Circulating tumour DNA: A non-invasive biomarker for melanoma

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\u27mar 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 \u3e1.0mm 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

The clinical utility of the Halosperm assay and the development of a simplified method of human semen storage for the testing of sperm DNA fragmentation

Male infertility is typically diagnosed upon routine semen analysis following the World Health Organisation’s (WHO) semen analysis manual. Recent editions of the manual have essentially changed the diagnosis of a semen sample, prompting debate between experts as to which edition should be followed. Deoxyribonucleic Acid (DNA) integrity analysis is proving to be a useful adjunct to semen analysis as 15% of infertile men have a normal semen analysis but they have an increased DNA fragmentation level (DFL) which has been associated with increased disease incidence in any resultant offspring. However, such tests are not endorsed by the WHO, possibly due to a lack of standardisation in the implementation, analysis and clinical interpretation of methods used to evaluate DNA integrity. Improved efficiency of testing is achieved by batch testing or sending samples to a central laboratory for analysis, requiring an effective storage system. Most current protocols for semen storage and related DNA integrity testing are complex, expensive and require specialised equipment. Nevertheless, since the Halosperm® G2 Kit, requires only standard laboratory equipment, a simple, convenient and stable storage method for the purpose of testing sperm DNA fragmentation would be advantageous. DNA has been successfully extracted from air‐dried semen and one particular study has investigated the use of air‐dried semen slides as a method of storage prior to DNA fragmentation testing, however, the effects of time and temperature on the integrity of spermatozoa DNA has not been considered. The first objective of this present study was to investigate the relationship between sperm DNA fragmentation (using the Halosperm® G2 Test Kit) and semen analysis results (measured according to the 4th and 5th Edition WHO semenanalysis manuals) to determine the clinical utility of the Halosperm assay. The second objective was to consider extrinsic effects on the DNA integrity of air‐dried semen in order to develop an alternative storage method for semen prior to DNA fragmentation testing using the Halosperm assay. A retrospective analysis was carried out on 905 consecutive semen samples with 4th and 5th Edition semen analysis and Halosperm result. Pearson correlations, analysis by ANOVA and post‐hoc testing by Tukey’s HSD were used for statistical analysis. Multiple aliquots of semen samples were prepared to achieve fresh, snap frozen and air‐dried samples. Samples were sequentially assessed for sperm DNA fragmentation using the Halosperm® G2 kit (Halotech DNA SL, Spain) and scored against 300 sperm, with fragmentation results ≥30% considered positive. Fragmentation levels were compared between the different protocols. Multiple aliquots of semen samples were then air‐dried to test the fragmentation levels between different slide types, reconstituting fluids, times and temperatures. Pearson’s correlation coefficient and paired t‐tests were used for statistical analysis. In summary, whilst significant associations exist between sperm DNA fragmentation and sexual abstinence, volume of the ejaculate, sperm concentration, normal sperm morphology and sperm motility, the Halosperm assay may provide an explanation for infertility where semen analysis cannot. Furthermore, air‐drying semen is a simple and stable storage method, for up to one month at ‐22 degrees, prior to DNA fragmentation testing with the Halosperm® G2 kit

The relationship between the halosperm assay and semen analysis performed according to the 4th and 5th Editions of the World Health Organization guidelines

Background: As a standard reference to evaluate male factor infertility, the majority of fertility laboratories use the 4th or 5th Editions of the World Health Organization’s semen analysis guidelines. Following the release of the 5th Edition, debate over its legitimacy has resulted in some laboratories using the 4th and others the 5th Edition. DNA integrity tests have been shown to be a valuable adjunct to semen analysis and have subsequently been adopted by many fertility laboratories. This study explored the prevalence of samples with high DNA fragmentation levels according to semen analysis categories using both the 4th and the 5th Edition reference ranges. Materials and Methods: The study included 905 consecutive semen samples from 863 infertile couples attending a fertility clinic. A semen analysis was conducted according to both the 4th and 5th Edition guidelines published by the World Health Organization. DNA damage was assessed using the Halosperm G2 test kit and expressed as a percentage DNA fragmentation level. Results: Alongside both the World Health Organization 4th and 5th Edition semen analysis criteria abnormal DNA fragmentation levels were more common in abnormal semen samples however elevated DNA fragmentation levels were also found in normal semen samples using the same criteria. Of the samples that were graded as normozoospermic according to the 5th Edition guidelines 16% were deemed to have elevated DNA fragmentation levels compared to 11.7% graded by the 4th Edition guidelines. The number of normozoospermic samples, graded according to the 5th Edition guidelines was significantly higher (n=697) than when the same samples were graded according to 4th Edition guidelines (n=385) (p=0.001). A significant proportion of samples with an abnormal DNA fragmentation level corresponding to the World Health Organisation 4th and 5th Edition criteria were evident in normozoospermic (

