Quantitative analysis of neuropilin-2 expression in pigmented lesions and melanoma
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The prevalence of skin cancer has been continuously increasing in the American population over the last 3 decades. Melanoma, the most serious and dangerous form, causes the most skin cancer related deaths. If melanoma is found early enough, it can be treated and cured. Current therapy for melanoma is based on the stage of the disease. It is crucial to identify and treat the melanoma before metastasis occurs as the risk of death greatly increases once the disease becomes widespread. The processes of angiogenesis and lymphangiogenesis as well as changes in the extracellular environment all play significant roles in the expansion of a primary melanoma and ultimately metastasis. Neuropilins, specifically Neuropilin-2 (NRP2), have been found to be associated with both angiogenesis and lymphangiogenesis and thus metastasis. Since it is critical to identify and treat melanoma before it advances to metastasis, the relationship between NRP2 and tumor progression may prove to be extremely valuable as a prognostic marker for primary melanoma tumors. In addition, it may serve to identify subsets of early stage tumors that would benefit from adjuvant therapy. This study aimed to demonstrate if there is a relationship between the level of expression of NRP2 in primary melanocytic lesions and the prognosis of melanoma, and determine the potential utility of NRP2 as a biomarker for melanoma. To do so, the levels of expression of NRP2 were analyzed in formalin-fixed, paraffin-embedded, tissue samples of nevi, primary melanomas, and metastatic melanomas. Sample RNA was extracted and converted to eDNA. Quantitative RT-PCR was then used to amplify eDNA and appropriate normalizations were performed. Luciferase RNA was added at the start of processing as an exogenous control and was used to correct for procedural variation between samples. Furthermore, Actin, beta (ACTB) and Melan-A were used as reference genes to correct for differences in tissue and lesion size respectively. To analyze the data, standard curves were developed for NRP2, ACTB, and Melan-A to calculate the copy numbers for each gene in each sample. When normalized by both ACTB and Melan-A, the differences in the level of expression of NRP2 between nevi and primary melanomas (p=0.94422), nevi and metastatic melanomas (p=0.29538), and primary melanomas and metastatic melanomas (p=0.32827) all failed to show statistical significance. In addition when normalized by only ACTB, there is no statistical significant difference in the level of expression of NRP2 between nevi and primary melanomas (p=0.21001), or primary and metastatic melanomas (p=0.30874). However, there is a difference between nevi and metastatic melanomas (p=0.00472) that is statistically significant when just normalized by ACTB. Furthermore, when just normalized by Melan-A, there is no significant difference in the level of expression of NRP2 between nevi and primary melanomas (p=0.3152) or primary melanomas and metastatic melanomas (p=0.14179). However, there is a statistically significant difference in the level of expression of NRP2 between nevi and metastatic melanomas (p=0.01736). The fact that there is a statistically significant difference (p less than 0.05) in the level of expression of NRP2 between nevi and primary melanomas demonstrates its potential ability to be used as a biomarker. However, the inability to establish statistical significance between sample groups may be due to the small sample size. Further studies with an increased sample size may better show the relationship between the levels of NRP2 and the stage of melanoma. Still, NRP2 shows promise as both a diagnostic and prognostic biomarker for melanoma.
Thesis (M.A.)--Boston University