Detection of clinical progression through plasma ctDNA in metastatic melanoma patients: a comparison to radiological progression.

BACKGROUND: The validity of circulating tumour DNA (ctDNA) as an indicator of disease progression compared to medical imaging in patients with metastatic melanoma requires detailed evaluation. METHODS: Here, we carried out a retrospective ctDNA analysis of 108 plasma samples collected at the time of disease progression. We also analysed a validation cohort of 66 metastatic melanoma patients monitored prospectively after response to systemic therapy. RESULTS: ctDNA was detected in 62% of patients at the time of disease progression. For 67 patients that responded to treatment, the mean ctDNA level at progressive disease was significantly higher than at the time of response (P < 0.0001). However, only 30 of these 67 (45%) patients had a statistically significant increase in ctDNA by Poisson test. A validation cohort of 66 metastatic melanoma patients monitored prospectively indicated a 56% detection rate of ctDNA at progression, with only two cases showing increased ctDNA prior to radiological progression. Finally, a correlation between ctDNA levels and metabolic tumour burden was only observed in treatment naïve patients but not at the time of progression in a subgroup of patients failing BRAF inhibition (N = 15). CONCLUSIONS: These results highlight the low efficacy of ctDNA to detect disease progression in melanoma when compared mainly to standard positron emission tomography imaging.

The Prognostic Impact of Circulating Tumour DNA in Melanoma Patients Treated with Systemic Therapies-Beyond BRAF Mutant Detection.

In this study, we evaluated the predictive value of circulating tumour DNA (ctDNA) to inform therapeutic outcomes in metastatic melanoma patients receiving systemic therapies. We analysed 142 plasma samples from metastatic melanoma patients prior to commencement of systemic therapy: 70 were treated with BRAF/MEK inhibitors and 72 with immunotherapies. Patient-specific droplet digital polymerase chain reaction assays were designed for ctDNA detection. Plasma ctDNA was detected in 56% of patients prior to first-line anti-PD1 and/or anti-CTLA-4 treatment. The detection rate in the immunotherapy cohort was comparably lower than those with BRAF inhibitors (76%, p = 0.0149). Decreasing ctDNA levels within 12 weeks of treatment was strongly concordant with treatment response (Cohen's k = 0.798, p < 0.001) and predictive of longer progression free survival. Notably, a slower kinetic of ctDNA decline was observed in patients treated with immunotherapy compared to those on BRAF/MEK inhibitors. Whole exome sequencing of ctDNA was also conducted in 9 patients commencing anti-PD-1 therapy to derive tumour mutational burden (TMB) and neoepitope load measurements. The results showed a trend of high TMB and neoepitope load in responders compared to non-responders. Overall, our data suggest that changes in ctDNA can serve as an early indicator of outcomes in metastatic melanoma patients treated with systemic therapies and therefore may serve as a tool to guide treatment decisions.

Detection of BRAF splicing variants in plasma-derived cell-free nucleic acids and extracellular vesicles of melanoma patients failing targeted therapy therapies.

The analysis of plasma circulating tumour nucleic acids provides a non-invasive approach to assess disease burden and the genetic evolution of tumours in response to therapy. BRAF splicing variants are known to confer melanoma resistance to BRAF inhibitors. We developed a test to screen cell-free RNA (cfRNA) for the presence of BRAF splicing variants. Custom droplet digital PCR assays were designed for the detection of BRAF splicing variants p61, p55, p48 and p41 and then validated using RNA from cell lines carrying these variants. Evaluation of plasma from patients with reported objective response to BRAF/MEK inhibition followed by disease progression was revealed by increased circulating tumour DNA (ctDNA) in 24 of 38 cases at the time of relapse. Circulating BRAF splicing variants were detected in cfRNA from 3 of these 38 patients; two patients carried the BRAF p61 variant and one the p55 variant. In all three cases the presence of the splicing variant was apparent only at the time of progressive disease. BRAF p61 was also detectable in plasma of one of four patients with confirmed BRAF splicing variants in their progressing tumours. Isolation and analysis of RNA from extracellular vesicles (EV) from resistant cell lines and patient plasma demonstrated that BRAF splicing variants are associated with EVs. These findings indicate that in addition to plasma ctDNA, RNA carried by EVs can provide important tumour specific information.

Circulating Tumor DNA Predicts Outcome from First-, but not Second-line Treatment and Identifies Melanoma Patients Who May Benefit from Combination Immunotherapy.

