Date of Award
Doctor of Philosophy
School of Medical and Health Sciences
Professor Melanie Ziman
Dr Elin Gray
Cutaneous melanoma accounts for 90% of all skin cancer deaths (Balch et al., 2010) and is responsible for 3.6% of deaths from cancer in Australia (Australian Institute of Health and Welfare, 2016). Whilst early detection and successful surgical removal of primary melanomas have improved survival rates (DeSantis et al., 2014), approximately 30% of these patients will have disease recurrence at some point in their lives (Soong et al., 1992; Soong et al., 1998). This is despite being considered disease free following treatment, which may have included surgical removal of the primary and/or its metastasis/es, radiation and/or systemic therapy. Whilst the risk of melanoma recurrence may correlate to some extent with the stage of the primary melanoma in terms of its size and thickness and whether it has metastasised (Shaw et al., 1987; Soong et al., 1992; Soong et al., 1998), recurrences occur even after thin melanomas (associated with low-risk for recurrence) that have been completely excised (Dalal et al., 2007; Jones et al., 2013; Leiter et al., 2012; Meier et al., 2002; Salama et al., 2013; Soong et al., 1998). Melanoma may recur at any point in time, even 10 or more years after a primary melanoma has been excised (Crowley et al., 1990; Dong et al., 2000; Hohnheiser et al., 2011; Kalady et al., 2003; Tsao et al., 1997). Recurrences may present in the same or in areas adjacent to the primary melanoma, however the majority of recurrences appear in lymph nodes or other organs, at which point the disease is among the most aggressive and treatment-resistant of all human cancers (Kenessey et al., 2012; Luke et al., 2017; Mocellin et al., 2013; Sanmamed et al., 2015; Ti'mar et al., 2013). In the metastatic setting, resective surgery of solitary metastases is associated with the most favourable outcome (Chua et al., 2010; Petersen et al., 2007; Sanki et al., 2009; Wasif et al., 2011), however systemic therapy options are dramatically improving survival of patients with unresectable metastases (Garbe et al., 2016). Overall, the greatest treatment efficacy is associated with a low disease burden at time of therapy (Hodi et al., 2010; Luke et al., 2017; McArthur et al., 2016; Sosman et al., 2011) and therefore early detection of melanoma recurrence is critical for improved survival.
To date, there are no reliable early markers of melanoma recurrence. Radiological imaging techniques and sentinel lymph node (SLN) biopsies (SLNB) are currently the methods employed to stage primary melanomas and detect metastases. Positron emission tomography (PET) with a labelled glucose analogue fluorine 18 fluorodeoxyglucose (18F-FDG) combined with computed tomography (CT) scans (FDG-PET/CT), are used routinely to determine disease burden. These have limited sensitivity however for the detection of early stage melanoma micro-metastases (Meyers et al., 2009; Pfannenberg et al., 2015), thus cannot provide timely clinical evidence of disease recurrence (Belhocine et al., 2002; Hindié et al., 2011; Krug et al., 2008). Fluorine 18 fluorodeoxyglucose Positron Emission Tomography combined with Computed Tomography (FDGPET/ CT) may be used routinely for monitoring of melanoma patients at high risk of disease recurrence, but it is expensive (Gellén et al., 2015) and subjects patients to excessive radiation exposure (Rueth et al., 2015). Whilst routine SLNBs offer a survival advantage in monitoring recurrence in patients with >1.0mm thick melanomas (Faries et al., 2017; Morton et al., 2014), they are relatively invasive for routine monitoring (Agnese et al., 2003; Lens et al., 2002). Early stage melanoma patients who are considered disease free and are not at high risk for a recurrence, are not routinely assessed by SLNB, or PET/CT or LNB, but rather by physical examinations (Australian Cancer Network Melanoma Guidelines Revision Working Party, 2008). Thus, an additional monitoring regime that can be performed regularly and in conjunction with physical examinations could lead to timely interventions resulting in improved treatment options that will positively impact on the patient’s quality of life and survival.
