Title: The landscape of prognostic outlier genesin high-risk prostate cancer

Shuang G Zhao1*, Joseph R Evans1*, Grace Sun1, Ashley Larm1, Victor Mondine1, Vishal Kothari1, Edward M Schaeffer2, Ashley Ross2, Eric A Klein MD3, Robert B Den4, Adam P Dicker4, R Jeffrey Karnes5, Nicholas Erho6, Paul L Nguyen7, Elai Davicioni6, and Felix Y Feng1,8,9

Supplemental Figure Legends

Supplemental Figure 1: Additional prognostic outlier genes. (A-D) Inverse waterfall plots of the expression profile across the discovery cohort of the three additional top prognostic outlier genes chosen for in vitro study: NVL (A), SMC4 (B), SQLE (C), and TPX2 (D). Black bars indicate patients who developed metastatic progression; gray bars indicate patients who did not develop metastatic progression. The dashed horizontal line indicates the outlier expression threshold.

Supplemental Figure 2: Additional prognostic outlier negative result examples. KLF2, LAMA5, and KLK3 show no outlier expression. RHOC and SERPINB3 show outlier expression that does not correlate with metastatic progression.

Supplemental Figure 3: Metastatic progression association in discovery cohort. Kaplan-Meier analysis of outlier status association with metastatic progression individually for NVL, SMC4, SQLE, and TPX2 in the discovery (MCI) cohort.

Supplemental Figure 4:Prognostic outlier score and overall survival.(A-B) Kaplan-Meier analysis of overall survival by prognostic outlier score of 0, 1-2, 3-4, or ≥5 in the discovery (MCI) cohort (A) and the pooled validation cohort (B). Multivariable logistic regression analysis results of prognostic outlier score (Outlier Score) association with 10-year overall survivalin the discovery (MCI) cohort (C) and the pooled validation cohort (D), including other clinicopathological variables Age, PSA, Gleason, surgical margin status (SMS), seminal vesicle invasion (SVI), extra-capsular extension (ECE), and lymph node involvement (LNI).

Supplemental Figure 5: Published prognostic instrument ROC AUC analysis. (A-C) ROCs and AUCs for published prognostic instruments Decipher (A), mCCP (B), and CAPRA-S (C) with prognostic outlier score integrated (blue line) or alone (red line). P-value is given for the difference between AUCs in each comparison.

Supplemental Table 1: Cohort clinical characteristics
Clinical Validation datasets
Mayo Discovery (n=545) / Mayo Validation (n=232) / Thomas Jefferson University (n = 130) / Cleveland Clinic (n =183)
Age at diagnosis (Years ± SD) / 65.3 ± 6.4 / 63.1 ± 7.4 / 60.0 ± 7.0 / 61.6 ± 6.3
Mean follow-up (Months ± SD) / 160.7 ± 56.2 / 80.6 ± 30.1 / 103.9 ± 58.9 / 116.6 ± 50.1
Pre-operative PSA
<10 / 282 (52%) / 126 (54%) / 83 (64%) / 127 (69%)
10 to 20 / 117 (22%) / 62 (27%) / 25 (19%) / 41 (23%)
>20 / 131 (24%) / 44 (19%) / 15 (11%) / 12 (7%)
Not available / 15 (3%) / 0 (0%) / 7 (5%) / 3 (1%)
Gleason score
6 / 60 (11%) / 17 (7%) / 17 (13%) / 25 (17%)
7 / 271 (49%) / 117 (50%) / 74 (57%) / 113 (62%)
8 / 68 (13%) / 39 (17%) / 22 (17%) / 23 (13%)
9 / 134 (24%) / 57 (25%) / 13 (10%) / 22 (12%)
10 / 9 (2%) / 1 (1%) / 2 (1.5%) / 0 (0%)
Not available / 3 (1%) / 1 (1%) / 2 (1.5%) / 0 (0%)
Tumor stage
I / 0 (0%) / 0 (0%) / 0 (0%) / 0 (0%)
II / 219 (40%) / 97 (42%) / 10 (8%) / 0 (0%)
III / 253 (46%) / 102 (44%) / 113 (87%) / 0 (0%)
IV / 0 (0%) / 0 (0%) / 7 (5%) / 0 (0%)
Not available / 73 (13%) / 33 (14%) / 0 (0%) / 183 (100%)
Surgical margin status
Negative / 279 (51%) / 99 (43%) / 31 (24%) / 92 (50%)
Positive / 266 (49%) / 133 (57%) / 99 (76%) / 91 (50%)
Extracapsular extension
Negative / 272 (50%) / 136 (59%) / 23 (18%) / 51 (28%)
Positive / 273 (50%) / 96 (41%) / 106 (81%) / 132 (72%)
Not available / 0 (0%) / 0 (0%) / 1 (1%) / 0 (0%)
Seminal vesicle invasion
Negative / 369 (68%) / 149 (64%) / 82 (63%) / 152 (83%)
Positive / 176 (32%) / 83 (36%) / 48 (37%) / 31 (17%)
Lymph node invasion
Negative / 472 (87%) / 199 (86%) / 128 (98%) / 183 (100%)
Positive / 73 (13%) / 33 (14%) / 2 (2%) / 0 (0%)
Metastatic progression
Yes / 212 (39%) / 75 (32%) / 10 (8%) / 49 (27%)
No / 333 (61%) / 157 (68%) / 120 (92%) / 134 (73%)

