Medical Brief: Melanoma Understanding the DNA Mutation Landscape

Medical Brief: Melanoma Understanding the DNA Mutation Landscape

Melanocytic tumors are a relatively common group of neoplasms in dogs that originate from a pigment producing cell or melanocyte from the epidermis, dermis or hair follicles.  Melanomas are the most common type of melanocytic tumor in the dog and affects approximately 19,000 dogs annually in the US, representing 7% of all malignant tumors in this species. Melanomas occurs in middle-aged to older dogs with mean age of 11.6 years, with no sex predisposition. Some breeds are more likely to develop melanoma in certain locations than others suggesting genetic factors are involved in melanoma development. The exposure to ultraviolet light is not a common etiology for most canine melanomas, as they occur predominantly in the mouth,  at the nail bed, or on the skin which is typically covered by hair. This situation is similar to that for mucosal melanomas in people 1–5.

Corresponding to the literature, the analysis of melanoma tumors submitted to the FidoCure® Platform for DNA sequencing reveal equal gender distribution (Figure 1) and a mean age of 10.87 years old. The most common presentation of melanomas in dogs is in the oral cavity, followed by the skin, nail bed, and eyes. This is similar to the cases submitted to FidoCure® for DNA sequencing: 34 mucosal, 10 cutaneous, 2 digit, 1 ocular, and interestingly 5 cases of visceral melanomas. The anatomical localization of the primary tumor is often correlated with prognosis. Mucosal melanomas are considered to have the worst prognosis followed by the digital and cutaneous forms. Oral mucosal melanomas are very aggressive, being both locally invasive and having a high metastatic rate to both regional lymph nodes and the lungs.  Affected dogs have clinical signs that are often associated with both the clinical stage and location of the tumor.  For example, dogs with oral melanomas frequently display dysphagia, halitosis, ptyalism, and oral bleeding  3,6,7.  The median survival time (MST) for dogs with oral melanoma is around 6 months, while it is 11.8 months for dogs with  cutaneous melanomas  2,6.

The preliminary diagnosis of canine melanomas can be obtained by fine-needle aspiration (FNA) and cytology analysis. Histopathology and immunohistochemistry confirm the diagnosis and provide important information about the prognosis. The immunostaining of Ki67, a biomarker indicating cellular proliferation, is useful to predict the biologic behavior of both oral  and cutaneous melanomas. Thoracic radiographs, CT scans and/or MRI scans are used to determine the clinical stage and to investigate the invasiveness of the primary tumor, the integrity of the bones of oral and nasal cavity in cases of oral melanomas, the presence or absence of lung metastases and whether  non palpable lymph nodes are enlarged 6,8,9.

The choice of treatment for melanomas in dogs is affected by several parameters, such as location, invasiveness and the clinical condition of the pet.  Surgical excision and radiation therapy are potential therapeutic options for the local disease. Surgical excision is often reserved for smaller, less invasive tumors that can be removed with complete margins, or for dogs that do not have access to radiation therapy. For invasive tumors and/or large tumors that cannot be completely resected, radiation therapy is often the best form of local therapy.  Similarly, when the melanoma affects the digit, surgical  amputation of the affected digit or digits is often the recommended local therapy. If the digit melanoma is too invasive or large and cannot be resected completely, then radiation therapy is an option for local disease control.   Radiation therapy, although effective at controlling local disease in patients with stage II and III disease, does not address the metastatic nature of malignant melanomas in dogs 2,6,10–15. Systemic therapy in the form of chemotherapy, (eg carboplatin), electrochemotherapy and COX-2 inhibitors are often recommended to address the systemic nature of this disease 6,16–2. Due to many melanomas being chemoresistant, the efficacy of adjuvant chemotherapy to treat melanoma is limited. Immunotherapy is another systemic adjuvant treatment option for dogs with melanoma. The Xenogeneic DNA vaccine, OnceptⓇ, may  increase overall survival when used in certain dogs with melanoma, either oral or digital 2,6,21–25.

