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.

This study examined the role of the ubiquitin E3-ligase RNF123 in modulating downstream NF-κB1 targets in glioblastoma (GB) tumor progression. Our findings revealed an oncogenic pathway (miR-155-5p-RNF123-NF-κB1-p50-SerpinE1) that may represent a new therapeutic target pathway for GB patients with isocitrate dehydrogenase 1 and 2 (IDH) WT (wild type). Mechanistically, we demonstrated that RNF123 is downregulated in IDH WT GB patients and leads to the reduction of p50 levels. RNA-sequencing, reverse-phase protein arrays, and in vitro functional assays on IDH WT GB cell lines with RNF123 overexpression showed that SerpinE1 was a downstream target that is negatively regulated by RNF123. SERPINE1 knockdown reduced the proliferation and invasion of IDH WT GB cell lines. Both SerpinE1 and miR-155-5p overexpression negatively modulated RNF123 expression. In clinical translational analysis, RNF123, SerpinE1, and miR-155-5p were all associated with poor outcomes in GB patients. Multivariable analysis in IDH WT GB patients showed that concurrent low RNF123 and high SerpinE1 was an independent prognostic factor in predicting poor overall survival (p < 0.001, hazard ratio (HR) = 2.93, 95% confidence interval (CI) 1.7–5.05), and an increased risk of recurrence (p < 0.001, relative risk (RR) = 3.56, 95% CI 1.61–7.83).

Primary cutaneous melanoma frequently metastasizes to distant organs including the brain. Identification of cell-free microRNAs (cfmiRs) found in the blood can be used as potential body fluid biomarkers for detecting and monitoring patients with melanoma brain metastasis (MBM). In this pilot study, we initially aimed to identify cfmiRs in the blood of MBM patients. Normal donors plasma (healthy, n = 48) and pre-operative MBM patients’ plasma samples (n = 36) were compared for differences in >2000 microRNAs (miRs) using a next generation sequencing (NGS) probe-based assay. A 74 cfmiR signature was identified in an initial cohort of MBM plasma samples and then verified in a second cohort of MBM plasma samples (n = 24). Of these, only 58 cfmiRs were also detected in MBM tissues (n = 24). CfmiR signatures were also found in patients who have lung and breast cancer brain metastasis (n = 13) and glioblastomas (n = 36) compared to MBM plasma samples. The 74 cfmiR signature and the latter cfmiR signatures were then compared. We found a 6 cfmiR signature that was commonly upregulated in MBM plasma samples in all of the comparisons, and a 29 cfmiR signature that distinguishes MBM patients from normal donors’ samples. In addition, we assessed for cfmiRs in plasma (n = 20) and urine (n = 14) samples collected from metastatic melanoma patients receiving checkpoint inhibitor immunotherapy (CII). Pre- and post-treatment samples showed consistent changes in cfmiRs. Analysis of pre- and post-treatment plasma samples showed 8 differentially expressed (DE) cfmiRs that overlapped with the 35 cfmiR signature found in MBM patients. In paired pre-treatment plasma and urine samples receiving CII 8 cfmiRs overlapped. This study identified specific cfmiRs in MBM plasma samples that may potentially allow for assessment of melanoma patients developing MBM. The cfmiR signatures identified in both blood and urine may have potential utility to assess CII responses after further validation.

Circulating tumor cells (CTCs) have been studied using multiple technical approaches for interrogating various cancers, as they allow for the real-time assessment of tumor progression, disease recurrence, treatment response, and tumor molecular profiling without the need for a tumor tissue biopsy. Here, we will review studies from the last 15 years on the assessment of CTCs in cutaneous melanoma patients in relation to different clinical outcomes. The focus will be on CTC detection in blood samples obtained from cutaneous melanoma patients of different clinical stages and treatments utilizing multiple platforms. Assessment of multiple molecular melanoma-associated antigen (MAA) markers by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was the most common assay allowing for the improvement of assay sensitivity, tumor heterogeneity, and to predict patient outcomes. Multicenter studies demonstrate the utility of CTC assays reducing the bias observed in single- center trials. The recent development of CTC enrichment platforms has provided reproducible methods. CTC assessment enables both multiple mRNAs and DNAs genomic aberration profiling. CTC provides specific important translational information on tumor progression, prediction of treatment response, and survival outcomes for cutaneous melanoma patients. The molecular studies on melanoma CTCs have provided and may set standards for other solid tumor CTC analyses.

Prostate cancer (PCa) is the most common cancer in men. Prostate-specific antigen screening is recommended for the detection of PCa. However, its specificity is limited. Thus, there is a need to find more reliable biomarkers that allow non-invasive screening for early-stage PCa. This study aims to explore urine microRNAs (miRs) as diagnostic biomarkers for PCa. We assessed cell-free miR (cfmiR) profiles of urine and plasma samples from pre- and post-operative PCa patients (n = 11) and normal healthy donors (16 urine and 24 plasma) using HTG EdgeSeq miRNA Whole Transcriptome Assay based on next-generation sequencing. Furthermore, tumor-related miRs were detected in formalin-fixed paraffin-embedded tumor tissues obtained from patients with localized PCa. Specific cfmiRs signatures were found in urine samples of localized PCa patients using differential expression analysis. Forty-two cfmiRs that were detected were common to urine, plasma, and tumor samples. These urine cfmiRs may have potential utility in diagnosing early-stage PCa and complementing or improving currently available PCa screening assays. Future studies may validate the findings.

The role of post-translational modifications (PTM) of the key epigenetic factor DNMT1 protein has not been well explored in cutaneous metastatic melanoma progression. The acetylated DNMT1 (ac-DNMT1) protein level was assessed using an anti-acetylated lysine antibody in a clinically annotated melanoma patient tumor specimen cohort. In this study, we showed that surgically resected tumors have significantly higher DNMT1 protein expression in metastatic melanoma (stage III metastasis n = 17, p = 0.0009; stage IV metastasis n = 164, p = 0.003) compared to normal organ tissues (n = 19). Additionally, reduced ac-DNMT1 protein levels were associated with melanoma progression. There was a significant inverse correlation between ac-DNMT1 and DNMT1 protein levels in stage IV metastatic melanoma (r = −0.18, p = 0.02, n = 164). Additionally, ac-DNMT1 protein levels were also significantly positively correlated with TIP60 (r = 0.6, p < 0.0001) and USP7 (r = 0.74, p < 0.0001) protein levels in stage IV metastatic melanoma (n = 164). Protein analysis in metastatic melanoma tumor tissues showed that with high ac-DNMT1 (p = 0.006, n = 59), or concurrent high ac-DNMT1 with low DNMT1 (p = 0.05, n = 27), or high TIP60 (p = 0.007, n = 41), or high USP7 (p = 0.01, n = 48) consistently showed better 4-year melanoma-specific survival (MSS). Multivariate Cox proportional hazard analysis showed that ac-DNMT1 level is a significant independent factor associated with MSS (HR, 0.994; 95% confidential interval (CI), 0.990–0.998; p = 0.002). These results demonstrated that low ac-DNMT1 levels may represent an important regulatory factor in controlling metastatic melanoma progression and a promising factor for stratifying aggressive stage IV metastasis.