The detection and analysis of mutant specific circulating tumour DNA (ctDNA) is an emerging tool for detection of residual disease and for prognosis and monitoring of different cancers (Bettegowda et al., 2014; Dawson et al., 2013; Gray et al., 2015; Spindler et al., 2012). There is however, limited use of ctDNA for monitoring of residual disease and recurrence in clinically disease free patients v (Oshiro et al., 2015; Tie et al., 2016) and to date, this has not been assessed in melanoma. In melanoma, mainly V-raf murine sarcoma viral oncogene homolog B1 (BRAF) and to some extent, neuroblastoma RAS viral oncogene (NRAS) mutant ctDNA are utilised to monitor patients during therapy in the research setting (Ascierto et al., 2013a; Girotti et al., 2015; Gray et al., 2015; Sanmamed et al., 2015; Santiago-Walker et al., 2015). Notably, telomerase reverse transcriptase (TERT) promoter mutations are present in 50-70% of melanomas and confer a significantly poorer prognosis if found concurrently with BRAF or NRAS mutations relative to the occurrence of each mutation alone. Thus, the ability to monitor patients at all disease stages for the presence of BRAF, NRAS as well as TERT mutant ctDNA, would be advantageous even in BRAF and NRAS wild-type patients.
The overall aim of this thesis was to further develop existing tools that could regularly, inexpensively and non-invasively monitor melanoma patients for melanoma recurrence. Firstly, we focused on increasing the number of patients that could be monitored through ctDNA analysis. To do this we developed a new and innovative ddPCR TERT mutation assay and investigated its sensitivity alongside current assays in detecting mutations in melanoma tissue containing a small fraction of tumour cells. The significance of ctDNA for patient monitoring relative to current methods of clinical monitoring was then investigated in relation to melanoma recurrence. Finally, we conducted a retrospective analysis of ctDNA levels relative to metabolic tumour burden (MTB) derived from FDG-PET/CT to determine the lower limit of disease burden detectable by ctDNA using ddPCR.
In the first study of this thesis, a novel droplet digital PCR (ddPCR) assay for the concurrent detection of C228T and C250T TERT promoter mutations was designed and developed to display a lower limit of detection (LOD) of 0.17%. The assay was validated using 22 matched plasma and vi tumour samples and showed a 68% concordance rate, with a sensitivity of 53% (95% CI, 27%- 79%) and a specificity of 100% (95% CI, 59%-100%). Plasma samples from 56 metastatic melanoma patients and 56 healthy controls were tested for TERT promoter mutations confirming a specificity of 100% (95% CI, 94%-100%). Importantly, we not only detected TERT mutant specific ctDNA in 4 BRAF mutant cases, but this assay allowed ctDNA quantification in 11 BRAF wild-type cases, which allows for an increased number of patients to be monitored using ctDNA.
To monitor patients for recurrence using ctDNA, the mutational profile must first be determined from a patient’s tumour. However, this may be difficult to obtain from tumours that have limited and/or low tumour cellularity and high heterogeneity, particularly when sourced from SLNB and fine needle aspiration biopsies of metastatic sites. Consequently, only limited, low-quality DNA may be isolated for use on different mutation detection platforms, each with varying analytical sensitivities. Limited previous studies focused predominantly on assessment of the BRAF V600 mutation (as the only actionable mutation), and, notably, in tumour samples with more than 50% cellularity. Given the prevalence of TERT promoter mutations which, together with BRAF and NRAS mutations provide prognostic significance, the ability to assess the presence of such mutations in patient tumours, at high sensitivity, would dramatically improve assessment of mutations. In the second study presented here, we evaluated the sensitivity of detection of BRAF, NRAS and TERT promoter mutations in 40 melanoma tissues, using ddPCR relative to Sanger sequencing and pyrosequencing. Tumour cellularity in our samples ranged from 5-50% (n=28) and 50-90% (n=12). Overall, ddPCR was the most sensitive, detecting one of the tested hotspot mutations in a total of 77.5% (31 of 40) of cases, including in 12.5% and 23% of samples deemed as wild-type by pyrosequencing and Sanger sequencing, respectively. The ddPCR sensitivity was particularly apparent among samples with less than 50% tumour cellularity. Therefore, implementation of ddPCR based assays could facilitate mutation detection of early stage tumours and support research aimed at using ctDNA to improve early detection of residual disease and disease recurrence or progression.
