factors were excluded. All tests of statistical significance were 2-sided at 5% level of significance. Combined model development and application (Metastatic Assay and CAPRA-S) A combined model using Metastatic Assay dichotomized calls and CAPRA-S dichotomized into Low risk (CAPRA-S: 0-5) and High risk (CAPRA-S: 6-10) was assessed in the resection validation cohort independently against biochemical and metastatic endpoints using Cox regression analysis (Supplementary Methods). Subjects were classified ‘Low risk’ given a combined model result Assay Negative/CAPRA-S low risk; otherwise subjects were labelled ‘High Risk’ (i.e. samples that were classified as Assay Negative/CAPRA-S high risk, Assay Positive/CAPRA-S low risk or Assay Positive/CAPRA-S high risk). See Supplemental Methods for additional experimental detail. RESULTS Molecular Subtyping and Identification of a Metastatic-subgroup in the Discovery cohort We hypothesized a molecular subgroup of poor prognosis primary prostate cancers would be transcriptionally similar to metastatic disease. To identify this subgroup, we measured gene expression in primary prostate cancers, primary prostate cancers with known concomitant metastases, metastatic lymph node samples and histologically confirmed normal prostate tissue (Supplementary Table 2). Unsupervised hierarchical clustering identified two sample groups and two gene clusters (Figure 1A). Importantly, one of the molecular subgroups (C1) demonstrated significant enrichment for primary cancers with known concomitant metastatic disease
Supplementary 4. Demographic and Clinical Characteristics of the Glinsky in silico validation cohort Covariate No. % Recurrence Recurrence 37 47 Non-Recurrence 42 53 Relapse Yes 37 47 No 42 53 Pre-operative PSA Median (Min - Max) 7.6(1.5 - 62.1) Gleason (Resection) <7 17 22 7 44 56 >7 18 22 Surgical Margins (SM) Positive 50 63 Negative 29 37 Lymph Node Invasion (LNI) Yes 3 4 No 76 96 Gleason Score (Biopsy) <7 37 47 7 32 41 >7 10 12 Age Median (Min - Max) 61.2(44.9 - 72.7)
Supplementary Table 6. Demographic and Clinical Characteristics of the MSKCC in silico validation cohort Covariate No. % Type of Tumour Primary 126 100 Biochemical Recurrence Yes 32 25 No 94 75 Metastatic Recurrence Yes 14 11 No 112 89 Site of Metastasis Bone 7 6 Local 2 2 RP node, Bone 1 1 Bone, Soft Tissue 1 1 RP node, Lung 1 1 Pelvic node 1 1 Lung 1 1 None 112 89 Age at Diagnosis Median (Min - Max) 57.6(37.3 - 72.78) Pre-operative PSA Median (Min - Max) 5.9(1.1 - 46.4) Pathological T1 71 56 T2 50 40 T3 5 4 Surgical Margins (SM) Positive 30 24 Negative 96 76 Seminal Vesicle Invasion (SVI) Yes 13 10 No 113 90 Lymph Node Invasion (LNI) No 97 77 Yes 6 5 Unknown 23 18 Race Black Non-Hispanic 23 18 White Non-Hispanic 94 75 Hispanic 3 2 Asian 2 2 Unknown 4 3 Gleason <7 40 32 7 72 57 >7 14 11 Supplementary Table 7. Demographic and Clinical characteristics of the Independent Resection Validation Cohort.
Supplementary Table 8 – contained within the Supplemental File (Tab 2) Supplementary 9. Demographic & Clinical Characteristics of the Methylation Cohort Covariate No. % Clinical Site Cambridge 5 23 Karolinska 17 77 Sample Type Primary Tumour 20 91 Metastatic Disease 2 8 Class Label/Subgroup C1 8 36 C2 14 64 Gleason Score 6 2 9 7 4 18 8 9 41 9 7 32 Stage T2 5 23 T3 14 64 Unknown 3 13 Supplementary 10 – contained within the Supplemental File (Tab 3) Supplementary Table 11 – contained within the Supplemental File (Tab 4) Supplementary Table 12 – contained within the Supplemental File (Tabs 5 & 6)
Supplementary Table 15. Performance metrics for metastatic recurrence for CAPRA-S and the Combined Model (CAPRA-S + Metastatic Assay) Metric Metastatic Assay CAPRA-S Combined Model Sensitivity 47.73% [32.46-63.31] 70.45% [54.80-83.24] 84.09% [69.93-93.36] Specificity 81.87% [75.69-87.03] 71.50% [64.58-77.75] 64.14% [53.88-68.06] Supplementary Table 14. Performance metrics for Biochemical recurrence (Glinsky) and Metastatic recurrence (Erho) for the Metastatic Assay Metric Metastatic Assay (Glinsky cohort) Metastatic (Erho cohort) Sensitivity 70.27% [56.41-80.00] 66.67% [52.44-76.63] Specificity 66.98% [60.51-71.36] 54.65% [50.43-57.38]