Supplemental materials

Supplemental File 1 : rdml-file containing miRNA expression data and clinical sample annotation for training and validation sets.

Supplemental File 2 : rdml-file containing mRNA expression data and clinical sample annotation for the first validation set.

Supplemental File 3 : rdml-file containing miRNA expression data and clinical sample annotation for FFPE sample set.

Supplemental Table 1 : Comparison of 4 multigene prognostic signatures by means of Kaplan-Meier statistics, multivariate analysis and performance in the first validation set.

59 mRNA 25 miRNA miRNA1 miRNA2 overall survival all patients 2.15E-11 9.98E-07 9.78E-05 3.10E-03 Kaplan -Meier HR patiens 3.99E-02 1.73E-01 6.84E-01 6.99E-01 p-value LR patients 2.43E-02 5.46E-01 8.01E-01 8.78E-01 performance (AUC) 0.84 0.74 0.71 0.66 odds ratio 8.45 2.98 2.99 2.50 multi-variate p-value 1.95E-03 9.25E-02 5.95E-02 1.19E-01 progression free survival all patients 2.67E-08 1.41E-05 1.54E-3 1.23E-03 Kaplan -Meier HR patiens 5.43E-02 1.79E-01 6.66E-01 6.48E-01 p-value LR patients 3.01E-01 1.13E-01 6.83E-01 1.09E-01 performance (AUC) 0.78 0.72 0.68 0.67 odds ratio 5.18 3.30 2.80 2.59 multi-variate p-value 6.81E-03 3.31E-02 4.73E-02 5.74E-02 1prognostic miRNA signature by Schulte et al., 2Prognostic miRNA signature by Bray et al., AUC: area under the curve, LR: low-risk patients, HR: high-risk patients, IR: intermediate-risk patients.

Supplemental Table 2: Ontology Biological Process terms associated with the mRNA and miRNA signatures.

mRNA enriched GO BP terms intracellular signaling cascade cellular process nervous system development communication signal transduction metabolic process primary metabolic process cell adhesion cell motion cell-cell adhesion cell cycle ectoderm development cellular glucose homeostasis metabolic process developmental process system development cellular amino acid and derivative metabolic process phosphate metabolic process apoptosis female gamete generation homeostatic process nucleobase, nucleoside, nucleotide and nucleic acid metabolic process linked signal transduction cell-matrix adhesion amino acid transport phosphate transport cytokinesis mitosis nucleobase, nucleoside, nucleotide and nucleic acid transport negative regulation of apoptosis

miRNA enriched GO BP terms actin cytoskeleton organization and biogenesis actin filament based process adaptive immune response adaptive immune response anatomical structure formation biosynthetic process camp mediated signaling cell cell signaling cell cycle checkpoint cell cycle

cell cycle phase cell cycle process cell division cell matrix adhesion cell substrate adhesion cell surface receptor linked signal transduction cellular biosynthetic process cellular response to stimulus chromatin assembly or disassembly chromatin modification chromatin remodeling organization and biogenesis chromosome segregation coagulation cytokinesis DNA damage checkpoint DNA damage response signal transduction DNA dependent DNA replication DNA integrity checkpoint DNA metabolic process DNA packaging DNA recombination DNA repair DNA replication endosome transport enzyme linked receptor protein signaling pathway epidermal growth factor receptor signaling pathway establishment and or maintenance of chromatin architecture G1 S transition of mitotic cell cycle G protein signaling coupled to camp nucleotide second messenger homophilic cell adhesion I kappab kinase NF KappaB cascade immune system process inflammatory response interphase interphase of mitotic cell cycle M phase M phase of mitotic cell cycle meiosis I meiotic cell cycle membrane fusion membrane organization and biogenesis mesoderm development metal ion transport microtubule cytoskeleton organization and biogenesis mitochondrion organization and biogenesis mitosis mitotic cell cycle mitotic cell cycle checkpoint mRNA metabolic process mRNA processing negative regulation of DNA metabolic process negative regulation of signal transduction neuron development neuron differentiation nucleobase nucleoside and nucleotide metabolic process one carbon compound metabolic process peptidyl tyrosine modification positive regulation of immune response positive regulation of signal transduction protein DNA complex assembly protein folding protein RNA complex assembly protein secretion ras protein signal transduction receptor mediated endocytosis regulation of anatomical structure morphogenesis regulation of blood pressure regulation of cell cycle regulation of cyclin dependent protein kinase activity regulation of DNA metabolic process regulation of DNA replication regulation of i kappab kinase nf kappab cascade regulation of immune response regulation of immune system process regulation of lymphocyte activation regulation of mitosis regulation of multicellular organismal process regulation of signal transduction response to DNA damage stimulus response to endogenous stimulus response to wounding ribonucleoprotein complex biogenesis and assembly RNA splicing RNA splicing via transesterification reactions small gtpase mediated signal transduction spliceosome assembly T cell activation tissue remodeling transcription from RNA polymerase III promoter translation transmembrane receptor protein tyrosine kinase signaling pathway tRNA metabolic process vasculature development vesicle mediated transport

