Background
Methods
Selection of candidate proteins
Clinical cohort and TMA construction
Immunohistochemistry
Antibody | Specificity | Clone | Dilution | Antigen retrieval | Incubation | Detection | Source | Cat # |
---|---|---|---|---|---|---|---|---|
ACPP ACPP | Polyclonal (rabbit) | - | 1:2500 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Sigma-Aldrich | HPA004335 |
ADAM9 | Polyclonal (rabbit) | - | 1:100 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Sigma-Aldrich | HPA004000 |
ALDH1A2 | Polyclonal (rabbit) | - | 1:1000 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Sigma-Aldrich | HPA010022 |
CASR | Polyclonal (rabbit) | - | 1:200 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Abcam | ab18200 |
CCND1 | Monoclonal (mouse) | DCS-6 | 1:50 | Decloaker/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Dako | M7155 |
CCPG1 | Polyclonal (rabbit) | - | 1:400 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Sigma-Aldrich | HPA026861 |
CD34 | Monoclonal (mouse) | QBEnd/10 | 1:4000 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Neomarkers | MS-363-P |
CD44 | Monoclonal (mouse) | SP37 | 1:400 | Steamer/Citrate pH 6 | O/N | Novocastra Novolink Polymer | Dako | M708201-2 |
CD44v6 | Monoclonal (mouse) | 2 F10 | 1:1000 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | R&D Systems | BBA13 |
CHGA | Monoclonal (mouse) | LK2H10 + PHE5 | 1:800 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Biocare Medical | CM010B |
CHMP1A | Polyclonal (rabbit) | - | 1:100 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Sigma-Aldrich | HPA006776 |
EI24 | Polyclonal (rabbit) | - | 1:8000 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Sigma-Aldrich | SAB1100756 |
ENO2 | Monoclonal (mouse) | 5E2 | 1:800 | No AR | 1 h | Novocastra Novolink Polymer | Leica | NCL-L-NSE2 |
GADD45B | Polyclonal (rabbit) | - | 1:25 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Sigma-Aldrich | HPA029816 |
HABPa
| NA (bovine) | - | 1:400 | No AR | O/N | VECTASTAIN Elite ABCb
| Calbiochem | 385911 |
HAS2 | Polyclonal (goat) | - | 1:1000 | Steamer/Citrate pH 6 | 1 h | VECTASTAIN Elite ABC | Santa Cruz | sc-34067 |
HES6 | Polyclonal (rabbit) | - | 1:1000 | Steamer/Citrate pH 6 | O/N | Novocastra Novolink Polymer | Abcam | ab66461 |
HMMR | Monoclonal (mouse) | 6B7D8 | 1:900 | Steamer/Citrate pH 6 | 1 h | Covance Ultra Streptavidin-HRP | ProMab | NA |
HOXC6 | Polyclonal (rabbit) | - | 1:200 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Santa Cruz | sc-66925 |
