Use of Circulating Tumour DNA (ctDNA) for Measurement of Therapy Predictive Biomarkers in Patients with Cancer

Biomarkers that predict likely response or resistance to specific therapies are critical in personalising treatment for cancer patients. Such biomarkers are now available for an increasing number of anti-cancer therapies, especially targeted therapy and immunotherapy. The gold-standard method for determining predictive biomarkers requires tumour tissue. Obtaining tissue, however, is not always possible and even if possible, the amount or quality of tissue obtained may be inadequate for biomarker analysis. Tumour DNA, however, can be released into the bloodstream, giving rise to what is referred to as circulating tumour DNA (ctDNA). In contrast to tissue, blood can be obtained from effectively all patients in a minimally invasive and safe manner. Other advantages of blood over tissue for biomarker testing include a shorter turn-around time and an ability to perform serial measurements. Furthermore, blood should provide a more complete profile of mutations present in heterogeneous tumours than a single-needle tissue biopsy. A limitation of blood vis-à-vis tissue, however, is lower sensitivity and, thus, the possibility of missing an actionable mutation. Despite this limitation, blood-based predictive biomarkers, such as mutant EGFR for predicting response to EGFR tyrosine kinase inhibitors in advanced non-small-cell lung cancer and mutant PIK3CA for predicting response to alpelisib in combination with fulvestrant in advanced breast cancer, may be used when tissue is unavailable. Although tissue remains the gold standard for detecting predictive biomarkers, it is likely that several further blood-based assays will soon be validated and used when tissue is unavailable or unsuitable for analysis.

Rapid Detection of Circulating Tumour DNA using Allele Specific Mini-Loop Mediated Isothermal Amplification

Isothermal amplification is an emerging approach for non-invasive, rapid and cost-effective real-time monitoring of cancer specific mutations through circulating tumour DNA (ctDNA). This study demonstrates a compact allele specific (AS) loop mediated isothermal amplification (LAMP) strategy, termed ‘AS-Mini-LAMP’, modelled using wild type (WT) and mutation specific reactions targeting the estrogen receptor ESR1 c.1138G>C (p.E380Q) missense mutation. Allele selectivity, encoded at the 5’-end of the forward and backward inner primers (FIP and BIP) promotes enhanced selectivity upon self-hybridisation, loop formation and self-primed exponential amplification. Inclusion of unmodified self-stabilising (USS) primers aimed to reduce the likelihood of non-specific allele amplification through competitive inhibition and to enhance reaction velocity through an assisted strand displacement ‘swarm’ priming effect. The two assays were optimised using short synthetic WT and E380Q mutant DNA templates, and subsequently validated to a limit of detection of 500 mutant copies in under 25 minutes in ddPCR-confirmed positive (20.7% variant allele frequency) and negative patient plasma cfDNA samples. These results demonstrate the ability of AS-Mini-LAMP to achieve sensitive and selective amplification of actionable mutations present within plasma ctDNA.

Gene Mutations in Circulating Tumour DNA as a Diagnostic and Prognostic Marker in Head and Neck Cancer—A Systematic Review

(1) Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is one of the most common malignancies globally. An early diagnosis of this disease is crucial, and the detection of gene mutations in circulating tumour DNA (ctDNA) through a liquid biopsy is a promising non-invasive diagnostic method. This review aims to provide an overview of ctDNA mutations in HNSCC patients and discuss the potential use of this tool in diagnosis and prognosis. (2) Methods: A systematic search for articles published in the English language between January 2000 and April 2021 in the Medline and Scopus databases was conducted. (3) Results: A total of 10 studies published in nine publications were selected and analysed. Altogether, 390 samples were obtained from HNSCC patients, and 79 control samples were evaluated. The most often explored gene mutation in ctDNA was TP53. (4) Conclusions: The examination of a larger group of gene mutations and the use of a combination of multiple detection methods contribute to a higher detection rate of mutated ctDNA. More studies are necessary to verify these conclusions and to translate them into clinical practice.