A Pilot Study Comparing the Efficacy of Lactate Dehydrogenase Levels Versus Circulating Cell-Free microRNAs in Monitoring Responses to Checkpoint Inhibitor Immunotherapy in Metastatic Melanoma Patients.

Serum lactate dehydrogenase (LDH) is a standard prognostic biomarker for stage IV melanoma patients. Often, LDH levels do not provide real-time information about the metastatic melanoma patients' disease status and treatment response. Therefore, there is a need to find reliable blood biomarkers for improved monitoring of metastatic melanoma patients who are undergoing checkpoint inhibitor immunotherapy (CII). The objective in this prospective pilot study was to discover circulating cell-free microRNA (cfmiR) signatures in the plasma that could assess melanoma patients' responses during CII. The cfmiRs were evaluated by the next-generation sequencing (NGS) HTG EdgeSeq microRNA (miR) Whole Transcriptome Assay (WTA; 2083 miRs) in 158 plasma samples obtained before and during the course of CII from 47 AJCC stage III/IV melanoma patients' and 73 normal donors' plasma samples. Initially, cfmiR profiles for pre- and post-treatment plasma samples of stage IV non-responder melanoma patients were compared to normal donors' plasma samples. Using machine learning, we identified a 9 cfmiR signature that was associated with stage IV melanoma patients being non-responsive to CII. These cfmiRs were compared in pre- and post-treatment plasma samples from stage IV melanoma patients that showed good responses. Circulating miR-4649-3p, miR-615-3p, and miR-1234-3p demonstrated potential prognostic utility in assessing CII responses. Compared to LDH levels during CII, circulating miR-615-3p levels were consistently more efficient in detecting melanoma patients undergoing CII who developed progressive disease. By combining stage III/IV patients, 92 and 17 differentially expressed cfmiRs were identified in pre-treatment plasma samples from responder and non-responder patients, respectively. In conclusion, this pilot study demonstrated cfmiRs that identified treatment responses and could allow for real-time monitoring of patients receiving CII.

RASSF8 regulates progression of cutaneous melanoma through nuclear factor-κb.

Our group previously demonstrated that the RASSF1 gene has a significant tumor suppressor role in cutaneous melanoma. The RASSF8 gene is a member of the N-terminal RASSF gene family. Previously, we identified RASSF8 (HOJ1, NCBI Gene ID:11228) expression in cutaneous melanoma; however the functional role of RASSF8 in melanoma is not known. RASSF8 expression was assessed in melanoma cell lines and tumors of different AJCC stages. Results indicated that RASSF8 expression was low in metastatic melanoma lines and decreased with melanoma progression. We then explored the mechanism of RASSF8 downregulation in melanoma by assessing methylation of RASSF8 and demonstrated that methylation of RASSF8 gene promoter was higher in advanced than in early stages melanomas. Functional activity of RASSF8 in melanoma lines by knockdown and overexpression of RASSF8 demonstrated that RASSF8 expression significantly inhibited cell growth, cell migration and invasion, whereas knockdown of RASSF8 expression significantly increased cell growth, cell migration and invasion of melanoma cells by increasing expression of P65 and its downstream target IL-6. Moreover RASSF8 was found to induce apoptosis in melanoma cells by activating the P53-P21 pathway, and also in vivo studies demonstrated that inhibiting RASSF8 increases the tumorigenic properties of human melanoma xenografts. These results suggest that RASSF8 plays a significant role in suppressing the progression of cutaneous melanoma.

Endosialin Expression in Metastatic Melanoma Tumor Microenvironment Vasculature: Potential Therapeutic Implications.

Ontuxizumab (MORAb-004) is a humanized recombinant antibody targeting endosialin (TEM-1, CD248). We conducted an analysis of endosialin expression in metastatic melanoma specimens using the anti-endosialin rat anti- MAb 9G5, in order to determine the potential of endosialin as a therapeutic target within the tumor microenvironment vasculature. Endosialin expression in paraffin-embedded archival tissue block (PEAT) melanoma tissues was assessed using immunohistochemistry (IHC) with the anti-endosialin, MAb 9G5, in the vessels of American Joint Commission on Cancer (AJCC) Stage III (n = 18) and Stage IV (n = 48) specimens. IHC for endosialin expression was further performed on a TMA that included 136 Stage IV and 33 paired Stage III melanoma specimens. BRAF mutation (mt) was also evaluated in individual melanoma specimens and as well as the TMA. Analysis showed 70 % of melanoma specimens (n = 46) were positive for endosialin expression. There was no significant difference in endosialin and BRAFmt expression between stages III vs. IV specimens. Endosialin expression was detected in 86 % (n = 117) of stage IV TMA specimens, while no expression was detected in 29 normal tissue controls. MAb 9G5 detects the presence of endosialin in the microenvironment tumor vasculature of most metastatic melanoma tissues, regardless of clinical stage and presence of BRAFmt. Endosialin may be a potential therapeutic target by virtue of its selective expression in metastatic melanoma relative to normal tissues.