HYAL1 | Polyclonal (rabbit) | - | 1:100 | Steamer/Citrate pH 6 | O/N | Novocastra Novolink Polymer | Sigma-Aldrich | HPA002112 |
IGF1 | Polyclonal (rabbit) | - | 1:400 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Millipore | 07-1411 |
IQCK | Polyclonal (rabbit) | - | 1:70 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Sigma-Aldrich | HPA026792 |
MAP4K4 | Polyclonal (rabbit) | - | 1:100 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Sigma-Aldrich | HPA008476 |
MKI67 | Monoclonal (mouse) | MM1 | Predilute | Decloaker/CCS1 | 32 min | Ventana ultraView Universal | Leica | ORG-8772 |
PAGE4 | Polyclonal (rabbit) | - | 1:2000 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Sigma-Aldrich | HPA023880 |
PLIN2 | Polyclonal (rabbit) | - | 1:100 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Abbiotec | 251533 |
PTEN | Polyclonal (rabbit) | - | 1:200 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Invitrogen | 18-0256 |
SIAH2 | Monoclonal (mouse) | 24E6H3 | 1:50 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Novus | NB110-88113 |
SMAD4 | Monoclonal (rabbit) | EP618Y | 1:100 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Millipore | 04-1033 |
SOX9 | Polyclonal (rabbit) | - | 1:200 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Sigma-Aldrich | HPA001758 |
SPP1 | Monoclonal (rabbit) | EPR3688 | 1:50 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Epitomics | 2671-1 |
SYP | Monoclonal (mouse) | 27G12 | 1:400 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Leica | NCL-SYNAP-299 |
TP53 | Monoclonal (mouse) | DO-7 | 1:40000 | Steamer/Citrate pH 6 | 1 h | Novocastra Novolink Polymer | Dako | M7001 |
Monoclonal (mouse) | Bp53-11 | Predilute | Decloaker/CCS1 | 16 min | Ventana ultraView Universal | Ventana | 760-2542 |
Slide digitization, annotation, and immunohistochemical quantification
Statistical analysis
Results
Immunohistochemical analysis of PCa related proteins
Clinicopathologic features of PCa patients and BF
Variable | N | % | HR (95% CI) | P-value |
---|---|---|---|---|
Total population | 160 | |||
Combined Gleason score | 0.034 | |||
3 + 3 | 50 | 31.3 | 0.09 (0.02-0.51) | |
3 + 4 | 63 | 39.4 | 0.17 (0.03-0.78) | |
4 + 3 | 34 | 21.3 | 0.17 (0.03-0.92) | |
4 + 4 | 8 | 5.0 | 0.11 (0.01-1.23) | |
4 + 5 | 5 | 3.1 | 1.00 | |
Primary Gleason pattern | 0.347 | |||
3 | 113 | 70.6 | 0.65 (0.27-1.58) | |
4 | 47 | 29.4 | 1.00 | |
Pathologic stage | 0.013 | |||
Extraprostatic extension (pT3+) | 29 | 18.1 | 3.13 (1.28-7.69) | |
Prostate-limited (pT2) | 131 | 81.9 | 1.00 | |