PURPOSE: We evaluated the predictive value of pretreatment ctDNA to inform therapeutic outcomes in patients with metastatic melanoma relative to type and line of treatment. EXPERIMENTAL DESIGN: Plasma circulating tumor DNA (ctDNA) was quantified in 125 samples collected from 110 patients prior to commencing treatment with immune checkpoint inhibitors (ICIs), as first- (n = 32) or second-line (n = 27) regimens, or prior to commencing first-line BRAF/MEK inhibitor therapy (n = 66). An external validation cohort included 128 patients commencing ICI therapies in the first- (N = 77) or second-line (N = 51) settings. RESULTS: In the discovery cohort, low ctDNA (≤20 copies/mL) prior to commencing therapy predicted longer progression-free survival (PFS) in patients treated with first-line ICIs [HR, 0.20; 95% confidence interval (CI) 0.07-0.53; P < 0.0001], but not in the second-line setting. An independent cohort validated that ctDNA is predictive of PFS in the first-line setting (HR, 0.42; 95% CI, 0.22-0.83; P = 0.006), but not in the second-line ICI setting. Moreover, ctDNA prior to commencing ICI treatment was not predictive of PFS for patients pretreated with BRAF/MEK inhibitors in either the discovery or validation cohorts. Reduced PFS and overall survival were observed in patients with high ctDNA receiving anti-PD-1 monotherapy, relative to those treated with combination anti-CTLA-4/anti-PD-1 inhibitors. CONCLUSIONS: Pretreatment ctDNA is a reliable indicator of patient outcome in the first-line ICI treatment setting, but not in the second-line ICI setting, especially in patients pretreated with BRAF/MEK inhibitors. Preliminary evidence indicated that treatment-naïve patients with high ctDNA may preferentially benefit from combined ICIs.

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 < 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.

Longitudinal Monitoring of ctDNA in Patients with Melanoma and Brain Metastases Treated with Immune Checkpoint Inhibitors.

PURPOSE: Brain involvement occurs in the majority of patients with metastatic melanoma. The potential of circulating tumor DNA (ctDNA) for surveillance and monitoring systemic therapy response in patients with melanoma brain metastases merits investigation. EXPERIMENTAL DESIGN: This study examined circulating BRAF, NRAS, and c-KIT mutations in patients with melanoma with active brain metastases receiving PD-1 inhibitor-based therapy. Intracranial and extracranial disease volumes were measured using the sum of product of diameters, and response assessment performed using RECIST. Longitudinal plasma samples were analyzed for ctDNA over the first 12 weeks of treatment (threshold 2.5 copies/mL plasma). RESULTS: Of a total of 72 patients, 13 patients had intracranial metastases only and 59 patients had concurrent intracranial and extracranial metastases. ctDNA detectability was 0% and 64%, respectively, and detectability was associated with extracranial disease volume (P < 0.01). Undetectable ctDNA on-therapy was associated with extracranial response (P < 0.01) but not intracranial response. The median overall survival in patients with undetectable (n = 34) versus detectable (n = 38) ctDNA at baseline was 39.2 versus 10.6 months [HR, 0.51; 95% confidence interval (CI), 0.28-0.94; P = 0.03] and on-therapy was 39.2 versus 9.2 months (HR, 0.32; 95% CI, 0.16-0.63; P < 0.01). CONCLUSIONS: ctDNA remains a strong prognostic biomarker in patients with melanoma with brain metastases, especially in patients with concurrent extracranial disease. However, ctDNA was not able to detect or monitor intracranial disease activity, and we recommend against using ctDNA as a sole test during surveillance and therapeutic monitoring in patients with melanoma.

Monitoring melanoma recurrence with circulating tumor DNA: a proof of concept from three case studies.

BACKGROUND: A significant number of melanoma patients experience recurrence to distant sites, despite having had surgical treatment of the primary lesion, with curative intent. Monitoring of patients for early evidence of disease recurrence would significantly improve management of the disease, allowing timely therapeutic intervention. Circulating tumor DNA (ctDNA) is becoming a well-recognized biomarker for monitoring malignancies and has, in a few studies, been shown to signify disease recurrence earlier than conventional methods. METHODS: We performed a retrospective analysis of plasma ctDNA using droplet digital PCR (ddPCR) in 30 primary melanoma patients with tumors harboring BRAF, NRAS or TERT promoter mutations. Mutant specific ctDNA, measured during clinical disease course, was compared with disease status in patients with confirmed disease recurrence (n = 3) and in those with no evidence of disease recurrence (n = 27). RESULTS: Mutant specific ctDNA was detected in all three patients with disease recurrence at the time of clinically confirmed progression. In one case, plasma ctDNA detection preceded clinical identification of recurrence by an interval of 4 months. CtDNA was not detected in patients who were asymptomatic and had no radiological evidence of recurrence. CONCLUSIONS: This study demonstrates promising results for the use of ctDNA as an informative monitoring tool for melanoma patients having undergone tumor resection of an early stage primary tumor. The clinical utility of ctDNA for monitoring disease recurrence warrants investigation in prospective studies as it may improve patient outcome.

