Advances in personalized targeted treatment of metastatic melanoma and non-invasive tumor monitoring

Despite extensive scientific progress in the melanoma field, treatment of advanced stage melanoma with chemotherapeutics and biotherapeutics has rarely provided response rates higher than 20%. In the past decade, targeted inhibitors have been developed for metastatic melanoma, leading to the advent of more personalized therapies of genetically characterized tumors. Here we review current melanoma treatments and emerging targeted molecular therapies. In particular we discuss the mutant BRAF inhibitors Vemurafenib and Dabrafenib, which markedly inhibit tumor growth and advance patients’ overall survival. However this response is almost inevitably followed by complete tumor relapse due to drug resistance hampering the encouraging initial responses. Several mechanisms of resistance within and outside the MAPK pathway have now been uncovered and have paved theway for clinical trials of combination therapies to try and overcome tumor relapse. It is apparent that personalized treatment management will be required in this new era of targeted treatment. Circulating tumor cells (CTCs) provide an easily accessible means of monitoring patient relapse and several new approaches are available for the molecular characterization of CTCs. Thus CTCs provide a monitoring tool to evaluate treatment efficacy and early detection of drug resistance in real time.We detail here how advances in the molecular analysis of CTCs may provide insight into new avenues of approaching therapeutic options that would benefit personalized melanoma management

Circulating tumour DNA (ctDNA) as a liquid biopsy for melanoma

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

SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes

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

Neuroendocrine and neurotrophic signaling in Huntington\u27s disease: Implications for pathogenic mechanisms and treatment strategies

Huntington\u27s disease (HD) is a fatal neurodegenerative disease caused by an extended polyglutamine tract in the huntingtin protein. Circadian, sleep and hypothalamic-pituitary-adrenal (HPA) axis disturbances are observed in HD as early as 15 years before clinical disease onset. Disturbances in these key processes result in increased cortisol and altered melatonin release which may negatively impact on brain-derived neurotrophic factor (BDNF) expression and contribute to documented neuropathological and clinical disease features. This review describes the normal interactions between neurotrophic factors, the HPA-axis and circadian rhythm, as indicated by levels of BDNF, cortisol and melatonin, and the alterations in these intricately balanced networks in HD. We also discuss the implications of these alterations on the neurobiology of HD and the potential to result in hypothalamic, circadian, and sleep pathologies. Measurable alterations in these pathways provide targets that, if treated early, may reduce degeneration of brain structures. We therefore focus here on the means by which multidisciplinary therapy could be utilised as a non-pharmaceutical approach to restore the balance of these pathways

Genetic analysis of heterogeneous subsets of circulating tumour cells from high grade serous ovarian carcinoma patients

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/EpCAMpos), mesenchymal (vimentinpos), and pseudoendothelial (CK/EpCAMpos plus CD31pos) 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/EpCAMpos cells analysed from the HGSOC patients, 2 of 3 cells showed diverse chromosomal CNAs. However, the 4 pseudoendothelial cells (CK/EpCAMpos plus CD31pos) 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/EpCAMpos cells that are not tumour cells in patients with HGSOC, which otherwise would be counted as CTCs

A diagnostic autoantibody signature for primary cutaneous melanoma

Melanoma is an aggressive form of skin cancer that is curable by surgical excision in the majority of cases, if detected at an early stage. To improve early stage melanoma detection, the development of a highly sensitive diagnostic test is of utmost importance. Here we aimed to identify antibodies to a panel of tumour associated antigens that can differentiate primary melanoma patients and healthy individuals. A total of 245 sera from primary melanoma patients and healthy volunteers were screened against a high-throughput microarray platform containing 1627 functional proteins. Following rigorous statistical analysis, we identified a combination of 10 autoantibody biomarkers that, as a panel, displays a sensitivity of 79%, specificity of 84% and an AUC of 0.828 for primary melanoma detection. This melanoma autoantibody signature may prove valuable for the development of a diagnostic blood test for routine population screening that, when used in conjunction with current melanoma diagnostic techniques, could improve the early diagnosis of this malignancy and ultimately decrease the mortality rate of patients

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 (

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 wildtype 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 \u3e T and chr5:1,295,250 C \u3e 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

Blood-based melanoma detection

How is melanoma usually detected? . .