Supplemental Table 2: Relation of prognostic outlier score to standard prognostic variables

Training cohort / Pooled validation cohort
Prognostic outlier score / 0
(n=359) / 1-2
(n=120) / 3-4
(n=27) / 5+
(n=39) / p-value / 0
(n=264) / 1-2
(n=186) / 3-4
(n=55) / 5+
(n=40) / p-value
Age (Years) / 65.3 +/- 6.4 / 65.2 +/- 6.1 / 64 +/- 7.3 / 67.1 +/- 6.4 / 0.27 / 61.7 +/- 6.9 / 61.9 +/- 7.1 / 62.2 +/- 6.8 / 62.2 +/- 7.7 / 0.92
NA / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
PSA
< 10 / 195 (54%) / 55 (46%) / 9 (33%) / 23 (59%) / 0.054 / 163 (62%) / 119 (64%) / 32 (58%) / 22 (55%) / 0.57
10-20 / 78 (22%) / 31 (26%) / 5 (19%) / 3 (8%) / 55 (21%) / 47 (25%) / 15 (27%) / 11 (28%)
> 30 / 79 (22%) / 30 (25%) / 11 (41%) / 11 (28%) / 38 (14%) / 18 (10%) / 8 (15%) / 7 (18%)
NA / 7 / 4 / 2 / 2 / 8 / 2 / 0 / 0
Gleason
<7 / 57 (16%) / 6 (5%) / 0 / 0 / 4.36E-15 / 39 (15%) / 19 (10%) / 3 (5%) / 0 / 4.93E-07
7 / 204 (57%) / 54 (45%) / 5 (19%) / 8 (21%) / 165 (63%) / 99 (53%) / 25 (46%) / 15 (38%)
8-10 / 98 (27%) / 60 (50%) / 22 (82%) / 31 (80%) / 59 (22%) / 68 (37%) / 27 (49%) / 25 (63%)
NA / 0 / 0 / 0 / 0 / 1 / 0 / 0 / 0
Tumor stage
II / 168 (47%) / 37 (31%) / 6 (22%) / 8 (21%) / 0.0067 / 55 (21%) / 40 (22%) / 7 (13%) / 5 (13%) / 0.15
III / 160 (45%) / 58 (48%) / 10 (37%) / 25 (64%) / 96 (36%) / 76 (41%) / 23 (42%) / 20 (50%)
IV / 0 / 0 / 0 / 0 / 6 (2%) / 0 / 1 (2%) / 0
NA / 31 / 25 / 11 / 6 / 107 / 70 / 24 / 15
PSM / 161 (45%) / 65 (54%) / 15 (56%) / 25 (64%) / 0.051 / 166 (63%) / 112 (60%) / 21 (38%) / 24 (60%) / 0.0088
NA / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
SVI / 87 (24%) / 55 (46%) / 14 (52%) / 20 (51%) / 5.54E-07 / 57 (22%) / 58 (31%) / 25 (46%) / 22 (55%) / 5.10E-06
NA / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
ECE / 161 (45%) / 68 (57%) / 15 (56%) / 29 (74%) / 0.0014 / 155 (59%) / 113 (61%) / 36 (66%) / 30 (75%) / 0.25
NA / 0 / 0 / 0 / 0 / 2 / 1 / 0 / 0
LNI / 31 (9%) / 25 (21%) / 11 (41%) / 6 (15%) / 1.21E-06 / 12 (5%) / 9 (5%) / 6 (11%) / 8 (20%) / 0.00095
NA / 0 / 0 / 0 / 0 / 0 / 1 / 0 / 0

