The role of cell-free circulating tumour DNA for detection of molecular residual disease and recurrence in head and neck cancer

Despite intensive therapies and improved treatment options, many patients with head and neck squamous cell carcinoma (HNSCC) develop tumour recurrences, metastases or a second primary tumour, so that the 5-year survival is still less than 50%. Treatment of HNSCC is mainly multimodal and surgical tumour excision is a crucial pillar of treatment in many cases. In order to achieve better treatment outcomes, new diagnostic and therapeutic approaches are needed that can benefit from the identification of new and personalised biomarkers.¹ The use of liquid biopsy to detect tumour cells and nucleic acids, proteins or metabolites is becoming increasingly important in this regard.^2,3 These are minimally invasive diagnostic procedures in which a wide variety of bioanalytes can be isolated and analysed from body fluids such as blood, urine, saliva or cerebrospinal fluid. These analytes include circulating tumour cells and circulating nucleic acids (including circulating tumour DNA (ctDNA), the tumour-derived fraction of cell-free DNA (cfDNA) in plasma) among others.

A single blood sample can contain a range of cell types and cell products originating from multiple tumour sites throughout the body, allowing both tumour burden and clonal evolution of disease to be investigated.⁴ In particular, the analysis of ctDNA as a potential biomarker for tumour detection and monitoring has become increasingly important for routine clinical practice. Until now, a limiting factor for the detection of genetic alterations in liquid biopsies has been the low proportion of tumour DNA, which is often less than 1% of the total circulating DNA. However, technological advances in recent years have significantly increased the sensitivity of various techniques.⁵ Primarily, next generation sequencing technologies are used to identify and evaluate tumour-specific point mutations, insertions, deletions, amplifications and translocations.^6–10

The advantage of a liquid biopsy is that it gives a continuous and representative insight into the genetic make-up of the primary tumour, but also its possible metastases, and, in contrast to tissue biopsies, it can be taken repeatedly without great effort or risk to the patient. A direct biopsy of the tumour tissue, on the other hand, is invasive and, as a snapshot of a single region, does not always take into account intratumoural heterogeneity, i.e. the coexistence of different cellular subclones, and the associated presence of possible mutations beneficial to the survival of the tumour cells. It has previously been shown that high levels of ctDNA correlate with shorter overall survival and tumour stage,¹¹ allowing ctDNA to be used to determine tumour burden and assess prognosis.^12,13 Monitoring of treatment response and tumour heterogeneity, potential development of resistance and possible early detection of recurrence or a second primary tumour are also among the core applications of ctDNA-based diagnostics for personalised treatment.

In collaboration with Inivata Ltd. our group at the Department of Otorhinolaryngology, Head and Neck Surgery at the Hospital of the Ludwig-Maximilians-University Munich, Germany, set up the Liquid BIOpsy for MiNimal RESidual DiSease Detection in Head and Neck Squamous Cell Carcinoma (LIONESS) study, an ongoing single-centre prospective cohort study of currently 17 patients with p16-negative HNSCC who had received primary surgical treatment with curative intent (Figure 1). We collected blood samples pre-operatively post-operatively, before start of adjuvant therapy (if any) and at follow-up visits and used RaDaR^TM, a highly sensitive personalised ctDNA assay, to monitor for molecular residual disease or recurrence. The aims of this study were firstly to determine whether post-operative ctDNA detection can act as a biomarker for surgical tumour clearance. Secondly, to evaluate the potential of personalised ctDNA analysis for early molecular-level detection of relapse prior to clinically confirmed recurrence. Whole exome sequencing (WES) was performed on FFPE tissue obtained from the primary tumours. For each patient tumour, up to 48 tumour-specific variants for RaDaR^TM assay design were selected to analyse plasma samples for evidence of minimal residual disease or recurrence. Variants were verified by deep sequencing of tumour tissue DNA and matched buffy coat DNA was sequenced to identify confounding clonal haematopoiesis of indeterminate potential (CHIP) mutations. In 5 case studies illustrating the use of ctDNA analysis in more detail (example shown in Figure 2), we demonstrate how ctDNA detection could have been applied to aid therapy planning and clinical decision-making. In post-surgery samples, we were able to detect ctDNA at levels as low as 0.0006% variant allele frequency. In all cases with clinical recurrence to date, ctDNA was detected prior to progression, with lead times, i.e. the interval between detection of the first ctDNA positive sample post-surgery and confirmation of clinical recurrence, ranging from 108 to 253 days. Limitations of our study include the relatively small number of patients (n=17) enrolled to date as well as the still on-going follow-up with the shortest follow-up length amounting to 10 months. However, a strength of our study includes the use of a personalised ctDNA assay design that is highly sensitive and specific and can be applied to all patients with available WES data.

In conclusion, our study illustrates the potential of ctDNA as a biomarker for monitoring of molecular residual disease as well as recurrence in patients with HNSCC and demonstrates the feasibility of personalised ctDNA assays for detection of disease post-treatment and with consequences for further therapy planning. Using such minimally invasive liquid biopsy-based tests, the use of burdensome and costly diagnostic tools such as CT or PET-CT for early detection of recurrences could be reduced in the future, enabling timely interventions, and significantly improving longitudinal tumour therapy and thus the quality of life of patients with head and neck cancer. 

In future, ctDNA-guided trials will need to further evaluate the role of ctDNA assays in clinical practice. This may especially be of relevance for the development of personalised treatment strategies for detection of molecular residual disease or recurrence in patients with early-stage HNSCC where adjuvant treatment may be considered optional.

References

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