Assessment of the clinical validity of ctDNA Analysis for melanoma management

Metastatic melanoma is responsible for almost 80% of all skin cancer-related deaths and the incidence of people affected continues to rise worldwide. The emergence of targeted therapy and immune-checkpoint inhibitors has improved the clinical management of melanoma, but durable survival benefit is only seen in a minority of patients. The use of these very expensive systemic therapies on all appropriate patients also poses a high economic burden on health systems across numerous countries. Currently, surveillance for treatment failure is not optimal. Thus, reliable and accurate biomarkers of patient disease status are urgently required. Circulating tumour DNA (ctDNA) analysis has emerged as a potential “liquid biopsy” for melanoma. Plasma-derived ctDNA are short DNA fragments released into the bloodstream by apoptotic tumour cells. Studies have shown that ctDNA levels in blood correlate with tumour burden and can comprehensively capture the molecular heterogeneity of melanoma metastases. Thus, ctDNA appears to be a viable biomarker for monitoring treatment response and disease progression in melanoma patients. However, further studies aimed at comparing ctDNA and current standard clinical assessments are needed to fully define its suitability as a complementary test to guide treatment decisions. This thesis aims to provide important information that will assist with the implementation of ctDNA as a biomarker for melanoma in the clinical management of the disease. This thesis is comprised of 7 chapters: a comprehensive literature review (Chapter 1. Introduction); a materials and methods chapter (Chapter 2); 4 results chapters (Chapter 3 – 6); and a final chapter with a general discussion of main findings and future directions (Chapter 7. General Discussion and Future Directions). The first chapter of the thesis includes a thorough review of the literature on ctDNA as a potential biomarker for melanoma disease (Chapter 1). This is then followed by a detailed description of our protocol for plasma ctDNA extraction and quantification using droplet digital PCR (Chapter 2). Using this methodology, we evaluated the ctDNA detection rate in untreated BRAF mutant melanoma patients, as a potential alternative to tumour genotyping (Chapter 3), where the potential economic benefit of implementing plasma ctDNA testing by ddPCR relative to tissue BRAF testing was also investigated. The study in Chapter 4 demonstrated that pre-treatment plasma ctDNA is predictive of patient outcomes in the first-line treatment setting. However, baseline ctDNA level was not predictive of outcomes in the second-line immunotherapy setting, especially in patients that were pre-treated with BRAF/MEK inhibitors. Moreover, we found preliminary evidence that patients with high pre-treatment ctDNA may benefit from combined anti-CTLA-4/anti-PD-1 therapy. Chapter 5 discusses the validity of ctDNA as a surveillance biomarker for melanoma. The kinetics of ctDNA decline were found delayed in patients treated with immunotherapy compared to those receiving MAPK inhibitors. Nonetheless, decreasing ctDNA levels within 12 weeks of immunotherapy or BRAF/MEK inhibitors was strongly concordant with treatment response and significantly associated with longer progression-free survival (PFS). Furthermore, exploratory analysis of nine patients commencing anti-PD- 1 therapy showed a trend of high tumour mutational burden (TMB) and neoepitope load in responders compared to non-responders. Chapter 6 evaluates the validity of ctDNA to accurately detect disease progression using both a retrospective and a prospective cohort of melanoma patients. The results indicated a moderate detection rate, suggesting that more sensitive methodologies are required to achieve a limit of detection comparable to current medical imaging. Finally, Chapter 7 provides a general discussion of the studies covered in this thesis. It underscores the clinical validity of ctDNA as a biomarker of prognosis and therapeutic response in melanoma patients, while highlighting important limitations inherent to ctDNA analysis that need to be thoroughly addressed before it can be successfully implemented in the clinic

