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Background: Immune checkpoint inhibition (ICI) has led to unprecedented outcomes for melanoma patients but is associated with toxicity. ICI resumption after high grade irAEs poses a significant challenge in the clinical management of melanoma patients and there are no biomarkers that can help identify patients that might benefit from resuming treatment. This study aims to determine if circulating tumor DNA (ctDNA) levels at the time of treatment-limiting irAE could guide treatment decisions in this clinical context., Methods: This is a retrospective exploratory biomarker study from 34 patients treated with combination ICI for stage IV melanoma. Patients had a treatment-limiting toxicity and a baseline plasma collection prior to commencing ICI and within 6 weeks of stopping therapy. Blood samples were tested for ctDNA at baseline and cessation therapy., Results: Median progression free survival (PFS) and overall survival (OS) have not been reached (24-month PFS rate 54% and OS rate 72.3%). PD occurred in 47% (16/34) of patients. Median PFS with detectable ctDNA from plasma collected at the time of toxicity was 6.5 months while not reached (NR) with undetectable levels (HR: 4.0, 95% CI 0.95-17.5, p=0.0023). Median OS with detectable ctDNA at cessation for toxicity was 19.4 months and NR for undetectable ctDNA (HR: 3.9, 95%CI 20.8-18.6, p=0.024). Positive ctDNA at the time of cessation was highly specific (specificity 0.94, 95% CI 0.74-0.99, PPV 0.88, 95% CI 0.53-0.99). However, ctDNA negativity has low sensitivity as a predictor of ongoing disease control (sensitivity 0.437, 95% CI 0.23-0.67). Notably, 4/9 (44%) ctDNA negative patients who had disease progression had brain only disease progression., Conclusions: Undetectable ctDNA and CR on imaging after stopping immunotherapy for toxicity results in high rates of long-term durable control. For patients with immunotherapy related toxicity, who have persistent ctDNA at 8 - 12 weeks, the risk of disease progression is significant., Competing Interests: LW is an Advisory Board Member for Bristol Myers Squibb, AstraZeneca, Roche, Merck Sharp and Dohme and Novartis and has received travel/accommodation support from Pierre Fabre, Merck Sharp & Dohme and Bristol Myers Squibb. MM is an Advisory Board Member melanoma or immuno-oncology for Bristol Myers Squibb, AstraZeneca, Roche, and Merck Sharp & Dohme and has received travel support from Merck Sharp & Dohme, AstraZeneca, and Bristol Myers Squibb. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Warburton, Reid, Amanuel, Calapre, Millward and Gray.)

Background: Approximately 50% of uveal melanoma (UM) patients will develop metastatic disease depending on the genetic features of the primary tumour. Patients need 3-12 monthly scans, depending on their prognosis, which is costly and often non-specific. Circulating tumour DNA (ctDNA) quantification could serve as a test to detect and monitor patients for early signs of metastasis and therapeutic response., Methods: We assessed ctDNA as a biomarker in three distinct UM cohorts using droplet-digital PCR: (A) a retrospective analysis of primary UM patients to predict metastases; (B) a prospective analysis of UM patients after resolution of their primary tumour for early detection of metastases; and (C) monitoring treatment response in metastatic UM patients., Results: Cohort A: ctDNA levels were not associated with the development of metastases. Cohort B: ctDNA was detected in 17/25 (68%) with radiological diagnosis of metastases. ctDNA was the strongest predictor of overall survival in a multivariate analysis (HR = 15.8, 95% CI 1.7-151.2, p = 0.017). Cohort C: ctDNA monitoring of patients undergoing immunotherapy revealed a reduction in the levels of ctDNA in patients with combination immunotherapy., Conclusions: Our proof-of-concept study shows the biomarker feasibility potential of ctDNA monitoring in for the clinical management of uveal melanoma patients., (© 2023. The Author(s).)

