Purpose of review Despite the availability of multiple targeted therapies, the 5-year survival rate of patients with metastatic clear cell renal cell carcinoma (ccRCC) rarely exceeds 10%. Recent insights into the mutational landscape and evolutionary dynamics of ccRCC have offered up a plausible explanation for these outcomes. The purpose of this review is to link the research findings to potential changes in clinical practice. Recent findings Intratumour heterogeneity (ITH) dominates the evolutionary landscape in ccRCC at the genetic, transcriptomic and proteomic level. Spatial and temporal separation of tumour subclones within the primary tumour as well as between primary and metastatic sites has been demonstrated at single nucleotide resolution. In the cases analysed to date, approximately two-thirds of somatic mutations are not shared between multiple biopsies from the same primary tumour. Very few of the key disease-driving events are shared across all primary tumour regions (with the exception of VHL and loss of chromosome 3p), whereas the majority are restricted to one or more tumour regions (TP53, SETD2, BAP1, PTEN, mTOR, PIK3CA and KDM5C). Summary ITH must be considered in the management of ccRCC with respect to diagnostic procedures, prognostic and predictive biomarkers and drug development.

Highlights from ASCO 2015 demonstrate the impasse we face in solid tumour oncology: the compelling novel immune and targeted therapies are often associated with cost–benefit ratios significantly above the thresholds for reimbursement. This is at least in part a consequence of our incomplete understanding of the mechanisms of response and resistance to these agents. For example, ipilimumab is associated with durable clinical benefit in 15%–20% of unselected advanced melanoma patients (∼£75 000 per patient treated), and while the responses to single-agent targeted therapies such as vemurafenib are higher, they are often relatively short-lived (∼£42 000 per median PFS of 6–7 months). New trial design strategies such as basket and umbrella studies have improved upon patient selection, but have not yielded detailed biological understanding of the drug targets, nor polygenic mechanisms of resistance within or between patients. Academically led studies have the opportunity and the responsibility to prioritize biological insights as trial end points, maximising research gain, increasing patient benefit/safety and ultimately, improving cost-effectiveness. Collection of tumour material is fundamental to these aims but the timing, handling and sample analysis are of critical importance (Figure ​(Figure11). Figure 1. A schematic for biological sample collection throughout the course of disease and treatment. TILs, tumour-infiltrating lymphocytes; cfDNA, cell-free tumour DNA; PBMCs, peripheral mononuclear blood cells; PK, pharmacokinetic; PD, pharmacodynamic; PDX, ... Resistance to targeted therapies can be mediated by pre-existing rather than de novo alterations. High resolution tracking of cancer cells in vitro demonstrated that only 10% of resistant clones arise de novo [1], while mathematical models of tumour growth suggest that radiographically detectable lesions harbour at least 10 resistant sub-clones [2]. Thus, comprehensive upfront tumour profiling could anticipate the genetic composition of such clone(s), while taking into account spatial and temporal tumour heterogeneity. Extensive sampling of metastatic sites at autopsy revealed 10 distinct PTEN alterations emerging under the selective pressure of PI(3)Kα inhibition [3], and five independent reversion events in a germline BRCA2 mutant carrier who progressed on olaparib and carboplatin [4]. Distinct mechanisms of BRAF and EGFR inhibitor resistance were detected across multiple metastases within individual patients with melanoma [5] and colorectal cancer [6], respectively. The benefit of combination strategies can be limited by excess toxicity (combined targeting of the PI3K and MAPK pathways [7]), cross-resistance (BRAF and MEK inhibitors in melanoma [8]) and the persistent role of intra-tumour heterogeneity (targeting of the T790M EGFR mutation in lung cancer [9]). Informed by pre-clinical models, such as discontinuous dosing in BRAF-mutant melanoma [10], academically led trials can address more finely tuned ways of managing treatment resistance. In colorectal cancer cell-free tumour DNA (cfDNA) shows pulsatile levels of mutant KRAS in response to intermittent EGFR inhibition [11], providing the molecular rationale for re-challenge with targeted therapy. Similar frameworks are required to prospectively evaluate alternative or sequential scheduling as well as the role of cfDNA in tracking tumour progression. PD-L1 expression, a putative predictive marker for PD1/PDL1 inhibition, is also spatially heterogeneous [12]. Genomic data are a promising alternative biomarker in this area [13]. Mutational data, integrated with HLA typing, and tumour and peripheral T-cell profiling can define individual neo-antigenic repertoires. Academically led studies of immunotherapeutic agents must evaluate the ability of this approach to predict responses, inform immunotherapy/targeted combinations, and ultimately, facilitate adoptive T-cell therapy. Non-genetic causes of treatment resistance have been largely overlooked but studies that incorporate longitudinal biological sample collection and novel imaging techniques are well placed to examine tumour drug exposure (including heterogeneity of drug distribution [14]) and individual variation in drug metabolising enzymes, receptors, and transporters. Patient-derived xenografts can provide a useful platform for investigating personalised therapy in co-clinical trials [15], but only if robustly characterised and used in the full knowledge of their limitations (e.g. immunosuppressed host, mouse stroma and disparities in tumour burden between mouse and patient). There clearly are challenges to implementation of such complex studies but they can be overcome through close interdisciplinary work of academic/clinical consortia as illustrated by the Lung TRACERx programme [16], the use of measures such as one-time consent [17], post-mortem studies and stakeholder engagement (patient and public). In summary, we argue for a change of emphasis in drug development from learning little from many patients towards biologically rich clinical studies focussed on gleaning the maximum amount of biological information that might inform drug response and resistance for every patient entered into academic trial protocols.

