Gene Expression Profiling in Melanoma for Development of Targeted Therapeutics

November 2014

Gene expression profiling in melanoma for development of targeted therapeutics

Abstract

Metastatic melanoma is a notoriously difficult cancer to treat with current therapeutic regimens eliciting a positive response in less than 20% of patients. The advent of high throughput sequencing technologies and bioinformatics has led researchers to begin to characterize previously unknown subtypes of melanomas based on their gene expression profiles in the hopes of eventually using tumour genetic signatures for diagnostic, prognostic and therapeutic purposes as well as in rational targeted-drug design. Genome-wide association studies have implicated a number of genes in melanoma tumourgenesis so far. Of note, are activating BRAFV600E/K mutations which are found in >66% of cutaneous melanomas. Three FDA-approved drugs targeting melanomas harboring these mutations have been somewhat effective in clinical trials, highlighting the prominent roles tumour profiling and targeted therapies are likely to play in cancer treatments in the future.

Melanoma

Melanoma is an aggressive form of malignant cancer arising from melanocytes. According to the Canadian Cancer Society, melanoma represents 3% of all diagnosed cancers and 1.5% of all cancer deaths and 75% of all skin cancer deaths [1]. Melanomas are classified into three categories based on their site of origin: mucosal, uveal and cutaneous melanomas. Mucosal melanomas arise from melanocytes in mucosal surfaces of the head and neck, gastrointestinal tract and genitourinary tract. Uveal melanomas arise from melanocytes in the uveal tract. Cutaneous melanomas, representing 90% of all melanomas, arise from melanocytes in the epidermal layer of the skin. Cutaneous melanomas can be further classified into superficial spreading melanomas, lentigo maligna melanomas, nodular melanomas, and acral letiginous melanomas. Superficial spreading melanomas develop from an existing mole and account for 70% of all melanomas. Lentigo maligna melanomas typically begin on skin chronically exposed to the sun such as the face and arms. Nodular melanomas, accounting for 15% of melanomas, are aggressive and tend to form rapidly. Acral letiginous melanomas develop on the palms of the hands, soles of the feet, or under the nail beds [2].

Important risk factors for melanoma development include: UV exposure, a large number of abnormal of moles, age, having fair skin and having light-coloured hair and eyes [2]. A family history of melanoma also increases risk, as 5-10% of melanomas are hereditary, 2% of melanomas are attributed to germline mutations in cyclin-dependent kinase inhibitor 2A (CDKN2A)[3].

Diagnosis of melanoma involves skin and lymph node biopsies and analysis of the biopsy sample using microscopy, immunohistochemistry and/or genetic screens. Imaging tests may also be used to determine the existence and extent of metastases. The results of clinical and laboratory tests allow the melanoma to be staged (I-IV) based on how widespread it is which allows an estimation of prognosis and helps determine the most appropriate therapeutic avenue to take[4].

Treatment options depend on the stage of the melanoma but may include surgery, targeted therapies or immuno-, chemo- or radiation-therapies. Early-stage tumours can often be treated with surgical excision alone. Treatment of metastasized melanomas often involves excision of, and radiation therapy against, the primary tumour as well as dissection of the lymph nodes. Immunotherapies using anti-tumour antibodies or cytokines may also be employed. Alkylating chemotherapy drugs such as dacarbazine and anti-microtubule chemotherapy drugs such as vinblastine are mainstay treatments for non-resectable, metastasize melanomas; however, they elicit tumour responses in only 10-15% of patients with no overall increase in survival and many side effects [5]. Five year survival rates in patients with stage IV melanoma are <10%[4]. Thus, there is clear need for tumour-specific therapeutic options.

Gene expression profiling of melanoma tumours:

It is now well established that individual tumours of the same type are genetically diverse, substantiating the fact that standard anatomical-based classification systems are proving to be inadequate diagnostic, prognostic and therapeutic predictors as is evidenced by the different therapeutic responses and disease outcomes of patients diagnosed with anatomically-similar malignancies. Fueled by recent advances in genomics, proteomics and transcriptomics, researchers are beginning to elucidate gene expression profiles of previously unknown subtypes of melanoma with the hopes of eventually using tumour genetic signatures for diagnostic, prognostic and therapeutic purposes as well as in rational targeted-drug design [2,4]. The process begins with high throughput sequencing of large cohorts of tumour samples. Bioinformatics techniques analyse and integrate the gene expression data with gene and network information found in gene databases in order to identify recurrent genetic aberrations and pathways potentially involved in tumourgenesis. The functional role of the putative gene(s) and/or pathway(s) are then assayed in order to establish their involvement in tumourgenesis.

Indeed, a number of seminal genome-wide association studies have identified key molecular players and pathways involved in tumourgenesis in certain melanomas [6]. Currently, the most clinically relevant discoveries are those involving disruptions to the RAS-RAF-MEK-ERK cell-signaling pathways that direct cellular activity in response to a variety of stimuli. The signaling cascade begins when an extracellular mitogen binds to a cell membrane receptor, activating Ras, a GTPase. Activated Ras, hydrolyzes GTP to GDP and activates the kinase activity of Raf kinase. Raf initiates a phosphorylation cascade causing the sequential phosphorylation and dephosphorylation of MEK and ERK (extracellular-signal-regulated kinases). ERK in turn phosphorylates a number of targets that regulate transcription and translation and ultimately control cell growth, proliferation and survival [7]. Davies et al. identified activating mutations in BRAF (one of three isoforms of Raf) in 66% of melanomas studied. 90% of the mutations result in the substitution of glutamic acid for valine at codon 600 (V600E) while the remaining 10% result in the substitution of lysine for valine at codon 600 (V600K). They determined that the V600E mutation in BRAF is found in its kinase domain and contributes to tumourgenesis by imparting a 10.7X increase in activation to the RAS-RAF-MEK-ERK pathway compared to wild-type BRAF [8]. BRAF V600E and V600K mutations are used to distinguish patients