Circulating tumour cells (CTCs) are heterogenous and contain genetic information from the tumour of origin. They bear specific intra- and extra-cellular protein markers aiding in their detection. However, since these markers may be shared with other rare cells in the blood, only genetic testing can confirm their malignancy. Herein, we analyse different CTC subsets using single cell whole genome DNA sequencing to validate their malignant origin. We randomly selected putative CTCs identified by immunostaining that were isolated from 4 patients with high grade serous ovarian cancer (HGSOC) and one with benign cystadenoma. We specifically targeted CTCs positive for epithelial (CK/EpCAM pos ), mesenchymal (vimentin pos ), and pseudoendothelial (CK/EpCAM pos plus CD31 pos ) markers. We isolated these cells and performed whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) for analysis of copy number alterations (CNA). Of the CK/EpCAM pos cells analysed from the HGSOC patients, 2 of 3 cells showed diverse chromosomal CNAs. However, the 4 pseudoendothelial cells (CK/EpCAM pos plus CD31 pos ) observed in the HGSOC cases did not carry any CNA. Lastly, two of the clusters of vimentin positive cells sequenced from those found in the benign cystadenoma case had CNA. Despite the low number of cells analysed, our results underscore the importance of genetic analysis of putative CTCs to confirm their neoplastic origin. In particular, it highlights the presence of a population of CK/EpCAM pos cells that are not tumour cells in patients with HGSOC, which otherwise would be counted as CTCs., (© 2023. The Author(s).)

Plasma circulating tumour DNA (ctDNA) has been suggested to be a viable biomarker of response to treatment in patients with high grade serous ovarian carcinoma (HGSOC). TP53 mutations are present in more than 90% of HGSOCs but somatic variants are distributed across all exonic regions of the gene, requiring next generation sequencing (NGS) technologies for mutational analysis. In this study, we compared the suitability of the Accel (Swift) and Oncomine (ThermoFisher) panels for identification of TP53 mutations in ctDNA of HGSOC patients (N = 10). Only 6 patients (60%) were found to have TP53 mutations using the ACCEL panel but the addition of molecular tags in the Oncomine panel improved ctDNA detection with at least one mutation detected in all cases (100%). Orthogonal validation of the 14 somatic variants found by Oncomine, using droplet digital PCR, confirmed 79% (11/14) of the identified mutations. Overall, the Oncomine panel with unique molecular identifiers (UMI) appears more useful for ctDNA analysis in HGSOC., (© 2023. The Author(s).)

Introduction: Biomarkers that predict the risk of immune-mediated adverse events (irAEs) among patients with non-small cell lung cancer (NSCLC) may reduce morbidity and mortality associated with these treatments., Methods: We carried out high resolution human leucocyte antigen (HLA)-I typing on 179 patients with NSCLC treated with anti-program death (PD)-1/program death ligand (PDL)-1. Toxicity data were collected and graded as per common terminology criteria for adverse event (CTCAE) v5.0. We used 14.8-week for landmark analysis to address lead-time bias to investigate the correlation between HLA-I/II zygosity, supertypes and alleles with irAE. Furthermore, we assessed the association for irAE with clinical benefit rate (CBR), progression-free survival (PFS) and overall survival (OS)., Results: Homozygosity at one or more HLA-I loci, but not HLA-II, was associated with a reduced risk of irAE (relative risk (RR) = 0.61, 95% CI 0.33-0.95, P = 0.035) especially pneumonitis or any grade 3 toxicity. Patients with HLA-A03 supertype had a higher risk of developing irAE (RR = 1.42, 95% CI 1.02-2.01, P = 0.039). The occurrence of any irAE was significantly associated with improved CBR (RR = 1.48, P < 0.0001), PFS (HR = 0.45, P = 0.0003) and OS (HR = 0.34, P < 0.0001)., Conclusions: Homozygosity at one or more HLA-I loci may serve as biomarker to predict patients who are unlikely to experience severe irAEs among patients with NSCLC and treated with anti-PD1/PDL1, but less likely to derive clinical benefit. Patients with HLA-I homozygous might benefit from additional therapy., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© 2021. The Author(s).)

Aims: Glioneuronal tumours, although rare, are an important cause of treatment-resistant epilepsy. Differential diagnosis of glioneuronal tumour subtypes is complicated by their variable histological appearance and the lack of pathognomonic histological or molecular biomarkers. In this study we have applied techniques available in a diagnostic laboratory setting to characterise molecular and cytogenetic abnormalities in a single institution cohort of glioneuronal tumours., Methods: A cohort of 29 glioneuronal tumours that included 21 gangliogliomas and 5 dysembryoplastic neuroepithelial tumours (DNETs) was analysed using low pass whole genome sequencing (WGS) and 2 multiplex ligation-dependent probe amplification (MLPA) central nervous system (CNS) tumour probesets., Results: Low pass WGS identified chromosomal or subchromosomal alterations in 15 specimens. The most common chromosomal alterations were gains of chromosome 7 (n = 8) and chromosome 16 (n = 3). The BRAF V600E mutation was detected by MLPA in 9/21 (42.9%) gangliogliomas and 2/2 pleomorphic xanthoastrocytomas (PXAs). Chromosome 7 gains detected by WGS were reflected in MLPAs by overall gains of chromosome 7 gene probes, including those for BRAF, KIAA1549 and EGFR, while an internal BRAF/MKRN1 duplication was detected in a single ganglioglioma. Homozygous CDKN2A/B loss was detected by MLPA in 3 gangliogliomas, with p16 immunohistochemistry supporting these results., Conclusions: The combination of low pass WGS and MLPA identifies multiple, diverse genetic and chromosomal alterations in glioneuronal tumours, irrespective of histological tumour grade., (Crown Copyright © 2021. Published by Elsevier GmbH. All rights reserved.)