Although the development of these adjuvant therapies has partially improved outcomes in dogs with melanomas, tumor heterogeneity and resistance mechanisms are still challenges and more effective therapies are needed, especially in dogs with metastatic disease. Genomic studies to discover novel mutations in canine melanomas are underway, allowing us to look for molecular markers associated with prognosis and potential therapeutic targets 2,26. Based on the FidoCure® data analysis, mucosal melanomas have a higher percentage of mutations compared to cutaneous melanomas (Figure 2). This contrast in the mutational landscape may contribute to the difference in the biologic behavior of these two types of melanoma.

According to the literature, alterations in TP53 and PTEN genes are commonly associated with canine melanomas. Studies have shown that mutations in TP53 occur in  8% to 19% of melanomas, while mutations in  PTEN are detected in 5% of tumors.26–28. These tumor suppressor genes are involved in cell cycle control, DNA repair, apoptosis, migration and growth. Mutations causing loss of function of these genes are related to cancer development in both humans and dogs6,29.  In the FidoCure® database (Figure 3), mutations in TP53 and PTEN are observed in 20% and 10% of the canine melanomas, respectively. The RAS family consists of NRAS, KRAS and HRAS oncogenes that activate the MAPK pathway and induce cell proliferation30. Although NRAS and KRAS are not as commonly mutated in canine melanomas, as they are in human melanomas, targeting PI3K/AKT/mTOR and MAPK pathways with rapamycin and a MEK1/2 inhibitor allowed a synergistic effect on growth inhibition in canine and human melanoma cell lines 6, 30, 31.


Analysis of the FidoCure® database identified KMT2C and KMT2D genes mutated in 46% and 24% of tumors, respectively, being KMT2C the major mutated gene in these melanoma cases. The KMT2 family of genes promote genome accessibility and transcription in the epigenetic context. These genes are linked to tumorigenesis, mutagenesis and immune tolerance when mutated. KMT2C and KMT2D genes are altered with loss of function in approximately 27% of human melanomas. Therapeutic intervention to inhibit the effect of loss-of-function of tumor suppressor genes still represent a challenge, but recent discoveries of KMT2C and KMT2D roles in cancer cells have been stimulating different therapeutic strategies. The loss of KMT2D in a Zebrafish model for human Kabuki syndrome, for example, results in hyperactivation of MEK and RAS/MAPK pathway. This activation can increase the chance of tumor to respond to BRAF inhibitors representing a treatment possibility of KMT2D mutated tumors 32–35.

Both NOTCH1 and ROS1 were the next most commonly mutated genes according to the FidoCure database, with 42% of the melanoma cases having a mutation in NOTCH1 and 42% of the melanomas cases having a mutation in ROS1. Mutations in NOTCH1 are also identified in 60% of human melanomas and this genomic alteration contributes to tumor growth by affecting survival properties and leading to an immunosuppressive tumor microenvironment. High expression of NOTCH1 has been identified in endothelial cells of human melanomas and correlates with shorter progression-free survival41. The potential development of NOTCH1 inhibitors could potentialize the efficacy of immunomodulators and decrease tumor proliferation36–40.

The ROS1 gene encodes the receptor tyrosine kinase ROS1, which is one of the subfamily of insulin receptor genes and its protein functions as a growth or differentiation factor receptor in the cell. This gene is involved in several activation of signaling pathways such as RAS-ERK and JAK/STAT3 (cell survival and proliferation), PI3K/AKT/mTOR (cell survival and growth) and VAV3/RHO (cell migration and invasiveness by actin remodeling) 42–45. Tumors like human melanoma are observed expressing altered ROS1 gene, as also seen in canine melanomas in the FidoCure®database as a third most commonly mutated gene.

Canine melanomas represent a challenging tumor to treat in veterinary medicine, due to the aggressiveness, heterogeneity and resistance mechanisms of the melanoma cells. Although many different therapeutic strategies have been tried, the rapid and frequent development of metastatic lesions is a major factor causing survival times for dogs with malignant melanoma often to be shorter than 12 months. The incorporation of DNA sequencing in the diagnostic cascade for canine malignant melanomas, may  reveal prognostic markers, potential therapeutic targets,  and abnormally activated pathways. This information may facilitate the development of more effective therapeutic options for this aggressive and heterogeneous tumor.

References

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