Locus-specific concordance of genomic alterations between tissue and plasma circulating tumor DNA in metastatic melanoma.

Circulating tumor DNA (ctDNA) may serve as a surrogate to tissue biopsy for noninvasive identification of mutations across multiple genetic loci and for disease monitoring in melanoma. In this study, we compared the mutation profiles of tumor biopsies and plasma ctDNA from metastatic melanoma patients using custom sequencing panels targeting 30 melanoma-associated genes. Somatic mutations were identified in 20 of 24 melanoma biopsies, and 16 of 20 (70%) matched-patient plasmas had detectable ctDNA. In a subgroup of seven patients for whom matching tumor tissue and plasma were sequenced, 80% of the mutations found in tumor tissue were also detected in ctDNA. However, TERT promoter mutations were only detected by ddPCR, and promoter mutations were consistently found at lower concentrations than other driver mutations in longitudinal samples. In vitro experiments revealed that mutations in promoter regions of TERT and DPH3 are underrepresented in ctDNA. While the results underscore the utility of using ctDNA as an alternative to tissue biopsy for genetic profiling and surveillance of the disease, our study highlights the underrepresentation of promoter mutations in ctDNA and its potential impact on quantitative liquid biopsy applications.

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' 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 (p < 0.001). CtDNA was not detectable in patients with an MTB of ≤10, defining this value as the lower limit of tumour burden that can be detected through ctDNA analysis by ddPCR. CONCLUSIONS: We showed that ctDNA levels measured by ddPCR correlate with MTB in treatment naïve metastatic melanoma patients and observed a limit in tumour size for which ctDNA cannot be detected in blood. Nevertheless, our findings support the use of ctDNA as a non-invasive complementary modality to functional imaging for monitoring tumour burden.

Droplet Digital PCR for Mutation Detection in Formalin-Fixed, Paraffin-Embedded Melanoma Tissues: A Comparison with Sanger Sequencing and Pyrosequencing.

The identification of somatic mutations is crucial for guiding therapeutic decisions about personalized melanoma treatment. However, genetic analysis of tumors is usually performed on limited and often low-quality DNA from tumors with low tumor cellularity and high tumor heterogeneity. Different mutation-detection platforms exist, with varying analytical sensitivities. Here we evaluated the detection of common mutations in BRAF, NRAS, and TERT promoter in 40 melanoma FFPE tissues using Droplet Digital (dd)PCR, and compared the results to the detection rates obtained by Sanger sequencing and pyrosequencing. The cellularity of tumors analyzed ranged from 5% to 50% (n = 28) and 50% to 90% (n = 12). Overall, droplet digital (dd)PCR was more sensitive, detecting mutations in 12.5% and 23% of tumors deemed as wild-type by pyrosequencing and Sanger sequencing, respectively. The increased sensitivity of ddPCR was more apparent among tumors with <50% tumor cellularity. Implementation of ddPCR-based assays may facilitate analysis of early-stage tumors and support research into improving outcomes in melanoma patients.

Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma.

BACKGROUND: Currently mainly BRAF mutant circulating tumor DNA (ctDNA) is utilized to monitor patients with melanoma. TERT promoter mutations are common in various cancers and found in up to 70% of melanomas, including half of BRAF wild-type cases. Therefore, a sensitive method for detection of TERT promoter mutations would increase the number of patients that could be monitored through ctDNA analysis. METHODS: A droplet digital PCR (ddPCR) assay was designed for the concurrent detection of chr5:1,295,228 C>T and chr5:1,295,250 C>T TERT promoter mutations. The assay was validated using 39 melanoma cell lines and 22 matched plasma and tumor samples. In addition, plasma samples from 56 metastatic melanoma patients and 56 healthy controls were tested for TERT promoter mutations. RESULTS: The established ddPCR assay detected TERT promoter mutations with a lower limit of detection (LOD) of 0.17%. Total concordance was demonstrated between ddPCR and Sanger sequencing in all cell lines except one, which carried a second mutation within the probe binding-site. Concordance between matched plasma and tumor tissue was 68% (15/22), with a sensitivity of 53% (95% CI, 27%-79%) and a specificity of 100% (95% CI, 59%-100%). A significantly longer PFS (p=0.028) was evident in ctDNA negative patients. Importantly, our TERT promoter mutations ddPCR assay allowed detection of ctDNA in 11 BRAF wild-type cases. CONCLUSIONS: The TERT promoter mutation ddPCR assay offers a sensitive test for molecular analysis of melanoma tumors and ctDNA, with the potential to be applied to other cancers.

Improving health Professional's 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.