Lymph node involvement | 0.018 | |||
Yes (pN1) | 5 | 3.1 | 4.46 (1.29-15.37) | |
No (pN0) | 155 | 96.9 | 1.00 | |
Surgical margin involvement | 0.022 | |||
Yes (pR1) | 53 | 33.1 | 2.82 (1.17-6.84) | |
No (pR0) | 107 | 66.9 | 1.00 | |
Non-Localized tumor Indicator* | 0.005 | |||
Yes | 67 | 41.9 | 4.21 (1.54-11.50) | |
No | 93 | 58.1 | 1.00 | |
N
|
Mean (SD)
|
HR (95%
CI)
|
P-value
| |
Age (years) | 160 | 60.8 (6.8) | 1.08 (1.01-1.16) | 0.035 |
Pre-operative PSA (ng/mL) | 148 | 7.2 (5.3) | 1.03 (0.98-1.09) | 0.238 |
Prognostic significance of proteins implicated in aggressive prostate cancer
N | Univariate | Multivariate* | |||
---|---|---|---|---|---|
Variable | HR (95% CI) | P-value | HR (95% CI) | P-value | |
ACPP | 139 | 0.93 (0.63-1.39) | 0.735 | 1.07 (0.59-1.93) | 0.827 |
ADAM9 | 152 | 0.98 (0.63-1.54) | 0.944 | 1.19 (0.75-1.91) | 0.462 |
ALDH1A2 | 141 | 0.79 (0.47-1.32) | 0.368 | 1.10 (0.62-1.95) | 0.737 |
CASR | 143 | 1.01 (0.65-1.57) | 0.949 | 0.98 (0.58-1.66) | 0.937 |
CCND1 (nuclear) | 142 | 1.38 (1.01-1.88) | 0.042 | 1.18 (0.87-1.61) | 0.296 |
CCPG1 | 143 | 1.19 (0.76-1.88) | 0.446 | 1.14 (0.69-1.86) | 0.611 |
CD34 (avg vess area) | 146 | 1.16 (0.74-1.83) | 0.515 | 1.02 (0.60-1.74) | 0.938 |
CD34 (avg vess per) | 146 | 1.42 (0.91-2.21) | 0.123 | 1.43 (0.84-2.43) | 0.188 |
CD34 (avg lum area) | 146 | 1.45 (0.95-2.22) | 0.086 | 1.37 (0.83-2.27) | 0.220 |
CD34 (avg vasc area) | 146 | 1.02 (0.66-1.60) | 0.915 | 0.89 (0.52-1.53) | 0.682 |
CD34 (MVD) | 146 | 1.11 (0.71-1.73) | 0.652 | 0.97 (0.56-1.67) | 0.901 |
CD34 | 146 | 1.11 (0.72-1.71) | 0.648 | 1.04 (0.63-1.71) | 0.881 |
CD44 | 148 | 1.07 (0.68-1.68) | 0.764 | 1.06 (0.70-1.60) | 0.786 |
CD44v6 | 143 | 1.23 (0.79-1.89) | 0.360 | 1.35 (0.84-2.16) | 0.217 |
CHGA | 136 | 0.50 (0.24-1.04) | 0.064 | 0.56 (0.26-1.21) | 0.139 |
CHMP1A | 146 | 1.31 (0.83-2.08) | 0.244 | 1.35 (0.84-2.20) | 0.218 |
EI24 (stroma) | 140 | 1.24 (0.85-1.82) | 0.265 | 1.36 (0.97-1.91) | 0.077 |
EI24 (tumor) | 140 | 1.32 (0.94-1.87) | 0.114 | 1.32 (0.97-1.79) | 0.077 |
ENO2 | 145 | 1.33 (0.92-1.93) | 0.135 | 1.48 (0.92-2.38) | 0.108 |
GADD45B | 148 | 1.11 (0.72-1.73) | 0.630 | 1.18 (0.78-1.77) | 0.435 |
HA (stroma) | 150 | 1.51 (0.99-2.29) | 0.055 | 1.43 (0.95-2.17) | 0.090 |
HAS2 | 148 | 1.27 (0.82-1.97) | 0.287 | 1.47 (0.94-2.30) | 0.090 |
HES6 | 140 | 0.80 (0.48-1.34) | 0.401 | 0.69 (0.38-1.27) | 0.238 |
HMMR | 139 | 2.01 (1.23-3.30) | 0.005 | 1.98 (1.20-3.29) | 0.008 |
HOXC6 | 142 | 1.22 (0.79-1.89) | 0.368 | 1.51 (1.00-2.28) | 0.050 |
HYAL1 | 147 | 1.42 (0.92-2.21) | 0.116 | 1.40 (0.87-2.25) | 0.163 |