Detection of ovarian cancer (OC) circulating tumour cells (CTCs) is primarily based on targeting epithelial markers, thus failing to detect mesenchymal tumour cells. More importantly, the immune checkpoint inhibitor marker PD-L1 has not been demonstrated on CTCs from OC patients. An antibody staining protocol was developed and tested using SKOV-3 and OVCA432 OC cell lines. We targeted epithelial (cytokeratin (CK) and EpCAM), mesenchymal (vimentin), and OC-specific (PAX8) markers for detection of CTCs, and CD45/16 and CD31 were used for the exclusion of white blood and vascular endothelial cells, respectively. PD-L1 was used for CTC characterisation. CTCs were enriched using the Parsortix™ system from 16 OC patients. Results revealed the presence of CTCs in 10 (63%) cases. CTCs were heterogeneous, with 113/157 (72%) cells positive for CK/EpCAM (epithelial marker), 58/157 (37%) positive for vimentin (mesenchymal marker), and 17/157 (11%) for both (hybrid). PAX8 was only found in 11/157 (7%) CTCs. In addition, 62/157 (39%) CTCs were positive for PD-L1. Positivity for PD-L1 was significantly associated with the hybrid phenotype when compared with the epithelial ( p = 0.007) and mesenchymal ( p = 0.0009) expressing CTCs. Characterisation of CTC phenotypes in relation to clinical outcomes is needed to provide insight into the role that epithelial to mesenchymal plasticity plays in OC and its relationship with PD-L1.

(1) Background: The stratification of uveal melanoma (UM) patients into prognostic groups is critical for patient management and for directing patients towards clinical trials. Current classification is based on clinicopathological and molecular features of the tumour. Analysis of circulating tumour cells (CTCs) has been proposed as a tool to avoid invasive biopsy of the primary tumour. However, the clinical utility of such liquid biopsy depends on the detection rate of CTCs. (2) Methods: The expression of melanoma, melanocyte, and stem cell markers was tested in a primary tissue microarray (TMA) and UM cell lines. Markers found to be highly expressed in primary UM were used to either immunomagnetically isolate or immunostain UM CTCs prior to treatment of the primary lesion. (3) Results: TMA and cell lines had heterogeneous expression of common melanoma, melanocyte, and stem cell markers. A multi-marker panel of immunomagnetic beads enabled isolation of CTCs in 37/43 (86%) patients with UM. Detection of three or more CTCs using the multi-marker panel, but not MCSP alone, was a significant predictor of shorter progression free ( p = 0.040) and overall ( p = 0.022) survival. (4) Conclusions: The multi-marker immunomagnetic isolation protocol enabled the detection of CTCs in most primary UM patients. Overall, our results suggest that a multi-marker approach could be a powerful tool for CTC separation for non-invasive prognostication of UM.

Tumor heterogeneity is a major obstacle to the success of cancer treatment. An accurate understanding and recognition of tumor heterogeneity is critical in the clinical management of cancer patients. Here, we utilized single-cell RNA sequencing (scRNA-seq) to uncover the intra- and intertumoral heterogeneity of liver metastases from a patient with metastatic uveal melanoma. The two metastases analyzed were largely infiltrated by noncancerous cells with significant variability in the proportion of different cell types. Analysis of copy-number variations (CNVs) showed gain of 8q and loss of 6q in both tumors, but loss of Chromosome 3 was only detected in one of the tumors. Single-nucleotide polymorphism (SNP) array revealed a uniparental isodisomy 3 in the tumor with two copies of Chromosome 3, indicating a regain of Chromosome 3 during the development of the metastatic disease. In addition, both tumors harbored subclones with additional CNVs. Pathway enrichment analysis of differentially expressed genes revealed that cancer cells in the metastasis with isodisomy 3 showed up-regulation in epithelial-mesenchymal transition and myogenesis related genes. In contrast, up-regulation in interferon signaling was observed in the metastasis with monosomy 3 and increased T-cell infiltrate. This study highlights the complexity and heterogeneity of different metastases within an individual case of uveal melanoma., (© 2021 Lin et al.; Published by Cold Spring Harbor Laboratory Press.)