Optimisation of the isolation and identification of circulating melanoma cells

Although melanoma is largely curable when detected in its earliest stages, it can metastasise to other tissues, drastically reducing survival rates. The most recent therapies used to treat metastatic melanoma are effective long-term in only 11 to 33% of patients. Our ability to monitor treatment failure is limited. New prognostic markers are urgently required to allow monitoring of treatment response and disease progression. Circulating tumour cells (CTCs) are released into the bloodstream by the tumours within a patient, this being a key step in melanoma spread. Since CTCs can be detected in the blood of metastatic melanoma patients, these cells can be used as a “liquid biopsy”, providing critical insight into each person’s melanoma biology. Melanoma CTCs have been described as very heterogeneous, hindering their isolation via commonly used CTC capturing methods. To address this, microfluidic devices have been developed to isolate viable CTCs from blood, independently of their marker expression. This study aimed to determine the effectiveness of two different microfluidic devices (Slanted and A5) in recovering melanoma cell lines, and their potential use in the isolation of CTCs from the blood of metastatic melanoma patients. It also aimed to study additional cancer or melanoma specific markers to be used in immunostaining protocols for detection of CTCs after microfluidic enrichment. The optimal isolation procedure was identified as two rounds of enrichment with the Slanted spiral device, after which we obtained a 3-log depletion of white blood cells and a recovery of over 60% when cells from two melanoma cell lines were spiked into blood samples from healthy volunteers. In addition, we optimised the detection of CTCs using four melanoma markers (gp100, Melan-A, s100 and MCSP) combined in a multimarker immunocytochemistry staining protocol. The optimised enrichment and detection procedures were validated in a cohort of ten metastatic melanoma patients. Results showed that 40% of the patients had one or more CTCs in their blood (1-4 CTCs/8 mL of blood). Furthermore, three additional markers (Vimentin, RANK, and ABCB5) were trialled so as to increase detection of highly heterogeneous melanoma CTCs in samples that have been processed through the Slanted microfluidic device. The improved enrichment and detection of CTCs in the blood of melanoma patients using the methods developed as part of this study will facilitate the molecular, genomic and functional characterisation of melanoma CTCs. This will ultimately improve our understanding of the biology of melanoma CTCs and their role in metastatic spread and treatment response

Síntesis y caracterización de reactivos para el diagnóstico de alergia a amoxicilina

[EN] The amoxicillin is one of the most beta-lactam antibiotics frequently involved in allergic reactions to pharmaceutical drugs. The current diagnostic techniques are based on in vivo tests, which are invasive and aggressive for the patients. Hence, the need arises to develop new diagnostic techniques more subtle that allow to quickly and easily detect the drug responsible for the allergic reaction. The intention of this project is to contribute to the development of a minimally intrusive in vitro assay. The technique is based on the detection and quantification of total IgEs and specific to amoxicillin IgEs using magnetic particles conjugated to anti-IgE antibodies. This methodology uses CD technology, creating a protein microarray with specific drug-protein conjugates for the detection of specific IgEs, and anti-IgE antibodies for total IgEs quantification.[ES] La amoxicilina es uno de los antibióticos betalactámicos más frecuentemente implicado en las reacciones alérgicas a fármacos. Las técnicas actuales de diagnóstico están basadas en ensayos in vivo, invasivos per se y agresivos para el paciente. Por ello, existe la necesidad de desarrollar nuevos métodos de diagnóstico, más sensibles que permitan una asignación rápida y sencilla del medicamento causante de la alergia. Este trabajo pretende contribuir al desarrollo de un ensayo in vitro mínimamente invasivo, basado en la detección y cuantificación de IgEs totales y específicas para amoxicilina utilizando partículas magnéticas conjugadas a anticuerpos anti-IgE. El método se basa en la tecnología de disco compacto, siguiendo un formato de micromatriz de proteínas utilizando conjugados específicos fármaco-proteína para la detección de IgEs específicas y anticuerpos anti-IgE para la cuantificación de IgEs totales.Marsavela, AG. (2014). SÍNTESIS Y CARACTERIZACIÓN DE REACTIVOS PARA EL DIAGNÓSTICO DE ALERGIA A AMOXICILINA. http://hdl.handle.net/10251/53327.TFG

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 \u3c 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

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

The prognostic impact of circulating tumour dna in melanoma patients treated with systemic therapies—beyond braf mutant detection

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. 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 \u3c 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

Isolation and detection of circulating tumour cells from metastatic melanoma patients using a slanted spiral microfluidic device

Circulating Tumour Cells (CTCs) are promising cancer biomarkers. Several methods have been developed to isolate CTCs from blood samples. However, the isolation of melanoma CTCs is very challenging as a result of their extraordinary heterogeneity, which has hindered their biological and clinical study. Thus, methods that isolate CTCs based on their physical properties, rather than surface marker expression, such as microfluidic devices, are greatly needed in melanoma. Here, we assessed the ability of the slanted spiral microfluidic device to isolate melanoma CTCs via label-free enrichment. We demonstrated that this device yields recovery rates of spiked melanoma cells of over 80% and 55%, after one or two rounds of enrichment, respectively. Concurrently, a two to three log reduction of white blood cells was achieved with one or two rounds of enrichment, respectively. We characterised the isolated CTCs using multimarker flow cytometry, immunocytochemistry and gene expression. The results demonstrated that CTCs from metastatic melanoma patients were highly heterogeneous and commonly expressed stem-like markers such as PAX3 and ABCB5. The implementation of the slanted microfluidic device for melanoma CTC isolation enables further understanding of the biology of melanoma metastasis for biomarker development and to inform future treatment approaches

Blood-based melanoma detection

How is melanoma usually detected? . .

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