Melanoma MicroRNA signature predicts post-recurrence survival

Abstract

Purpose: To identify a melanoma microRNA (miRNA) expression signature that is predictive of outcome and then evaluate its potential to improve risk stratification when added to the standard-of-care staging criteria.

Experimental design: Total RNA was extracted from 59 formalin-fixed paraffin-embedded melanoma metastases and hybridized to miRNA arrays containing 911 probes. We then correlated miRNA expression with post-recurrence survival and other clinicopathologic criteria.

Results: We identified a signature of 18 miRNAs whose overexpression was significantly correlated with longer survival, defined as more than 18 months post-recurrence survival. Subsequent cross-validation showed that a small subset of these miRNAs can predict post-recurrence survival in metastatic melanoma with an estimated accuracy of 80.2% (95% confidence interval, 79.8-80.6%). In contrast to standard-of-care staging criteria, a six-miRNA signature significantly stratified stage III patients into "better" and "worse" prognostic categories, and a multivariate Cox regression analysis revealed the signature to be an independent predictor of survival. Furthermore, we showed that most miRNAs from the signature also showed differential expression between patients with better and worse prognoses in the corresponding paired primary melanoma.

Conclusions: MiRNA signatures have potential as clinically relevant biomarkers of prognosis in metastatic melanoma. Our data suggest that molecularly based models of risk assessment can improve the standard staging criteria and support the incorporation of miRNAs into such models.

Figure 1

Identification of miRNAs differentially expressed in melanoma patients with longer post-recurrence survival (≥ 1.5 years). A. Mean normalized fluorescence for each microRNA (black dots) indicating expression levels as measured by microarray in patients surviving less than 1.5 years post-surgical resection compared to patients surviving 1.5 years or longer. MiRNAs significantly elevated in the longer survival group are indicated as red dots (FDR < 5%). Diagonal lines demarcate 1.5-fold difference levels in expression between the two groups. B. Real time PCR validation of microarray data. Expression levels of miRNAs were measured by real time RT-PCR in a subset on 10 samples, including 5 patients who survived at least 1.5 years after their recurrence and 5 patients who died in less than 1.5 years post-recurrence. Cycle threshold (Ct) values for each miRNA were normalized versus the housekeeping small RNA RNU44 (ΔCt) and represented as 2−ΔCt. The x axis shows 7 individual miRNA species examined both by microarray and real-time PCR; the y axis represents the mean value of the ratio of expression of the 7 miRNA species in microarray and real-time PCR (qRT-PCR).

Figure 2

Kaplan-Meier estimates of post-recurrence survival stratified by (A) stage at recurrence, (B) site of metastasis, (C) “better prognosis” and “worse prognosis” groups predicted by the pre-validated miRNA signature, and (D) “Better prognosis” and “worse prognosis” groups predicted by the optimal predictor of post-recurrence survival that combines the pre-validated miRNA signature and stage.

Figure 3

Expression of predictor miRNAs in paired primary-metastatic melanomas relative to their levels in congenital nevi. Box-plots in logarithmic scale represent the expression of the 6 miRNAs most commonly included in the ‘predictor’ set, measured by quantitative PCR in paired primary-metastatic samples, relative to their respective levels in congenital nevi. Data was normalized to small nuclear RNA RNU44. The "box" part contains the middle 50% of the data, the line in the box indicates the median value, and the whiskers indicate the minimum and maximum data values.