Integrated Assessment of Circulating Cell-Free MicroRNA Signatures in Plasma of Patients with Melanoma Brain Metastasis

Abstract

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.

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

“…Only plasma samples were included in the study and from this point on all the samples will be referred to as plasma. The samples were all analyzed using the HTG EdgeSeq miR WTA [ 32 ]. All of the patients analyzed had a median follow-up of 9.7 months and received CII (ipilimumab, nivolumab, pembrolizumab, or the combination of ipilimumab and nivolumab) as first line treatment.…”

“…In evaluating the utility of cfmiRs, it is important to find cfmiRs that have applicability in real-time monitoring of melanoma patient’s disease status before and during CII(s) to evaluate response. Recently, we have shown that specific cfmiR patterns found in MBM patients’ plasma may have utility in monitoring melanoma patients undergoing treatment [ 32 ]. Our hypothesis is that specific cfmiRs have a better utility compared to serum LDH levels in the assessment of melanoma patients undergoing CII.…”

“…Also, significant efforts have been made in determining circulating cell-free miRs (cfmiRs) and exosomal miRs [ 29 , 31 ]. Recently, by using HTG EdgeSeq miR WTA, we found cfmiR signatures in plasma samples from patients with melanoma brain metastasis (MBM) [ 32 ]. Furthermore, we unraveled common cfmiR signatures in pre-operative plasma samples taken from stage III and IV melanoma patients receiving CIIs [ 32 ].…”

“…Recently, by using HTG EdgeSeq miR WTA, we found cfmiR signatures in plasma samples from patients with melanoma brain metastasis (MBM) [ 32 ]. Furthermore, we unraveled common cfmiR signatures in pre-operative plasma samples taken from stage III and IV melanoma patients receiving CIIs [ 32 ]. The advantage of using HTG EdgeSeq miR WTA to study cfmiRs is that we can directly profile and quantify >2000 miRs found in plasma samples by next-generation sequencing (NGS) to identify signature patterns [ 32 ].…”

“…Furthermore, we unraveled common cfmiR signatures in pre-operative plasma samples taken from stage III and IV melanoma patients receiving CIIs [ 32 ]. The advantage of using HTG EdgeSeq miR WTA to study cfmiRs is that we can directly profile and quantify >2000 miRs found in plasma samples by next-generation sequencing (NGS) to identify signature patterns [ 32 ]. Moreover, compared to other cfNA assays, the assay requires a minimal amount of plasma and no tedious extraction procedures.…”