Gabriela Gremel

Application of Sequencing, Liquid Biopsies, and Patient-Derived Xenografts for Personalized Medicine in Melanoma

UNLABELLEDnTargeted therapies and immunotherapies have transformed melanoma care, extending median survival from ∼9 to over 25 months, but nevertheless most patients still die of their disease. The aim of precision medicine is to tailor care for individual patients and improve outcomes. To this end, we developed protocols to facilitate individualized treatment decisions for patients with advanced melanoma, analyzing 364 samples from 214 patients. Whole exome sequencing (WES) and targeted sequencing of circulating tumor DNA (ctDNA) allowed us to monitor responses to therapy and to identify and then follow mechanisms of resistance. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and inhibitor-resistant BRAF-mutant tumors, which were then validated in patient-derived xenografts (PDX). We also developed circulating tumor cell-derived xenografts (CDX) as an alternative to PDXs when tumors were inaccessible or difficult to biopsy. Thus, we describe a powerful technology platform for precision medicine in patients with melanoma.nnnSIGNIFICANCEnAlthough recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments.

Distinct subclonal tumour responses to therapy revealed by circulating cell-free DNA

The application of precision medicine requires in-depth characterisation of a patients tumours and the dynamics of their responses to treatment. We used next-generation sequencing of cfDNA to monitor therapy responses of a metastatic vaginal mucosal melanoma and show that cfDNA can be used to monitor tumour evolution and subclonal responses to therapy even when biopsies are not available.

Circulating tumor DNA predicts survival in patients with resected high risk stage II/III melanoma

Abstract Background Patients with high-risk stage II/III resected melanoma commonly develop distant metastases. At present, we cannot differentiate between patients who will recur or those who are cured by surgery. We investigated if circulating tumor DNA (ctDNA) can predict relapse and survival in patients with resected melanoma. Patients and methods We carried out droplet digital polymerase chain reaction to detect BRAF and NRAS mutations in plasma taken after surgery from 161 stage II/III high-risk melanoma patients enrolled in the AVAST-M adjuvant trial. Results Mutant BRAF or NRAS ctDNA was detected (≥1 copy of mutant ctDNA) in 15/132 (11%) BRAF mutant patient samples and 4/29 (14%) NRAS mutant patient samples. Patients with detectable ctDNA had a decreased disease-free interval [DFI; hazard ratio (HR) 3.12; 95% confidence interval (CI) 1.79–5.47; Pu2009<u20090.0001] and distant metastasis-free interval (DMFI; HR 3.22; 95% CI 1.80–5.79; Pu2009<u20090.0001) versus those with undetectable ctDNA. Detectable ctDNA remained a significant predictor after adjustment for performance status and disease stage (DFI: HR 3.26, 95% CI 1.83–5.83, Pu2009<u20090.0001; DMFI: HR 3.45, 95% CI 1.88–6.34, Pu2009<u20090.0001). Five-year overall survival rate for patients with detectable ctDNA was 33% (95% CI 14%–55%) versus 65% (95% CI 56%–72%) for those with undetectable ctDNA. Overall survival was significantly worse for patients with detectable ctDNA (HR 2.63; 95% CI 1.40–4.96); Pu2009=u20090.003) and remained significant after adjustment for performance status (HR 2.50, 95% CI 1.32–4.74, Pu2009=u20090.005). Conclusion ctDNA predicts for relapse and survival in high-risk resected melanoma and could aid selection of patients for adjuvant therapy. Clinical trial number ISRCTN 81261306

Plasma total cell-free DNA (cfDNA) is a surrogate biomarker for tumour burden and a prognostic biomarker for survival in metastatic melanoma patients

Introduction Tumour burden is a prognostic biomarker in metastatic melanoma. However, tumour burden is difficult to measure and there are currently no reliable surrogate biomarkers to easily and reliably determine it. The aim of this study was to assess the potential of plasma total cell free DNA as biomarker of tumour burden and prognosis in metastatic melanoma patients. Materials and methods A prospective biomarker cohort study for total plasma circulating cell-free DNA (cfDNA) concentration was performed in 43 metastatic melanoma patients. For 38 patients, paired blood collections and scan assessments were available before treatment and at first response evaluation. Tumour burden was calculated as the sum of volumes from three-dimensional radiological measurements of all metastatic lesions in individual patients. Results Baseline cfDNA concentration correlated with pre-treatment tumour burden (ρ = 0.52, P < 0.001). Baseline cfDNA levels correlated significantly with hazard of death and overall survival, and a cut off value of 89 pg/μl identified two distinct prognostic groups (HR = 2.22 for high cfDNA, P = 0.004). Patients with cfDNA ≥89 pg/μl had shorter OS (10.0 versus 22.7 months, P = 0.009; HR = 2.22 for high cfDNA, P = 0.004) and the significance was maintained when compared with lactic dehydrogenase (LDH) in a multivariate analysis. We also found a correlation between the changes of cfDNA and treatment-related changes in tumour burden (ρ = 0.49, P = 0.002). In addition, the ratio between baseline cfDNA and tumour burden was prognostic (HR = 2.7 for cfDNA/tumour volume ≥8 pg/(μl*cm3), P = 0.024). Conclusions We have demonstrated that cfDNA is a surrogate marker of tumour burden in metastatic melanoma patients, and that it is prognostic for overall survival.

New insights into naevoid melanomas: a clinicopathological reassessment

Because the term ‘naevoid melanoma’ has variable clinical and pathological interpretations, we aimed to clarify the features of melanomas referred to as naevoid.

Abstract 5024: Implications of MAPK pathway inhibition on monocytes and tumor-associated macrophages in melanoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnPlasticity in T-cell populations during response and onset of resistance to targeted therapy has been well documented. More recently, macrophage-derived tumor necrosis factor has been described to drive resistance to MAPK pathway inhibition, yet little is known on the adaptation of tumor-associated macrophage populations to treatment and disease progression in human melanoma.nnTo test for alterations in macrophage abundance following resistance to MAPK pathway inhibition, we applied a fluorescence-based co-staining strategy on melanoma specimens, representing tissue collected from patients before treatment and at relapse. In addition, we established in vitro co-culture systems to study the paracrine effects of melanoma cells on various aspects of monocyte differentiation and macrophage activation in the background of MAPK inhibition and resistance.nnResistance to targeted therapy was found to be associated with an increase in the number of tumor-resident macrophages. In vitro co-culture of melanoma cell lines with monocytes isolated from the peripheral blood of healthy donors or with CSF1-differentiated macrophages in the presence or absence of MAPK inhibition led to profound changes in monocyte/macrophage marker expression, indicating a direct interplay between the two cell populations upon MAPK pathway inhibition. Our data show that macrophages play an integral role in the changing immune-microenvironment during the course of MAPK pathway inhibition. Elucidation of mechanisms driving these processes will result in strategies to optimize treatment combinations and consecutive treatment outcomes for melanoma patients.nnNote: This abstract was not presented at the meeting.nnCitation Format: Gabriela Gremel, Maria Romina Girotti, Amaya Viros, Garry Ashton, Helen Bradley, Elena Galvani, Rebecca Lee, Alberto Fusi, Paul Lorigan, Richard Marais. Implications of MAPK pathway inhibition on monocytes and tumor-associated macrophages in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5024. doi:10.1158/1538-7445.AM2015-5024