Cancer metastasis is the main reason for the high mortality in patients, contributing to 90% of cancer-related deaths. Biomarkers for early detection and therapeutic monitoring are essential to improve cancer outcomes. Circulating tumour cells (CTCs) arise from solid tumours and are capable of metastatic dissemination via the bloodstream or lymphatic system. Thus, CTCs can potentially be developed as a minimally invasive biomarker for early detection and therapeutic monitoring. Despite its clinical potential, research on CTCs remains limited, and this is likely due to their low numbers, short half-life, and the lack of robust methods for their isolation. There is also a need for molecular characterisation of CTCs to identify tumour-specific features, such as epigenetic signatures of metastasis. This review provides an overview of the epigenetic landscape of CTCs. We discuss the role of epigenetic modifications in CTC dissemination,metastatic tumour formation and progression and highlight its clinical implications., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose: We examined changes in plasma creatine kinase (CK) activity, hydroxyproline and cell-free DNA (cfDNA) concentrations in relation to changes in maximum voluntary isometric contraction (MVIC) torque and delayed-onset muscle soreness (DOMS) following a session of volume-matched higher- (HI) versus lower-intensity (LI) eccentric cycling exercise., Methods: Healthy young men performed either 5 × 1-min HI at 20% of peak power output (n = 11) or 5 × 4-min LI eccentric cycling at 5% of peak power output (n = 9). Changes in knee extensor MVIC torque, DOMS, plasma CK activity, and hydroxyproline and cfDNA concentrations before, immediately after, and 24-72 h post-exercise were compared between groups., Results: Plasma CK activity increased post-exercise (141 ± 73.5%) and MVIC torque decreased from immediately (13.3 ± 7.8%) to 48 h (6.7 ± 13.5%) post-exercise (P < 0.05), without significant differences between groups. DOMS was greater after HI (peak: 4.5 ± 3.0 on a 10-point scale) than LI (1.2 ± 1.0). Hydroxyproline concentration increased 40-53% at 24-72 h after both LI and HI (P < 0.05). cfDNA concentration increased immediately after HI only (2.3 ± 0.9-fold, P < 0.001), with a significant difference between groups (P = 0.002). Lack of detectable methylated HOXD4 indicated that the cfDNA was not derived from skeletal muscle. No significant correlations were evident between the magnitude of change in the measures, but the cfDNA increase immediately post-exercise was correlated with the maximal change in heart rate during exercise (r = 0.513, P = 0.025)., Conclusion: Changes in plasma hydroxyproline and cfDNA concentrations were not associated with muscle fiber damage, but the increased hydroxyproline in both groups suggests increased collagen turnover. cfDNA may be a useful metabolic-intensity exercise marker.

In recent years, circulating tumor DNA (ctDNA) has emerged as a promising prognostic and monitoring biomarker of various cancers, including melanoma. However, sensitive methods are required for its preservation, isolation, and detection. Here we describe a sensitive method for plasma ctDNA isolation using a column-based extraction kit, followed by quantification using a single mutational target with a droplet digital PCR system. This sensitive protocol has been successfully used to quantify diverse mutations present in plasma-derived ctDNA from cancer patients. The full procedure, from blood processing to the analysis of results, takes approximately a day of work.

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.

Immunotherapy is an important and established treatment option for patients with advanced melanoma. Initial anti-PD1 trials arbitrarily defined a two-year treatment duration, but a shorter treatment duration may be appropriate. In this study, we retrospectively assessed 70 patients who stopped anti-PD1 therapy in the absence of progressive disease (PD) to determine clinical outcomes. In our cohort, the median time on treatment was 11.8 months. Complete response was attained at time of anti-PD1 discontinuation in 61 (87%). After a median follow up of 34.2 months (range: 2-70.8) post discontinuation, 81% remained disease free. Using ddPCR, we determine the utility of circulating tumour DNA (ctDNA) to predict progressive disease after cessation ( n = 38). There was a significant association between presence of ctDNA at cessation and disease progression ( p = 0.012, Fisher's exact test) and this conferred a negative and positive predictive value of 0.82 (95% CI: 0.645-0.930) and 0.80 (95% CI 0.284-0.995), respectively. Additionally, dichotomised treatment-free survival in patients with or without ctDNA at cessation was significantly longer in the latter group ( p < 0.001, HR: 0.008, 95% CI: 0.001-0.079). Overall, our study confirms that durable disease control can be achieved with cessation of therapy in the absence of disease progression and undetectable ctDNA at cessation was associated with longer treatment-free survival.