Acral melanoma is a subtype of melanoma with distinct epidemiological, clinical and mutational profiles. To define the genomic alterations in acral melanoma, we conducted whole‐genome sequencing and SNP array analysis of five metastatic tumours and their matched normal genomes. We identified the somatic mutations, copy number alterations and structural variants in these tumours and combined our data with published studies to identify recurrently mutated genes likely to be the drivers of acral melanomagenesis. We compared and contrasted the genomic landscapes of acral, mucosal, uveal and common cutaneous melanoma to reveal the distinctive mutational characteristics of each subtype.

When therapy leads to cancer metastasis, knowing where else to target in the pathway may be the key to successful treatment. Blocking Melanoma Metastasis Although inhibitors of the mutant BRAF kinase are effective in some melanoma patients, intrinsic or acquired resistance to the drug is common. Furthermore, the growth of melanoma tumors with concomitant mutations in guanosine triphosphatase RAS, which activated kinases in the RAF family, is paradoxically accelerated by BRAF inhibition. RAF is the first kinase in a three-kinase cascade [the RAF–MEK (mitogen-activated protein kinase kinase)–ERK (extracellular signal–regulated kinase) pathway] that is involved in cell proliferation. Using proteomics, patient material, and mouse models, Sanchez-Laorden et al. found that BRAF inhibition paradoxically stimulated MEK and ERK signaling to induce metastasis of melanoma cells with mutant BRAF, resistance to a BRAF inhibitor, or mutant RAS. Combined treatment with a MEK inhibitor prevented BRAF inhibitor–induced metastasis in mice. Thus, combination therapies may be best to prevent both primary tumor growth and drug-induced metastasis. Melanoma is a highly metastatic and lethal form of skin cancer. The protein kinase BRAF is mutated in about 40% of melanomas, and BRAF inhibitors improve progression-free and overall survival in these patients. However, after a relatively short period of disease control, most patients develop resistance because of reactivation of the RAF–ERK (extracellular signal–regulated kinase) pathway, mediated in many cases by mutations in RAS. We found that BRAF inhibition induces invasion and metastasis in RAS mutant melanoma cells through a mechanism mediated by the reactivation of the MEK (mitogen-activated protein kinase kinase)–ERK pathway, increased expression and secretion of interleukin 8, and induction of protease-dependent invasion. These events were accompanied by a cell morphology switch from predominantly rounded to predominantly elongated cells. We also observed similar responses in BRAF inhibitor–resistant melanoma cells. These data show that BRAF inhibitors can induce melanoma cell invasion and metastasis in tumors that develop resistance to these drugs.

We used a combination of whole-genome sequencing and in vitro validation to show that mutations that activated at least two pro-growth/survival pathways mediated intrinsic resistance to BRAF inhibition in a melanoma patient. These data demonstrate how in-depth analysis can reveal intrinsic resistance to standard of care, providing an opportunity for alternative therapeutic strategies for patients who are likely to fail first-line treat-575 ment.