Improving health professional\u27s knowledge of hepatitis B using cartoon based learning tools: a retrospective analysis of pre and post tests

Background: Hepatitis B serology is complex and a lack of knowledge in interpretation contributes to the inadequate levels of screening and referral for highly effective hepatitis antiviral treatments. This knowledge gap needs to be addressed so that current and future healthcare professionals are more confident in the detection and assessment of hepatitis B to improve the uptake of treatment and reduce long-term complications from the disease. Cartoons have been used effectively as a teaching tool in other settings and were considered as a potentially useful teaching aid in explaining hepatitis B serology. This study examines the impact of cartoons in improving healthcare professionals’ knowledge. Methods: A cartoon based learning tool designed to simplify the complexities of hepatitis B serology was developed as part of an online learning program for medical practitioners, nurses and students in these professions. A retrospective analysis was carried out of pre and post online test results. Results: An average improvement of 96% of correct answers to case study questions in hepatitis B serology was found across all ten questions following the use of an online cartoon based learning tool. Conclusion: The data indicates a significant improvement of participants’ knowledge of hepatitis B serology from pre-test to post-test immediately following an online cartoon based learning tool. However, further research is required to measure its long term impact

Vasculogenic mimicry in malignant mesothelioma: an experimental and immunohistochemical analysis

SummaryVasculogenic mimicry, the process in which cancer cells form angiomatoid structures independent of or in addition to host angiogenesis has been recorded in several otherwise non-endothelial malignant neoplasms. This study describes evidence of routine vascular mimicry by human mesothelioma cell lines in vitro, when the cell lines are cultured alone or co-cultured with human umbilical vascular endothelial cells, with the formation of angiomatoid tubular networks. Vasculogenic mimicry is also supported by immunohistochemical demonstration of human-specific anti-mitochondria antibody labelling of tumour-associated vasculature of human mesothelioma cells xenotransplanted into nude mice, and by evidence of vascular mimicry in some biopsy samples of human malignant mesotheliomas. These studies show mosaic interlacing of cells that co-label or label individually for immunohistochemical markers of endothelial and mesothelial differentiation. If vascular mimicry in mesothelioma can be characterised more fully, this may facilitate identification of more specific and targeted therapeutic approaches such as anti-angiogenesis in combination with chemotherapy and immunotherapy or other therapeutic approaches

Cartoons for e-health informatics

Not only is Hepatitis B serology often misunderstood because of its complex serological implications, but advances in medical science have revolutionised screening and treatment of hepatitis B. To maximise such evolution however, this new information must be relayed effectively and efficiently to current and future medical professionals. Cartoons have been well regarded as a teaching tool in a variety of different settings as is the use of web based technology. Therefore the delivery of a cartoon based learning tool, accessed via on-line learning modules was considered a novel and potentially effective way of disseminating new knowledge. To increase health professionals’ understanding of hepatitis B serology and skill in interpreting the tests that indicate the appropriate treatment, a cartoon series was developed. The cartoons are located on an online educational website and include characters that represent the different antibodies and antigens associated with hepatitis B. The cartoon characters are involved in a series of adventures that represent the various phases of hepatitis B infection, and the paper describes their development. Subsequent research demonstrated that exposure to the online cartoon based learning tool indicates that they are a fun and useful way to increase knowledge

Stopping targeted therapy for complete responders in advanced BRAF mutant melanoma