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., (©2020 American Association for Cancer Research.)

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., Competing Interests: CONFLICTS OF INTEREST The following authors have received travel support from: MAK [Merck Sharp and Dohme (MSD), Bristol-Myers Squibb (BMS) and Merck Serono], TMM [BMS, Novartis, AstraZeneca (AZ)] and ESG [MSD]. The following authors sit on advisory boards for: TMM [BMS, MSD, Novartis, AZ]; MSC [Sanofi, Nektar, Merck Serono BMS, MSD, Novartis, Amgen, Pierre Fabre, Ideaya]; AMM [BMS, MSD, Novartis, Roche, Pierre-Fabre], GVL [Aduro, Amgen, Array, BMS, MSD, Novartis, Pierre-Fabre, Oncosec, Roche] and MM [BMS, AZ, Roche, MSD]., (Copyright: © 2020 Clark et al.)

Background: We aimed to assess the impact of genomic human leukocyte antigen (HLA)-I/II homozygosity on the survival benefit of patients with unresectable locally advanced, metastatic non-small lung cancer treated by single-agent programmed cell death protein-1/programmed death ligand 1 (PD1/PDL1) inhibitors., Methods: We collected blood from 170 patients with advanced lung cancer treated with immunotherapy at two major oncology centers in Western Australia. Genomic DNA was extracted from white blood cells and used for HLA-I/II high-resolution typing. HLA-I/II homozygosity was tested for association with survival outcomes. Univariable and multivariable Cox regression models were constructed to determine whether HLA homozygosity was an independent prognostic factor affecting Overall Survival (OS) and Progression Free Survival (PFS). We also investigated the association between individual HLA-A and -B supertypes with OS., Results: Homozygosity at HLA-I loci, but not HLA-II, was significantly associated with shorter OS (HR=2.17, 95% CI 1.13 to 4.17, p=0.02) in both univariable and multivariable analysis. The effect of HLA-I homozygosity in OS was particularly relevant for patients with tumors expressing PDL1 ≥50% (HR=3.93, 95% CI 1.30 to 11.85, p<0.001). The adverse effect of HLA-I homozygosity on PFS was only apparent after controlling for interactions between PDL1 status and HLA-I genotype (HR=2.21, 95% CI 1.04 to 4.70, p=0.038). The presence of HLA-A02 supertype was the only HLA-I supertype to be associated with improved OS (HR=0.56, 95% CI 0.34 to 0.93, p=0.023)., Conclusion: Our results suggest that homozygosity at ≥1 HLA-I loci is associated with short OS and PFS in patients with advanced non-small cell lung cancer with PDL1 ≥50% treated with single-agent immunotherapy. Carriers of HLA-A02 supertype reported better survival outcomes in this cohort of patients., Competing Interests: Competing interests: MM sits on advisory boards for Merck Sharp and Dohme (MSD), Bristol-Myers Squibb (BMS) and AstraZeneca (AZ). MAK sits on advisory boards of MSD, BMS and Merck Serono. ESG has received travel support from MSD., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Circulating tumour cells (CTCs) can be assessed through a minimally invasive blood sample with potential utility as a predictive, prognostic and pharmacodynamic biomarker. The large heterogeneity of melanoma CTCs has hindered their detection and clinical application., Methods: Here we compared two microfluidic devices for the recovery of circulating melanoma cells. The presence of CTCs in 43 blood samples from patients with metastatic melanoma was evaluated using a combination of immunocytochemistry and transcript analyses of five genes by RT-PCR and 19 genes by droplet digital PCR (ddPCR), whereby a CTC score was calculated. Circulating tumour DNA (ctDNA) from the same patient blood sample, was assessed by ddPCR targeting tumour-specific mutations., Results: Our analysis revealed an extraordinary heterogeneity amongst melanoma CTCs, with multiple non-overlapping subpopulations. CTC detection using our multimarker approach was associated with shorter overall and progression-free survival. Finally, we found that CTC scores correlated with plasma ctDNA concentrations and had similar pharmacodynamic changes upon treatment initiation., Conclusions: Despite the high phenotypic and molecular heterogeneity of melanoma CTCs, multimarker derived CTC scores could serve as viable tools for prognostication and treatment response monitoring in patients with metastatic melanoma.

