Early Postoperative Circulating miR-483-5p Is a Prognosis Marker for Adrenocortical Cancer

Abstract

We have previously identified serum miR-483-5p as a preoperative diagnosis and prognosis biomarker for adrenocortical cancer (ACC). Here, we aimed to determine whether circulating miR-483-5p levels measured 3 months post-operatively distinguished patients with good prognosis (no recurrence for at least 3 years; NR3yrs) from patients with poor prognosis (recurrence or death within 3 years after surgery; R < 3yrs). We conducted a single-center retrospective analysis using sera from 48 patients with ACC that were initially non-metastatic and treated by surgery. Sera sampled within 3 months after surgery were available in 26 patients. MiR-483-5p absolute circulating levels were measured using quantitative PCR. Thirteen patients showed a recurrence before 3 years (=R < 3yrs). Thirteen patients showed no recurrence within 3 years, including 11 patients with a follow-up longer than 3 years (=NR3yrs). Serum miR-483-5p levels were higher in R < 3yrs than in NR3yrs: 1,541,990 ± 428,377 copies/mL vs. 388,457 ± 62,169 copies/mL (p = 0.002). Receiver operating characteristic analysis showed that a value of 752,898 copies/mL distinguished R < 3yrs from NR3yrs with 61.5% sensitivity (CI 31.6-86.1) and 100% specificity (CI 71.5-100) with an area under the curve of 0.853. Patients with a value below this threshold had a significantly longer recurrence-free and overall survival. In multivariate analysis, miR-483-5p provided the single best prognostic value for recurrence-free survival (RFS) (hazard ratio (HR) for recurrence 5.98, p < 0.011) but not for overall survival. Our study suggests that serum miR-483-5p is a potent early post-operative biomarker for ACC prognosis that might be a better predictor of RFS than currently used markers.

Keywords: adrenocortical carcinoma; biomarker; circulating microRNA; early prognosis; miR-483-5p; recurrence.

Figure 1

Schematic diagram of the study population. Details are described in the text (“Patients” section). R < 3yrs: patients with recurrence occurring before 3 years post-surgery; NR3yrs: patients with no recurrence within 3 years post-surgery.

Figure 2

Post-operative serum levels of miR-483-5p in ACC patients. (A) Comparison of miR-483-5p levels in NR3yrs (n = 11) and R < 3yrs groups (n = 13). Cycle threshold (Ct) values were converted to absolute number of copies/mL using a dilution series of a known input quantity of synthetic target miRNA run simultaneously with the experimental samples. Statistically significant difference was assessed using Mann–Whitney test (p = 0.0025). Black circles and triangles: patients who received mitotane adjuvant therapy after serum sampling. White circles in the NR3yrs group: Patients 5 and 8 received mitotane adjuvant therapy 0.9 and 1.8 months before serum sampling, respectively. Red symbols: patients with no mitotane therapy throughout their follow-up. (B) Comparison of miR-483-5p levels in NR3yrs and R < 3yrs patients in function of ENSAT stage (stage I: n = 2; stage II: n = 12; stage III: n = 10); ns: non-significant. The lines within the scatter plot represent the mean ± S.E.M of miRNA copy number/mL.

Figure 3

Receiver operating characteristic (ROC) curve analysis for miR-483-5p absolute copy number in ACC patients. Three months-post-operative serum samples can discriminate between NR3yrs and R < 3yrs patients with significant accuracy. The area under the curve (AUC), the 95% confidence interval (CI) and the p value are indicated.

Figure 4

Time to recurrence or follow-up with no recurrence in function of miR-483-5p copy number.

Figure 5

Kaplan–Meier survival analysis of all 26 patients (NR3yrs n = 11, R < 3yrs n = 13 and two patients with no recurrence and follow-up <3 years) according to miR-483-5p circulating levels. (A) Recurrence-free survival (RFS). High levels of miR-483-5p (>752,898 copies/mL cut-off value) within the 3-month post-surgery period predict a shorter RFS (Log-rank p = 0.0005). (B) Overall survival. High levels of miR-483-5p (>752,898 copies/mL cut-off value) within the 3-month post-surgery period predict a shorter OS (Log-rank p = 0.007).

Figure 6

Kaplan–Meier survival analysis according to European Network for the Study of Adrenal Tumors (ENSAT) stage II and stage III (24 patients). No significant differences were observed between ENSAT stage II and stage III for Recurrence-free survival (A) or Overall survival (B) predictions in this cohort.