NA=data missing. PSM=positive surgical margins. SVI=seminal vesicle invasion. ECE=extracapsular extension. LNI=lymph node involvement.

Supplemental Table 3: Prognostic outlier score prognostic association analysis
Discovery (MCI)
Univariate / Multivariate
p-value / OR [95%CI] / p-value / OR [95%CI]
Outlier Score
(per outlier) / 0.00 / 1.72 [1.47 - 2.00] / 0.00 / 1.47 [1.26 - 1.71]
Age (per year) / 0.69 / 1.01 [0.98 - 1.03] / 0.80 / 1.00 [0.96 - 1.03]
PSA (per unit) / 0.00 / 1.02 [1.01 - 1.03] / 0.24 / 1.01 [1.00 - 1.02]
Gleason (per unit) / 0.00 / 2.41 [1.98 - 2.94] / 0.00 / 1.81 [1.45 - 2.27]
SMS / 0.02 / 1.51 [1.05 - 2.16] / 0.66 / 0.91 [0.59 - 1.40]
SVI / 0.00 / 2.86 [1.96 - 4.18] / 0.07 / 1.59 [0.96 - 2.64]
ECE / 0.00 / 1.89 [1.32 - 2.72] / 0.86 / 0.96 [0.61 - 1.51]
LNI / 0.00 / 3.05 [1.84 - 5.05] / 0.60 / 1.19 [0.63 - 2.25]
Validation
Univariate / Multivariate
p-value / OR [95%CI] / p-value / OR [95%CI]
Outlier Score
(per outlier) / 0.00 / 1.58 [1.30 - 1.93] / 0.00 / 1.50 [1.22 - 1.84]
Age (per year) / 0.38 / 1.02 [0.98 - 1.07] / 0.72 / 1.01 [0.96 - 1.07]
PSA (per unit) / 0.01 / 1.04 [1.01 - 1.07] / 0.01 / 1.04 [1.01 - 1.07]
Gleason (per unit) / 0.00 / 2.29 [1.64 - 3.21] / 0.01 / 1.69 [1.16 - 2.47]
SMS / 0.42 / 0.79 [0.44 - 1.41] / 0.97 / 1.01 [0.51 - 2.02]
SVI / 0.02 / 2.02 [1.11 - 3.67] / 0.78 / 1.11 [0.54 - 2.25]
ECE / 0.03 / 2.15 [1.08 - 4.29] / 0.26 / 1.61 [0.70 - 3.68]
LNI / 0.26 / 2.07 [0.59 - 7.33] / 0.85 / 1.17 [0.22 - 6.28]
Multivariable logistic regression analysis results of prognostic outlier score (Outlier Score) association with 10-year metastatic progression in the discovery (MCI) cohort and the pooled validation cohort, including other clinicopathological variables Age, PSA, Gleason, surgical margin status (SMS), seminal vesicle invasion (SVI), extra-capsular extension (ECE), and lymph node involvement (LNI).