Analysis of specific somatic copy number alterations (SCNAs) using multiplex ligation-dependent probe amplification (MLPA) is used routinely as a prognostic test for uveal melanoma (UM). This technique requires relatively large amounts of input DNA, unattainable from many small fine-needle aspirate biopsy specimens. Herein, we compared the use of MLPA with whole-genome amplification (WGA) combined with low-pass whole-genome sequencing (LP-WGS) for detection of SCNA profiles in UM biopsy specimens. DNA was extracted from 21 formalin-fixed, paraffin-embedded UM samples and SCNAs were assessed using MLPA and WGA followed by LP-WGS. Cohen's κ was used to assess the concordance of copy number calls of each individual chromosome arm for each patient. MLPA and WGA/LP-WGS detection of SCNAs in chromosomes 1p, 3, 6, and 8 were compared and found to be highly concordant with a Cohen's κ of 0.856 (bias-corrected and accelerated 95% CI, 0.770-0.934). Only 13 of 147 (8.8%) chromosomal arms investigated resulted in discordant calls, predominantly SCNAs detected by WGA/LP-WGS but not MLPA. These results indicate that LP-WGS might be a suitable alternative or adjunct to MLPA for the detection of SCNAs associated with prognosis of UM, for cases with limiting tissue or DNA yields., (Copyright © 2020 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Liquid biopsies hold the potential to inform cancer patient prognosis and to guide treatment decisions at the time when direct tumor biopsy may be impractical due to its invasive nature, inaccessibility and associated complications. Specifically, circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have shown promising results as companion diagnostic biomarkers for screening, prognostication and/or patient surveillance in many cancer types. In ovarian cancer (OC), CTC and ctDNA analysis allow comprehensive molecular profiling of the primary, metastatic and recurrent tumors. These biomarkers also correlate with overall tumor burden and thus, they provide minimally-invasive means for patient monitoring during clinical course to ascertain therapy response and timely treatment modification in the context of disease relapse. Here, we review recent reports of the potential clinical value of CTC and ctDNA in OC, expatiating on their use in diagnosis and prognosis. We critically appraise the current evidence, and discuss the issues that still need to be addressed before liquid biopsies can be implemented in routine clinical practice for OC management., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Circulating tumour DNA (ctDNA) has emerged as a promising blood-based biomarker for monitoring disease status of patients with advanced cancers. The presence of ctDNA in the blood is a result of biological processes, namely tumour cell apoptosis and/or necrosis, and can be used to monitor different cancers by targeting cancer-specific mutation., Case Presentation: We present the case of a 67 year old Caucasian male that was initially treated with BRAF inhibitors followed by anti-CTLA4 and then anti-PD1 immunotherapy for metastatic melanoma but later developed colorectal cancer. The kinetics of ctDNA derived from each cancer type were monitored targeting BRAF V600R (melanoma) and KRAS G13D (colon cancer), specifically reflected the status of the patient's tumours. In fact, the discordant pattern of BRAF and KRAS ctDNA was significantly correlated with the clinical response of melanoma to pembrolizumab treatment and progression of colorectal cancer noted by PET and/or CT scan. Based on these results, ctDNA can be used to specifically clarify disease status of patients with metachronous cancers., Conclusions: Using cancer-specific mutational targets, we report here for the first time the efficacy of ctDNA to accurately provide a comprehensive outlook of the tumour status of two different cancers within one patient. Thus, ctDNA analysis has a potential clinical utility to delineate clinical information in patients with multiple cancer types.

The analysis of circulating tumor DNA provides a minimally invasive molecular interrogation that has the potential to guide treatment selection and disease monitoring. Here, the authors evaluated a custom UltraSEEK melanoma panel for the MassARRAY system, probing for 61 mutations over 13 genes. The analytical sensitivity and clinical accuracy of the UltraSEEK melanoma panel was compared with droplet digital PCR. The blinded analysis of 68 mutations detected in 48 plasma samples from stage IV melanoma patients revealed a concordance of 88% between the two platforms. Further comparison of both methods for the detection of BRAF V600E mutations in 77 plasma samples demonstrated a Cohen's κ of 0.826 (bias-corrected and accelerated 95% CI, 0.669-0.946). These results indicate that the UltraSEEK melanoma panel is as sensitive as droplet digital PCR for the detection of circulating tumor DNA in this cohort of patients but highlight the need for detected variants to be confirmed orthogonally to mitigate any false-positive results. The MassARRAY system enables rapid and sensitive genotyping for the detection of multiple melanoma-associated mutations in plasma., (Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

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., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

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.