UNLABELLED Uveal melanoma, the most common eye malignancy, causes severe visual morbidity and is fatal in approximately 50% of patients. Primary uveal melanoma can be cured by surgery or radiotherapy, but the metastatic disease is treatment refractory. To understand comprehensively uveal melanoma genetics, we conducted single-nucleotide polymorphism arrays and whole-genome sequencing on 12 primary uveal melanomas. We observed only approximately 2,000 predicted somatic single-nucleotide variants per tumor and low levels of aneuploidy. We did not observe an ultraviolet radiation DNA damage signature, but identified SF3B1 mutations in three samples and a further 15 mutations in an extension cohort of 105 samples. SF3B1 mutations were associated with good prognosis and were rarely coincident with BAP1 mutations. SF3B1 encodes a component of the spliceosome, and RNA sequencing revealed that SF3B1 mutations were associated with differential alternative splicing of protein coding genes, including ABCC5 and UQCC, and of the long noncoding RNA CRNDE. SIGNIFICANCE Our data show that despite its dismal prognosis, uveal melanoma is a relatively simple genetic disease characterized by recurrent chromosomal losses and gains and a low mutational burden. We show that SF3B1 is recurrently mutated in uveal melanoma, and the mutations are associated with aberrant alternative splicing.

Mucosal melanoma displays distinct clinical and epidemiological features compared to cutaneous melanoma. Here we used whole genome and whole exome sequencing to characterize the somatic alterations and mutation spectra in the genomes of ten mucosal melanomas. We observed somatic mutation rates that are considerably lower than occur in sun‐exposed cutaneous melanoma, but comparable to the rates seen in cancers not associated with exposure to known mutagens. In particular, the mutation signatures are not indicative of ultraviolet light‐ or tobacco smoke‐induced DNA damage. Genes previously reported as mutated in other cancers were also mutated in mucosal melanoma. Notably, there were substantially more copy number and structural variations in mucosal melanoma than have been reported in cutaneous melanoma. Thus, mucosal and cutaneous melanomas are distinct diseases with discrete genetic features. Our data suggest that different mechanisms underlie the genesis of these diseases and that structural variations play a more important role in mucosal than in cutaneous melanomagenesis. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Introduction: Aberrant activation of RAF signalling is a frequent finding in human cancers. BRAF is the only RAF family member that is commonly mutated, whilst CRAF and ARAF play important roles in the signal transduction from mutant RAS. BRAF-specific inhibitors have been more effective in the treatment of BRAF-mutant melanoma than BRAF-mutant thyroid and colorectal cancers. Areas covered: The review summarises the experience with RAF kinase inhibitors, including efficacy, modes of acquired resistance, and the mechanism behind the progression of pre-malignant RAS-mutant lesions observed with RAF kinase inhibitors. The authors review all the completed and ongoing Phase I or II clinical trials of RAF kinase inhibitors and discuss in detail the rationale behind the combinatorial approaches. Expert opinion: The success of RAF kinase inhibitors has demonstrated the necessity of genotype-driven treatment selection for cancer patients. The spectrum of responses in different tumour types is explained by feedback events that are determined by cell lineage. Dissection of these events and the mechanisms of acquired resistance will determine the appropriate combination therapies. Ongoing characterisation of RAS-MAPK regulation in malignant cells may aid the development of novel agents that have greater potency for the inhibition of activated RAF kinase, and lesser propensity for promotion of RAS-mutant tumours.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC We generated melanoma cell lines resistant to BRAF inhibitors and show that the EGF receptor (EGFR)-SRC family kinase (SFK)-STAT3 signaling pathway was upregulated in these cells. In addition to driving proliferation of resistant cells, this pathway also stimulated invasion and metastasis. EGFR inhibitors cooperated with BRAF inhibitors to block the growth of the resistant cells in vitro and in vivo, and monotherapy with the broad specificity tyrosine kinase inhibitor dasatinib blocked growth and metastasis in vivo. We analyzed a tumor from a BRAF mutant patient with intrinsic resistance to vemurafenib. Vemurafenib treatment induced SFK activation in this tumor and we show that dasatinib blocked its growth and metastasis in immunocompromised mice. Our data shows that BRAF inhibitor-mediated activation of EFGR/SFK/STAT3 signaling can mediate resistance in BRAF mutant melanoma patients. We describe two treatments that appear to overcome this resistance and could deliver therapeutic efficacy in drug-resistant BRAF mutant melanoma patients. Citation Format: Maria R. Girotti, Malin Pedersen, Berta Sanchez-Laorden, Amaya Viros, Samra Turajlic, Dan Niculescu-Duvaz, Alfonso Zambon, John Sinclair, Andrew Hayes, Martin Gore, Caroline Springer, James Larkin, Claus Jorgensen, Richard Marais. Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3404. doi:10.1158/1538-7445.AM2013-3404