first systematic review of methodological factors that influence recovery and quantification of cfDNA, enabling recommendations to be made that will support standardization of methodological approaches towards development of blood-based cancer tests

Harmonizing Cell-Free DNA Collection and Processing Practices through Evidence-Based Guidance.

Circulating cell-free DNA (cfDNA) is rapidly transitioning from discovery research to an important tool in clinical decision making. However, the lack of harmonization of preanalytic practices across institutions may compromise the reproducibility of cfDNA-derived data and hamper advancements in cfDNA testing in the clinic. Differences in cellular genomic contamination, cfDNA yield, integrity, and fragment length have been attributed to different collection tube types and anticoagulants, processing delays and temperatures, tube agitation, centrifugation protocols and speeds, plasma storage duration and temperature, the number of freeze-thaw events, and cfDNA extraction and quantification methods, all of which can also ultimately impact subsequent downstream analysis. Thus, there is a pressing need for widely applicable standards tailored for cfDNA analysis that include all preanalytic steps from blood draw to analysis. The NCI's Biorepositories and Biospecimen Research Branch has developed cfDNA-specific guidelines that are based upon published evidence and have been vetted by a panel of internationally recognized experts in the field. The guidelines include optimal procedures as well as acceptable alternatives to facilitate the generation of evidence-based protocols by individual laboratories and institutions. The aim of the document, which is entitled "Biospecimen Evidence-based Best Practices for Cell-free DNA: Biospecimen Collection and Processing," is to improve the accuracy of cfDNA analysis in both basic research and the clinic by improving and harmonizing practices across institutions

Factors that influence quality and yield of circulating-free DNA: A systematic review of the methodology literature

Background Circulating-free DNA (cfDNA) is under investigation as a liquid biopsy of cancer for early detection, monitoring disease progression and therapeutic response. This systematic review of the primary cfDNA literature aims to identify and evaluate factors that influence recovery of cfDNA, and to outline evidence-based recommendations for standardization of methods. Methods A search of the Ovid and Cochrane databases was undertaken in May 2018 to obtain relevant literature on cfDNA isolation and quantification. Retrieved titles and abstracts were reviewed by two authors. The factors evaluated include choice of specimen type (plasma or serum); time-to-processing of whole blood; blood specimen tube; centrifugation protocol (speed, time, temperature and number of spins); and methods of cfDNA isolation and quantification. Findings Of 4,172 articles identified through the database search, 52 proceeded to full-text review and 37 met the criteria for inclusion. A quantitative analysis was not possible, due to significant heterogeneity in methodological approaches between studies. Therefore, included data was tabulated and a textual qualitative synthesis approach was taken. Interpretation This is the first systematic review of methodological factors that influence recovery and quantification of cfDNA, enabling recommendations to be made that will support standardization of methodological approaches towards development of blood-based cancer tests

Theoretical Perspectives on Gender and Development

This core module was collaboratively developed and written by the Centre for Gender and Development Studies at the University of the West Indies, the International Women's Tribune Centre in New York, and the Summer Institute for Gender and Development (a joint project of Saint Mary's and Dalhousie Universities in Canada) through a project managed and co-ordinated by COL and funded with the aid of a grant from Canada's International Development Research Centre. This module focuses on the theoretical justification for examining the specific roles and contributions of women to development initiatives