Supplemental Table 3: mRNAs from the 59 mRNA expression classifier that are predicted to be targeted by a miRNA of the 25 miRNA expression signature, according to at least one of the four target prediction databases (TargetScan, miRDB, Diana, Microcosm) (number of databases) miRNAs higher expressed mRNAs lower expressed in low-risk patients in low risk-patients hsa-miR-190 PRAME (2), SLC25A5 (2), MRPL3 (1) hsa-miR-204 PRAME (3), SLC6A8 (2), NHLH2 (2), PAICS (2) hsa-miR-26a CDKN3 (1), NCAN (1), PRAME (1) hsa-miR-30c AHCY (1), NHLH2 (1) hsa-miR-326 CDCA5 (2), NCAN (2), BIRC5 (1), MCM2 (1) hsa-miR-485-5p CDCA5 (2), MYCN (2), SNAPC1 (2), PAICS (2), BIRC5 (2) hsa-miR-488 MYCN (2), PAICS (2), AHCY (2), TYMS (1) hsa-miR-500 NCAN (2), MCM2 (1) hsa-miR-542-3p BIRC5 (3), SLC6A8 (2), NCAN (2) miRNAs higher expressed mRNAs lower expressed in high-risk patients in high risk-patients HIVEP2 (3), PRKACB (2), EPB41L3 (1), ARHGEF7 (1), CHD5 hsa-miR-18a (1) CLSTN1 (3), PRKACB (2), DPYSL3 (2), CADM1 (2), MAP2K4 hsa-miR-192 (1), WSB1 (1), PDE4DIP (1), EPN2 (1), CHD5 (1), CAMTA1 (1) CAMTA1 (4), CHD5 (3), ULK2 (2), EPB41L3 (2), MAPT (2), hsa-miR-19a MAP2K4 (2), PRKACB (2), GNB1 (2), EPN2 (2), PDE4DIP (1), HIVEP2 (1) EPHA5 (3), CAMTA2 (2), PTPRF (2), CHD5 (2), MAP7 (2), CLSTN1 (2), PIK3R1 (2), CAMTA1 (2), PDE4DIP (1), MAPT hsa-miR-20b (1), GNB1 (1), PTN (1), MAP2K4 (1), ELAVL4 (1), PRKACB (1), ARHGEF7 (1) MAP2K4 (3), PLAGL1 (1), EPN2 (1), PIK3R1 (1), CD44 (1), hsa-miR-25 NRCAM (1), CAMTA1 (1) CHD5 (2), PRDM2 (2), PTPRF (2), CLSTN1 (1), PTPRN2 (1), hsa-miR-345 PTN (1), ARHGEF7 (1), PDE4DIP (1), PTPRH (1) PTPRF (3), PIK3R1 (3), PRDM2 (2), PTPRN2 (2), CLSTN1 hsa-miR-572 (1), FYN (1), DDC (1), PRKCZ (1) CAMTA2 (3), EPHA5 (3), PTPRF (2), MAP7 (2), CLSTN1 (2), PIK3R1 (2), CAMTA1 (2), PDE4DIP (1), MAPT (1), GNB1 (1), hsa-miR-93 PTN (1), MAP2K4 (1), ELAVL4 (1), CHD5 (1), PRKACB (1), ARHGEF7 (1) DDC (2), PDE4DIP (1), ULK2 (1), ELAVL4 (1), PLAT (1), PLAGL1 (1), MAPT (1), CAMTA2 (1), WSB1 (1), DPYSL3 (1), hsa-miR-320 NRCAM (1), CHD5 (1), HIVEP2 (1), MAP7 (1), CAMTA1 (1), MTSS1 (1), FYN (1), INPP1 (1), PRKCZ (1)

Supplemental Figure 1: (A) Standard deviation of Cq-values for from the 59 mRNA signature and miRNAs from the 25 miRNA signature. Each dot represents the standard deviation of the Cq-values of a mRNA or miRNA measured in 262 patients. The average standard deviation for the 59 mRNAs is significanly higher than that for the 25 miRNAs (Mann-Whitney, p < 0.0001). (B) Expression fold change between patients that died from disease and patients that are alive (with a follow-up > 36 months) for the 59 mRNAs and 25 miRNAs. The average expression fold change for the mRNAs is significantly higher comapred to the miRNAs (Mann-Whitney, p < 0.0001).

A B