Purpose: To evaluate the feasibility of using circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) for the management of uveal melanoma (UM)., Patients and Methods: Low-coverage whole-genome sequencing was used to determine somatic chromosomal copy number alterations (SCNAs) in primary UM tumors, ctDNA, and whole-genome amplified CTCs. CTCs were immunocaptured using an antimelanoma-associated chondroitin sulfate antibody conjugated to magnetic beads and immunostained for melanoma antigen recognised by T cells 1 (MART1)/glycoprotein 100 (gp100)/S100 calcium-binding protein β (S100β). ctDNA was quantified using droplet digital polymerase chain reaction assay for mutations in the GNAQ , GNA11 , PLCβ4 , and CYSLTR2 genes., Results: SCNA analysis of CTCs and ctDNA isolated from a patient with metastatic UM showed good concordance with the enucleated primary tumor. In a cohort of 30 patients with primary UM, CTCs were detected in 58% of patients (one to 37 CTCs per 8 mL of blood), whereas only 26% of patients had detectable ctDNA (1.6 to 29 copies/mL). The presence of CTCs or ctDNA was not associated with tumor size or other prognostic markers. However, the frequent detection of CTCs in patients with early-stage UM supports a model in which CTCs can be used to derive tumor-specific SCNA relevant for prognosis. Monitoring of ctDNA after treatment of the primary tumor allowed detection of metastatic disease earlier than 18 F-labeled fluorodeoxyglucose positron emission tomography in two patients., Conclusion: The presence of CTCs in localized UM can be used to ascertain prognostic SCNA, whereas ctDNA can be used to monitor patients for early signs of metastatic disease. This study paves the way for the analysis of CTCs and ctDNA as a liquid biopsy that will assist with treatment decisions in patients with UM., Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Aaron BeasleyNo relationship to discloseTimothy IsaacsTravel, Accommodations, Expenses: PfizerMuhammad A. KhattakHonoraria: MSD Oncology, Novartis, Merck Serono Consulting or Advisory Role: Bristol-Myers Squibb, Merck Serono Speakers' Bureau: Merck Serono, MSD Oncology, Novartis Research Funding: MSD Oncology Travel, Accommodations, Expenses: MSD Oncology, Amgen, Merck SeronoJames B. FreemanNo relationship to discloseRichard AllcockNo relationship to discloseFred K. ChenSpeakers' Bureau: Bayer HealthCare Pharmaceuticals Research Funding: Novartis (Inst) Travel, Accommodations, Expenses: Bayer HealthCare Pharmaceuticals, AllerganMichelle R. PereiraNo relationship to discloseKyle YauNo relationship to discloseJacqueline BentelNo relationship to discloseTersia VermeulenNo relationship to discloseLeslie CalapreNo relationship to discloseMichael MillwardConsulting or Advisory Role: Roche, Bristol-Myers Squibb, AstraZeneca, Merck Sharp & Dohme, Novartis, Boehringer Ingelheim Travel, Accommodations, Expenses: Roche, Merck Sharp & Dohme, Bristol-Myers Squibb, AstraZenecaMelanie R. ZimanResearch Funding: Merck Sharp & DohmeElin S. GrayResearch Funding: Merck Sharp & Dohme Patents, Royalties, Other Intellectual Property: Provisional patent on a blood test to detect melanoma based on auto-antibody detection Travel, Accommodations, Expenses: Bio-Rad Laboratories, (© 2018 by American Society of Clinical Oncology.)

Circulating tumour DNA (ctDNA) has emerged as a promising blood-based biomarker for monitoring disease status of patients with advanced cancers. In melanoma, ctDNA has been shown to have clinical value as an alternative tumour source for the detection clinically targetable mutations for the assessment of response to therapy. This review provides a critical summary of the evidence that gives credence to the utility of ctDNA as a biomarker for monitoring of disease status in advanced melanoma and the steps required for its implementation into clinical settings., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: CONFLICTS OF INTEREST No author has any conflicts of interest to declare.

Background: Exposure to heat stress after UVB irradiation induces a reduction of apoptosis, resulting in survival of DNA damaged human keratinocytes. This heat-mediated evasion of apoptosis appears to be mediated by activation of SIRT1 and inactivation of p53 signalling. In this study, we assessed the role of SIRT1 in the inactivation of p53 signalling and impairment of DNA damage response in UVB plus heat exposed keratinocytes., Results: Activation of SIRT1 after multiple UVB plus heat exposures resulted in increased p53 deacetylation at K382, which is known to affect its binding to specific target genes. Accordingly, we noted decreased apoptosis and down regulation of the p53 targeted pro-apoptotic gene BAX and the DNA repair genes ERCC1 and XPC after UVB plus heat treatments. In addition, UVB plus heat induced increased expression of the cell survival gene Survivin and the proliferation marker Ki67. Notably, keratinocytes exposed to UVB plus heat in the presence of the SIRT1 inhibitor, Ex-527, showed a similar phenotype to those exposed to UV alone; i.e. an increase in p53 acetylation, increased apoptosis and low levels of Survivin., Conclusion: This study demonstrate that heat-induced SIRT1 activation mediates survival of DNA damaged keratinocytes through deacetylation of p53 after exposure to UVB plus heat.