In the third paper presented here, we assessed the sensitivity of ctDNA to detect disease recurrence. A cohort of 139 patients diagnosed with AJCC stages 0-III in the preceding 10 years were enrolled in the study between January 2015 and February 2017. A blood sample was collected at enrolment and on average 11 months thereafter. Patients were followed up for disease progression for a median time of 50.2 months. From the remaining cohort, three patients developed metastatic disease. The median follow-up from diagnosis of the primary tumour to stage IV disease was 34.4 months. The remaining patients had no clinical evidence of disease recurrence at last follow-up or at death from other causes. We analysed the primary tumour of 37 patients for mutations in BRAF, NRAS and TERT, and identified mutations in 30 patients (three patients with recurrence and 27 patients without recurrence). Using our proven, highly sensitive ddPCR tests we analysed BRAF, NRAS and TERT promoter mutated ctDNA in all available blood samples. Three serial plasma samples were available for each of the three patients who had recurred. CtDNA was detected at the time of radiological or biopsy confirmation of metastases in all three patients. Moreover, ctDNA was detectable in earlier plasma samples from one of the three patients; in this one patient, ctDNA was detected four months prior to clinical detection of gastric and ileum metastases by gastroscopy and biopsy. We detected no mutant specific ctDNA at any time point in the patients without recurrence. Whilst this data is limited because of the limited number of patients and the limited rates of recurrence in early disease stages (2.15%), it provides proof of concept that ctDNA may be a valuable tool to monitor early disease recurrence. Additionally, our assessments were limited by our knowledge of the level of sensitivity of the ctDNA analyses. There was therefore, a robust need to understand the correlation between ctDNA levels and the patient’s tumour burden as assessed by metabolic activity using PET.
Given that the metabolic activities of tumours are measured routinely during clinical disease monitoring by assessment of FDG uptake using PET/CT (Larson et al., 1999), we hypothesised that if ctDNA levels correlate with metabolic tumour burden (MTB) derived from FDG-PET/CT scans in melanoma patients, we could determine the limit of detection (LOD) of ctDNA to signify disease recurrence which would indicate the limitations of ctDNA as a biomarker to identify low disease burden. Thus, the indications of ctDNA in the clinical setting will be more clearly identified OR, the need to improve the sensitivity of ctDNA is therefore apparent. Consequently, in the fourth paper of this thesis, we conducted a retrospective analysis of the ctDNA levels in 32 stage IV melanoma patients with active disease prior to systemic therapy. Corresponding FDG-PET/CT scans were examined and the MTB was determined from metabolic tumour volume (MTV) and tumour lesion glycolysis (TLG) (Larson et al., 1999; Winther-Larsen et al., 2017). Within this cohort of patients, ctDNA was detected in 72% of cases with the number of mutated copies per mL of plasma ranging from 1.6 to 52,440. A significant correlation between the MTB and allele frequency was found (P
Overall, ctDNA tests were developed to monitor TERT promoter mutations in cell free DNA (cfDNA) in addition to those currently available for BRAF and NRAS therefore maximising the number of patients whose disease status can be monitored using ctDNA. We also demonstrated that ddPCR is a highly sensitive method for detection of BRAF, NRAS and TERT promoter mutations in tumour tissue. Using these tests, we identified a strong correlation between the level of ctDNA and metabolic tumour burden, suggesting, for the first time in melanoma, that ctDNA reflects melanoma disease burden. We also detected ctDNA in early stage melanoma patients that suffered disease recurrence. Prospective studies are now warranted to serially assess the amount of ctDNA after resective surgery to determine if the presence of ctDNA can detect residual disease, and whether ix rising levels of ctDNA in the blood can detect disease recurrence earlier than current clinical methods. This will ultimately provide a sensitive method with which to monitor patients, to ensure timely, earlier interventions thereby improving melanoma survival rates.
Access to Chapters 3, 4 and 5 of this thesis is not available.
McEvoy, A. C. (2018). Circulating tumour DNA: A non-invasive biomarker for melanoma. https://ro.ecu.edu.au/theses/2064
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