Novel Regional Approaches To In-transit Melanoma: Two-pronged Approach For Maximal Drug Efficacy
Jamie L. Kaplan, M.D, Antonio J. Forte, MD, PhD, Emmanuel Gabriel, MD, PhD.
Mayo Clinic Florida, Jacksonville, FL, USA.
PURPOSE: For patients with advanced melanoma, including in-transit (IT) disease, there are limited effective local/intra-lesional therapies. Intra-tumoral phenylephrine and other agents combined with chemotherapy have been utilized for the treatment of cutaneous head/neck SCC with significant local tumor response. Additionally, it is known that melanoma lacks HER2 expression, and thus, anti-HER2 antibodies are not part of their management. In these studies, we combined phenylephrine with melphalan to determine if phenylephrine could augment chemotherapeutic responses, as well as sought to induce neu-expression (mouse homologue of HER2) as a potential target for anti-neu antibody.
METHODS: Mice bearing B16 melanoma tumors were treated with intra-tumoral injection of melphalan with/without phenylephrine. Treatment consisted of twice-weekly injections for 4 weeks. Tumor volumes and event-free survival were recorded. Kaplan-Meier methods were used for time-to-event analysis. Immunohistochemistry was performed on harvested tumors to analyze the immune cell population. Additionally, RT-PCR was used to construct a rat neu-expressing vector using a lentivirus-vector (pLenti6.3). Neu-expressing pLenti6.3 was transfected in-vitro into B16 mouse melanoma cells. Neu-expression was detected and quantified by Western blot analysis and flow cytometry.
RESULTS: Kaplan-Meier analysis showed the highest event-free rate with the combination treatment of intra-tumoral melphalan plus phenylephrine, which was statistically significant compared to melphalan alone, p<0.01. IHC analysis of harvested tumors showed increased infiltration of a CD11b+ cell population, suggesting an immune-mediated response to B16 treated with intra-tumoral melphalan and phenylephrine. After transfection of our lentivirus-vector into B16, Western blot analysis qualitatively confirmed expression of neu. The neu-expressing cell line was designated B16/neu. As a positive control, we confirmed HER2 expression following transfection with our established lentivirus-vector encoding HER2. We quantitatively detected 50% expression of neu among B16/neu.
CONCLUSION: Both pre-clinical studies demonstrate promising results. The addition of phenylephrine to intra-tumoral melphalan injection significantly increased tumor responses and results suggest there may be an immune-mediated response with increased infiltration of CD11b+ macrophages, which warrants further studies. Current ongoing experiments using inoculated B16/neu will determine whether anti-neu antibody will generate anti-tumor responses against neu protein. Future experiments will combine anti-neu antibody with immune checkpoint blockade to determine if responses can be further augmented.
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