MicroRNA paraffin-based studies in osteosarcoma reveal reproducible independent prognostic profiles at 14q32

Abstract

Background: Although microRNAs (miRNAs) are implicated in osteosarcoma biology and chemoresponse, miRNA prognostic models are still needed, particularly because prognosis is imperfectly correlated with chemoresponse. Formalin-fixed, paraffin-embedded tissue is a necessary resource for biomarker studies in this malignancy with limited frozen tissue availability.

Methods: We performed miRNA and mRNA microarray formalin-fixed, paraffin-embedded assays in 65 osteosarcoma biopsy and 26 paired post-chemotherapy resection specimens and used the only publicly available miRNA dataset, generated independently by another group, to externally validate our strongest findings (n = 29). We used supervised principal components analysis and logistic regression for survival and chemoresponse, and miRNA activity and target gene set analysis to study miRNA regulatory activity.

Results: Several miRNA-based models with as few as five miRNAs were prognostic independently of pathologically assessed chemoresponse (median recurrence-free survival: 59 months versus not-yet-reached; adjusted hazards ratio = 2.90; P = 0.036). The independent dataset supported the reproducibility of recurrence and survival findings. The prognostic value of the profile was independent of confounding by known prognostic variables, including chemoresponse, tumor location and metastasis at diagnosis. Model performance improved when chemoresponse was added as a covariate (median recurrence-free survival: 59 months versus not-yet-reached; hazard ratio = 3.91; P = 0.002). Most prognostic miRNAs were located at 14q32 - a locus already linked to osteosarcoma - and their gene targets display deregulation patterns associated with outcome. We also identified miRNA profiles predictive of chemoresponse (75% to 80% accuracy), which did not overlap with prognostic profiles.

Conclusions: Formalin-fixed, paraffin-embedded tissue-derived miRNA patterns are a powerful prognostic tool for risk-stratified osteosarcoma management strategies. Combined miRNA and mRNA analysis supports a possible role of the 14q32 locus in osteosarcoma progression and outcome. Our study creates a paradigm for formalin-fixed, paraffin-embedded-based miRNA biomarker studies in cancer.

Figure 1

miRNAs associated with recurrence and survival. miRNAs significantly associated with (A) recurrence and (B) survival (P < 0.01). Color map displays univariate HRs for recurrence. Bold text denotes miRNAs located at 14q32. FDR, false discovery rate; HR, hazard ratio.

Figure 2

Recurrence risk prediction. (A, D) Kaplan-Meier analysis of recurrence risk (supervised principal components analysis for the 22 and 5 miRNA profiles). (B, E) Kaplan-Meier analysis of recurrence risk for the 22 and 5 miRNA profiles in addition to chemoresponse as a clinical covariate in the model. (C, F) Kaplan-Meier analysis of recurrence risk using both 22 and 5 miRNA profiles and chemoresponse as categorical variables (three-group analysis). (G) 22 miRNA profile. (H) 5 miRNA profile.

Figure 3

miRNAs are prognostic of recurrence and survival in an independent external dataset. Our prognostic miRNAs were used to generate models of OS in an independent external validation dataset. Of the 22 miRNA profile, 18 miRNAs could be mapped on the platform used in the external dataset. (A-C) These overlapping miRNAs (as well as smaller subsets of this profile) were used to generate survival risk prediction models. A consistent discriminatory trend was observed in the external dataset, despite a smaller sample size, fewer events and different array platform.

Figure 4

Recurrence risk prediction in relevant homogeneous patient subsets and using miRNA gene targets. (A) Kaplan-Meier recurrence analysis with the five miRNA profile in the non-metastatic (only) subset of the cohort. (B) Kaplan-Meier recurrence analysis with the five miRNA profile in the subset of patients that received MAP (only). (C) Kaplan-Meier recurrence analysis using a subset of gene targets of prognostic miRNAs.