IGF1 | 147 | 1.57 (1.02-2.42) | 0.039 | 1.67 (1.10-2.52) | 0.015 |
IQCK | 149 | 0.90 (0.58-1.40) | 0.651 | 0.90 (0.59-1.38) | 0.625 |
MAP4K4 | 145 | 1.44 (0.91-2.28) | 0.121 | 1.76 (1.08-2.86) | 0.024 |
MKI67 (nuclear) | 142 | 1.43 (1.04-1.96) | 0.026 | 1.29 (0.93-1.79) | 0.125 |
PAGE4 | 145 | 0.68 (0.45-1.03) | 0.069 | 0.88 (0.57-1.35) | 0.566 |
PLIN2 | 115 | 1.21 (0.71-2.09) | 0.481 | 1.07 (0.62-1.86) | 0.801 |
PTEN | 144 | 0.83 (0.54-1.27) | 0.393 | 0.84 (0.51-1.38) | 0.493 |
SIAH2 (nuclear) | 144 | 1.46 (1.07-1.99) | 0.016 | 1.37 (0.94-2.02) | 0.104 |
SMAD4 | 130 | 1.76 (1.15-2.71) | 0.010 | 1.73 (1.11-2.70) | 0.016 |
SOX9 | 145 | 1.39 (0.90-2.16) | 0.137 | 1.45 (0.93-2.27) | 0.101 |
SPP1 | 128 | 1.07 (0.62-1.84) | 0.805 | 0.76 (0.44-1.33) | 0.338 |
SYP | 133 | 0.88 (0.48-1.63) | 0.681 | 0.95 (0.53-1.70) | 0.869 |
TP53_Lab1 (nuclear) | 148 | 1.23 (0.83-1.83) | 0.305 | 1.19 (0.78-1.82) | 0.431 |
TP53_Lab2 (nuclear) | 143 | 0.97 (0.63-1.50) | 0.906 | 0.93 (0.60-1.44) | 0.741 |
Multiple imputations and multibiomarker modeling of prostate cancer aggressiveness
Variable | Top 2 | Top 3 | Top 4 | Top 5 |
---|---|---|---|---|
ACPP | 0 | 0 | 0 | 0 |
ADAM9 | 0 | 0 | 0 | 0 |
ALDH1A2 | 0 | 0 | 0 | 0 |
CASR | 0 | 0 | 0 | 0 |
CCND1 (nuclear) | 0 | 0 | 0 | 0 |
CCPG1 | 0 | 0 | 0 | 0 |
CD34 (avg vess area) | 0 | 0 | 0 | 0 |
CD34 (avg vess per) | 0 | 1 | 4 | 5 |
CD34 (avg lum area) | 0 | 1 | 3 | 3 |
CD34 (avg vasc area) | 0 | 0 | 0 | 0 |
CD34 (MVD) | 0 | 0 | 0 | 0 |
CD34 | 0 | 0 | 0 | 0 |
CD44 | 0 | 0 | 0 | 0 |
CD44v6 | 0 | 2 | 2 | 2 |
CHGA | 0 | 1 | 1 | 2 |
CHMP1A | 0 | 0 | 0 | 0 |
EI24 (stroma) | 0 | 0 | 0 | 0 |
EI24 (tumor) | 0 | 0 | 0 | 0 |
ENO2 | 0 | 0 | 1 | 1 |
GADD45B | 0 | 0 | 0 | 0 |
HA (stroma) | 1 | 1 | 1 | 3 |
HAS2 | 0 | 0 | 0 | 0 |
HES6 | 0 | 0 | 0 | 0 |
HMMR | 2 | 4 | 6 | 6 |
HOXC6 | 0 | 0 | 0 | 0 |
HYAL1 | 0 | 0 | 0 | 0 |
IGF1 | 0 | 0 | 0 | 0 |
IQCK | 0 | 0 | 0 | 0 |
MAP4K4 | 0 | 0 | 0 | 0 |
MKI67 (nuclear) | 0 | 0 | 0 | 0 |
PAGE4 | 0 | 0 | 0 | 0 |
PLIN2 | 0 | 0 | 0 | 0 |
PTEN | 0 | 0 | 0 | 0 |
SIAH2 (nuclear) | 7 | 10 | 10 | 10 |
SMAD4 | 4 | 7 | 7 | 8 |
SOX9 | 0 | 0 | 0 | 0 |
SPP1 | 0 | 0 | 0 | 0 |
SYP | 0 | 0 | 0 | 0 |
TP53_Lab1 (nuclear) | 0 | 0 | 0 | 0 |
TP53_Lab2 (nuclear) | 0 | 0 | 0 | 0 |
3-year recurrence | 5-year recurrence | |||
---|---|---|---|---|
AUC (95% CI) | P-value | AUC (95% CI) | P-value | |
Unadjusted AUC | 0.708 (0.581-0.882) | 0.004 | 0.721 (0.592-0.880) | 0.002 |
Cross-validation adjusted AUC | 0.685 (0.499-0.871) | 0.052 | 0.702 (0.533-0.866) | 0.024 |