Abstract P2-01-04: A method for comprehensive genomic analysis of cell free DNA

Abstract Circulating tumor DNA (ctDNA) is released from tumor tissue into the blood, carries tumor specific genetic alterations, and can be analyzed through noninvasive "liquid biopsy" approaches to identify genetic alterations in cancer patients. Liquid biopsies offer a considerable advantage as they may eliminate the need for invasive tissue biopsies and allow for the detection of alterations in multiple metastatic lesions throughout the course of therapy. However, the fraction of ctDNA obtained from a blood sample is often very low (&amp;lt;1.0%) and can be difficult to detect. Additionally, most methods to evaluate circulating tumor DNA (ctDNA) interrogate single hot spot mutations or few genetic alterations. The next generation of ctDNA assays must interrogate multiple gene regions from a single sample with high precision and accuracy and need to evaluate all forms of actionable genomic alterations including point mutations, amplifications, and translocations. To address these issues, we have developed a ctDNA approach called PlasmaSelect to detect somatic sequence mutations, amplifications and translocations at low allele frequencies in the circulation of cancer patients. Utilizing digital genomic approaches, PlasmaSelect achieves high sensitivity and specificity while interrogating &amp;gt;250,000 nucleotides spanning 63 well-established cancer genes. In addition to sequence mutations in the entire coding region of 18 genes and the exons of 40 genes that are frequently mutated in cancer, PlasmaSelect also performs a comprehensive genomic analysis of amplifications in 57 genes and translocations in 10 genes significant in cancer tumorigenesis. To evaluate the PlasmaSelect approach, we performed dilution series using tumor-derived DNA, containing well-characterized somatic mutations, in the presence of wild-type DNA. PlasmaSelect was able to detect genetic alterations with high specificity and a lower level of detection of 0.10% for sequence mutations and translocations, as well as a focal amplification of ERBB2 with a lower level of detection of 0.20%. We evaluated the clinical utility of PlasmaSelect for detection of genetic alterations in the plasma and matched tissue biopsy specimens from late stage cancer patients. These analyses demonstrated high concordance between the somatic sequence mutations, amplifications, and translocations identified in the tumor sample and those identified directly in the plasma, including alterations in both driver genes as well as those related to acquired resistance to targeted therapies. PlasmaSelect provides a non-invasive platform to enable liquid biopsy detection of clinically relevant genetic alterations across a large number of genomic loci. Citation Format: Parpart-Li S, Angiuoli SV, Chesnick B, Galens K, Jones S, Kadan M, Kann L, Lytle K, Murphy D, Nesselbush M, Phallen J, Riley D, Shukla M, Zhang T, Husain H, Velculescu V, Diaz, Jr LA, Sausen M. A method for comprehensive genomic analysis of cell free DNA. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-01-04.</jats:p

Abstract 619: Identification of clinically actionable genomic alterations in the tumor and circulation of pancreatic cancer patients

Abstract Pancreatic adenocarcinoma has the worst overall mortality of any solid tumor, with only 6% of patients surviving after 5 years. To evaluate the clinical implications of genomic alterations in this low cellularity tumor type, we deeply sequenced the genomes of 134 enriched pancreatic adenocarcinomas from patients who underwent potentially curative resections. Given the low neoplastic cellularity of pancreatic cancers, we enriched for neoplastic cells either by macrodissection of primary tumors or by flow-sorting of tumor nuclei, and performed deep sequencing (high coverage) of these enriched samples using next-generation sequencing approaches. We obtained a total of &amp;gt;1Tb of sequence data, resulting in an average coverage within the target regions of &amp;gt;200-fold for each tumor analyzed by whole-exome sequencing and &amp;gt;750-fold for each tumor analyzed by targeted cancer gene sequencing. These approaches allowed us to identify sequence changes, including single base and small insertion or deletion mutations, as well as copy number alterations in &amp;gt;20,000 genes in the whole-exome analyses and in 116 specific genes in the targeted analyses. These analyses revealed that somatic mutation of chromatin remodeling genes were associated with improved progression-free and overall survival. Alterations in genes with potential clinical utility were observed in a majority of cases and included alterations of AKT1, AKT2, BRCA2, ERBB2, KIT, and PIK3CA. Non-invasive liquid biopsy analyses were performed before and after surgery to evaluate the presence of circulating tumor DNA in the plasma of 83 patients. Through these approaches, we were able to diagnose early stage pancreatic tumors in the majority of patients and to detect the presence of circulating tumor DNA prior to clinical relapse. These observations provide genetic markers of clinical outcome in pancreatic cancer and suggest new avenues for personalized therapy. Citation Format: Mark Sausen, Jillian Phallen, Vilmos Adleff, Siân Jones, Rebecca J. Leary, Karli Lytle, Sonya Parpart-Li, Derek Murphy, Michael T. Barrett, David C. Linehan, Anirban Maitra, Ralph Hruban, Daniel D. Von Hoff, Julia S. Johansen, Luis A. Diaz, Jeffrey A. Drebin, Victor E. Velculescu. Identification of clinically actionable genomic alterations in the tumor and circulation of pancreatic cancer patients. [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 619. doi:10.1158/1538-7445.AM2015-619</jats:p