Stephen Fox is Director of Pathology at the Peter MacCallum Cancer Centre and Professorial Fellow at the University of Melbourne. He holds Honours degree and Medical degrees from the University of Bristol, UK and a DPhil in Medicine from the University of Oxford. He has Fellowships of both the Royal College of Pathologists Australasia (RCPA) and UK and is also a Founding Fellow of the Faculty of Science (RCPA). He is an NHMRC Practioner Fellow and a Fellow of the Australian Academy of Health and Medical Sciences. His current focus is development of diagnostic predictive markers of response to therapies in several tumour types using protein and DNA-based assays.

Title: Using ctDNA for Precision Cancer Medicine: Is this the Future?

Abstract:

Molecular characterisation of DNA extracted from tumour biopsies to identify driver mutations has since its inception been the Gold standard for precision cancer medicine. There have been a number of notable successes using this approach for diagnosis, prognosis and for therapeutic stratification. Indeed, there are now many standard of care drugs in a number of tumour types matched to their genetic alterations that have led to improved quality of life, prolonged relapse-free and overall survival. Even tumours previously considered to have a dismal prognosis like lung cancer have become a poster child for this approach with a succession of effective targeted therapies against mutations in EGFR, ALK, ROS1, RET, HER2, Ex14 MET. Furthermore, serial testing is increasingly being advocated on progression to identify resistant mutations that have emerged with initial therapies that can then be targeted again with second and third generation drug regimens.

Nevertheless, in several tumour streams including lung, it is recognised that this approach is problematic for many patients. Notwithstanding the morbidity and sometimes mortality associated with biopsy procedures, it has been shown that biopsies are not possible or are non-informative in approximately 20% of patients due to lesions being inaccessible or insufficient availability of neoplastic tissue after standard histopathology. It is also increasingly clear that biopsies taken in the past at diagnosis with continual tumour evolution are unlikely to represent the underlying genetic structure of the current circumstances of the patient. Furthermore, lung cancers are highly heterogeneous both within an individual lesion and between lesions and a “representative biopsy” might not give the genomic resolution required to fully understand the likely response of any individual tumour to a targeted therapy. Thus, other approaches have been explored not least the so called liquid biopsy.

This term has been attributed to Pantel and Alix-Panabieres for a blood test that could be used as a surrogate for a tissue biopsy. There are many obvious advantages that make this approach attractive for patients such as its convenience, minimal risk and less expensive collection, and importantly, as it is likely to represent the genetic burden of all lesions, it might provide more complete information than a single lesional biopsy. It also can also be easily performed serially unlike tissue biopsies. Although liquid biopsy includes measurement of proteins, tumour markers, circulating tumour cells, exosomes, tumour educated platelets I will focus solely on circulating tumour DNA (ctDNA).

ctDNA is a promising area for the clinical management of patients with potential roles in screening, diagnosis, prognosis, monitoring and treatment. Although there is increasing adoption of ctDNA in the clinic, this should be tempered with robust assessment to ensure that each test it is truly ready for clinical use. Thus, the analytical performance and validation, the clinical validation, the clinical utility and ethical, legal and social implications, the so called ACCE framework should be applied. Technical considerations from sample, (plasma rather than serum), collection tubes (proprietary that stabilise the ctDNA for days vs standard that need processing within 4hr) and extraction methodologies all have significant influences on the assay performance. Extraction methods should be directed against small fragments as plasma DNA has a peak of ~166 bp with smaller peaks occurring at 10 bp periodicity from cleavage patterns by apoptosis and packing around nucleosomes. The performance of any assay technology itself needs to be fully understood together with the technical parameters and input DNA requirements. These are wide and varied and have their own inherent advantages and disadvantages with differing sensitivities and specificities. As with any technique the assay needs to be able to identify the relevant mutation type whether be SNV, indel, copy number or structural variants, and the sensitivity, specificity (and associated cost of each) of the individual genomic changes taken into account. Emerging evidence shows significant discordance between ctDNA assays and tissue specimens and between the results between different ctDNA assays. Thus, ASCO/CAP to date have taken a conservative view of the clinical utility of these assays and support an algorithm of ctDNA testing in a limited number of clinical circumstances with reflex tissue testing if the ctDNA test is negative. Further work is needed to understand the clinical utility of ctDNA testing. This is becoming more important with the emergence of using ctDNA to acquire ancillary information such as tumour mutational burden and microsatellite instability to determine suitability for immunotherapies or mutational signatures that might help appropriate treatment selection e.g. homologous recombination signatures for PARP inhibitors.