Background: UV radiation induces significant DNA damage in keratinocytes and is a known risk factor for skin carcinogenesis. However, it has been reported previously that repeated and simultaneous exposure to UV and heat stress increases the rate of cutaneous tumour formation in mice. Since constant exposure to high temperatures and UV are often experienced in the environment, the effects of exposure to UV and heat needs to be clearly addressed in human epidermal cells., Methods: In this study, we determined the effects of repeated UVB exposure 1 kJ/m(2) followed by heat (39 °C) to human keratinocytes. Normal human ex vivo skin models and primary keratinocytes (NHEK) were exposed once a day to UVB and/or heat stress for four consecutive days. Cells were then assessed for changes in proliferation, apoptosis and gene expression at 2 days post-exposure, to determine the cumulative and persistent effects of UV and/or heat in skin keratinocytes., Results: Using ex vivo skin models and primary keratinocytes in vitro, we showed that UVB plus heat treated keratinocytes exhibit persistent DNA damage, as observed with UVB alone. However, we found that apoptosis was significantly reduced in UVB plus heat treated samples. Immunohistochemical and whole genome transcription analysis showed that multiple UVB plus heat exposures induced inactivation of the p53-mediated stress response. Furthermore, we demonstrated that repeated exposure to UV plus heat induced SIRT1 expression and a decrease in acetylated p53 in keratinocytes, which is consistent with the significant downregulation of p53-regulated pro-apoptotic and DNA damage repair genes in these cells., Conclusion: Our results suggest that UVB-induced p53-mediated cell cycle arrest and apoptosis are reduced in the presence of heat stress, leading to increased survival of DNA damaged cells. Thus, exposure to UVB and heat stress may act synergistically to allow survival of damaged cells, which could have implications for initiation skin carcinogenesis.

Metastatic melanoma is a highly heterogeneous tumor; thus, methods to analyze tumor-derived cells circulating in blood should address this diversity. Taking this into account, we analyzed, using multiparametric flow cytometry, the co-expression of the melanoma markers melanoma cell adhesion molecule and melanoma-associated chondroitin sulphate proteoglycan and the tumor-initiating markers ATP-binding cassette sub-family B member 5 (ABCB5), CD271, and receptor activator of NF-κβ (RANK) in individual circulating tumor cells (CTCs) from 40 late-stage (III-IV) and 16 early-stage (I-II) melanoma patients. CTCs were heterogeneous within and between patients, with limited co-expression between the five markers analyzed. Analysis of patient matched blood and metastatic tumors revealed that ABCB5 and RANK subpopulations are more common among CTCs than in the solid tumors, suggesting a preferential selection for these cells in circulation. Pairwise comparison of CTC subpopulations longitudinally before and 6-13 weeks after treatment initiation showed that the percentage of RANK(+) CTCs significantly increased in the patients undergoing targeted therapy (N=16, P<0.01). Moreover, the presence of ⩾5 RANK(+) CTCs in the blood of patients undergoing targeted therapies was prognostic of shorter progression-free survival (hazards ratio 8.73, 95% confidence interval 1.82-41.75, P<0.01). Taken together, our results provide evidence of the heterogeneity among CTC subpopulations in melanoma and the differential response of these subpopulations to targeted therapy.

Recent evidence suggests that heat stress may also be a risk factor of skin carcinogenesis. Heat stress causes activation of heat shock proteins (HSPs), chaperone proteins which prevent cells from undergoing apoptosis and ensuring their cellular function. However, HSPs recruitment may also have deleterious effects particularly if the cells rescued from apoptosis carry oncogenic mutations. We hypothesise that exposures to both heat and UV induce skin cancer(s) by concomitant expression of HSPs and oncogenic mutant proteins. Here we review studies demonstrating that heat stress-activated heat shock proteins such as HSP72 and HSP90 can influence signalling pathways such as MAPK, JNK and p53, which are all involved in regulating cell proliferation, survival and apoptosis., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)