BRAF inhibitors revolutionised the management of melanoma patients and although resistance occurs, there is a subgroup of patients who maintain durable disease control. For those cases with durable complete response (CR) it is not clear whether it is safe to cease therapy. Here we identified 13 patients treated with BRAF +/− MEK inhibitors, who cease therapy after prolonged CR (median = 34 months, range 20–74). Recurrence was observed in 3/13 (23%) patients. In the remaining 10 patients with sustained CR off therapy, the median follow up after discontinuation was 19 months (range 8–36). We retrospectively measured ctDNA levels using droplet digital PCR (ddPCR) in longitudinal plasma samples. CtDNA levels were undetectable in 11/13 cases after cessation and remained undetectable in patients in CR (10/13). CtDNA eventually became detectable in 2/3 cases with disease recurrence, but remained undetectable in 1 patient with brain only progression. Our study suggests that consideration could be given to ceasing targeted therapy in the context of prolonged treatment, durable response and no evidence of residual disease as measured by ctDNA

Role of serum vascular endothelial growth factor (VEGF) as a potential biomarker of response to immune checkpoint inhibitor therapy in advanced melanoma: results of a pilot study

Background: The development of biomarkers predictive of response to immune checkpoint inhibitor (ICI) therapies in advanced melanoma is an area of great interest in oncology. Our study evaluated the potential role of serum vascular endothelial growth factor (VEGF) as a predictive biomarker of clinical benefit and response to treatment with ICIs. Methods: Pre-treatment peripheral blood samples were obtained from advanced melanoma patients undergoing ICI therapy as monotherapy or in combination at two tertiary care hospitals in Western Australia. Serum VEGF levels were correlated with response to therapy and survival outcomes. Results: Serum VEGF samples were collected from a total of 130 patients treated with ICI therapy (pembrolizumab 73, ipilimumab 15, and ipilimumab/nivolumab combination 42). Median serum VEGF level was significantly higher in the non-responders (82.15 pg/mL) vs. responders (60.40 pg/mL) in the ipilimumab monotherapy cohort (P \u3c 0.0352). However, no difference was seen in VEGF levels between non-responders and responders in pembrolizumab and ipilimumab/nivolumab treated patients. Conclusions: The results of our study confirm previous observations that that high pre-treatment serum VEGF levels in advanced melanoma patients may predict poor response to ipilimumab. However, serum VEGF is not predictive of outcome in patients treated with anti-PD-1 agents alone or in combination with ipilimumab

Correlation between circulating tumour DNA and metabolic tumour burden in metastatic melanoma patients

Background: Circulating tumour DNA (ctDNA) may serve as a measure of tumour burden and a useful tool for non-invasive monitoring of cancer. However, ctDNA is not always detectable in patients at time of diagnosis of metastatic disease. Therefore, there is a need to understand the correlation between ctDNA levels and the patients\u27 overall metabolic tumour burden (MTB). Methods: Thirty-two treatment naïve metastatic melanoma patients were included in the study. MTB and metabolic tumour volume (MTV) was measured by 18F-fluoro-D-glucose positron emission tomography/computed tomography (FDG PET/CT). Plasma ctDNA was quantified using droplet digital PCR (ddPCR). Results: CtDNA was detected in 23 of 32 patients. Overall, a significant correlation was observed between ctDNA levels and MTB (

PD-L1 expression on circulating tumor cells may be predictive of response to Pembrolizumab in advanced melanoma: Results from a pilot study

BACKGROUND: PD-1 inhibitors are routinely used for the treatment of advanced melanoma. This study sought to determine whether PD-L1 expression on circulating tumor cells (CTCs) can serve as a predictive biomarker of clinical benefit and response to treatment with the PD-1 inhibitor pembrolizumab. METHODS: Blood samples were collected from patients with metastatic melanoma receiving pembrolizumab, prior to treatment and 6-12 weeks after initiation of therapy. Multiparametric flow cytometry was used to identify CTCs and evaluate the expression of PD-L1. RESULTS: CTCs were detected in 25 of 40 patients (63%). Patients with detectable PD-L1 CONCLUSION: Our results reveal the potential of CTCs as a noninvasive real-time biopsy to evaluate PD-L1 expression in patients with melanoma. PD-L1 expression on CTCs may be predictive of response to pembrolizumab and longer PFS. IMPLICATIONS FOR PRACTICE: The present data suggest that PD-L1 expression on circulating tumor cells may predict response to pembrolizumab in advanced melanoma. This needs further validation in a larger trial and, if proven, might be a useful liquid biopsy tool that could be used to stratify patients into groups more likely to respond to immunotherapy, hence leading to health cost savings