Leveraging the Fragment Length of Circulating Tumour DNA to Improve Molecular Profiling of Solid Tumour Malignancies with Next-Generation Sequencing: A Pathway to Advanced Non-invasive Diagnostics in Precision Oncology?

Abstract

Circulating cell-free DNA (ccfDNA) has emerged as a promising diagnostic tool in oncology. Identification of tumour-derived ccfDNA (i.e. circulating tumour DNA [ctDNA]) provides non-invasive access to a malignancy's molecular landscape to diagnose, inform therapeutic strategies, and monitor treatment efficacy. Current applications of ccfDNA to detect somatic mutations, however, have been largely constrained to tumour-informed searches and identification of common mutations because of the interaction between ctDNA signal and next-generation sequencing (NGS) noise. Specifically, the low allele frequency of ctDNA associated with non-metastatic and early-stage lesions may be indistinguishable from artifacts that accrue during sample preparation and NGS. Thus, using ccfDNA to achieve non-invasive and personalized molecular profiling to optimize individual patient care is a highly sought goal that remains limited in clinical practice. There is growing evidence, however, that further advances in the field of ccfDNA diagnostics may be achieved by improving detection of somatic mutations through leveraging the inherently shorter fragment lengths of ctDNA compared to non-neoplastic ccfDNA. Here, the origins and rationale for seeking to improve the mutation-based detection of ctDNA by using ccfDNA size profiling are reviewed. Subsequently, in vitro and in silico methods to enrich for a target ccfDNA fragment length are detailed to identify current practices and provide perspective into the potential of using ccfDNA size profiling to impact clinical applications in oncology.

Improving Fetal Fraction of Noninvasive Prenatal Screening Samples Collected in EDTA-Gel Tubes Using Gel Size Selection

“…4,10e13 The size selection appeared also to improve the detection of rare mutated alleles in the context of cancer. 14,25 Tumor circulating cell-free DNA was also shown to be shorter than cfDNA from healthy cells. 26 For NIPS or tumor cfDNA analysis, different systems were used to isolate short fragments with electrophoresis using precast gels, such as 2% Egel EX from Invitrogen (Thermofisher), 2% to 3% agarose cassettes from BluePippin (Sage Bioscience Q20 ), and 3% agarose cassettes from Yourgene Health Canada.…”

“…Different instruments were used to isolate these short fragments with electrophoresis using precast gels. 14 To obtain cfDNA, plasma must be prepared with caution to avoid contamination with genomic DNA from white blood cells. Blood samples need to be processed immediately after collection, and an additional high-speed (16,000 Â g) centrifugation step for plasma preparation is required.…”

Deciphering Tumour Heterogeneity: From Tissue to Liquid Biopsy

“…Recent progress has been made in order to improve the detection performance of ctDNA-based approaches through the development of novel ultra-sensitive techniques or in silico error-suppression tools [154]. Some groups also proposed to more easily discriminate ctDNA from total cfDNA based on the profiling of ctDNA-specific features (methylation profile and fragment length) [155,156]. It also appears difficult in some cases to distinguish tumour-specific signals from physiological events that can appear with ageing.…”

Liquid biopsies to predict CDK4/6 inhibitor efficacy and resistance in breast cancer

“…This, in turn, is reflected in the physical characteristics of the cfDNA fragments and their distribution over the genome (i.e., fragmentation features), revealing information about cell and tissue of origin. Early work on cfDNA fragments revealed differences in fragment length, a phenomenon that multiple studies have leveraged to enrich ctDNA and improve the accuracy of cancer detection [112][113][114][115][116][117] .…”