Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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A direct plasma assay of circulating microRNA-210 of hypoxia can identify early systemic metastasis recurrence in melanoma patients.

Author(s): Ono S, Oyama T, Lam S, Chong K, Foshag LJ, Hoon DS

Publication: Oncotarget, 2015, Vol. 6, Page 7053-64

PubMed ID: 25749524 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate if miR-210 could be amplified directly from plasma without further purification and if miR-210 levels were correlated with disease stage and patient outcome. Levels of miR-210 in formalin-fixed paraffin-embedded primary tumor and metastasis were also compared. Blood was collected in the initial study from six healthy donors and stage III (n=20) and IV (n=26) melanoma patients with current disease into sodium citrate tubes. To investigate differences in miR-210 levels in disease-free patients, sodium-citrate blood was collected from 35 healthy donors and 60 stage III and 70 stage IV disease-free melanoma patients. To investigate the association of miR-210 levels and disease-free survival, sodium-citrate blood was obtained at multiple timepoints including 13-95 days post-surgery from 88 stage III patients and segregated to 46 patients with reoccurrence <2 years after surgery and 42 patients with no reoccurrence >5 years after surgery. Blood was centrifuged at 1,300 × g for 10 min to obtain plasma and stored in liquid nitrogen. Proteins in plasma were deactivated and solubilized with Tween-20 and the specimen was centrifuged at 9,000 x g for 5 min and then purified with Centri-Sep 8-well strips kit before real-time PCR amplification of miR-210. FFPE specimens included primary tumors (n=21), metastasis from lymph node (n=43), lung (n=16), bowel (n=12), liver (n=6), distant skin (n=3), brain (n=2) and others (n=5) but no details of processing were included. RNA was extracted from ten 10 µm-thick sections, reverse transcribed using qScript cDNA Synthesis Kit, and quantified using the real-time PCR-based PerfeCTa microRNA Assay.

   Summary of Findings:

   Although miR-210 was detectable in all specimens regardless of the inclusion of a Centri-Sep purification after reverse transcription, the CT values were significantly higher when amplification was conducted without use of a Centri-Sep column (P<0.001). Importantly, higher levels of miR-210 were found in plasma from patients with Stage III and Stage IV disease than in plasma from healthy patients (P=0.002 and P=0.001, respectively) and in plasma from stage IV patients than Stage III patients (P=0.008). miR-210 levels were also significantly higher in the plasma from disease-free melanoma patients than healthy controls (P<0.001). Using plasma direct amplification found increased miR-210 levels prior to clinical recurrence (P=0.012) and higher levels of miR-210 were associated with lower disease-free survival (P<0.001) and melanoma-specific survival (P<0.001). Importantly, unlike miR-210 levels in plasma LDH levels were not a significant predictor of disease free survival (P=0.081) and using a combination of LDH and plasma miR-210 levels was not a significantly better predictor of disease-free survival than miR-210 levels alone. miR-210 levels were higher in lymph node and distant organ metastasis compared to the primary tumor (P=0.002 and P<0.001, respectively).

   Biospecimens

   • Bodily Fluid - Plasma
   • Tissue - Skin
   • Tissue - Lung
   • Tissue - Small Bowel
   • Tissue - Lymph Node
   • Tissue - Brain
   • Tissue - Liver

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Melanoma
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Healthy Metastatic melanoma Stage III melanoma Stage IV melanoma
   Biospecimen Acquisition	Biospecimen location	Primary tumor Lymph node Distant metastasis
   Real-time qRT-PCR Specific	Template modification	Not purified Purified with Centri-Sep
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-221
   Analyte Extraction and Purification	Analyte purification	None Purified with Centri-Sep column after reverse transcription

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