Investigating Serum and Tissue Expression Identified a Cytokine/Chemokine Signature as a Highly Effective Melanoma Marker
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Serum Expression: Single-Molecule Analysis of the Cytokines/Chemokines in Melanoma Patients vs. Controls
2.2. Serum Expression: Analysis of Paired Molecules by a Correlation Matrix
2.3. Serum Expression: Profile Analysis by SVM
2.4. Tissues Expression: Single-Molecule Analysis of 27 Cytokines/Chemokines in Melanoma Patients and Controls
2.5. Tissue Expression: Analyzing Paired Molecules by a Matrix Correlation
2.6. Tissue Expression: Profile Analysis by SVM
2.7. Results Validation
3. Discussion
4. Materials and Methods
4.1. Patients Selection and Recruitment
4.2. Serum Handling
4.3. Cytokines Quantification in Sera Samples
4.4. Serum Expression Data
4.5. Tissue Expression Data from GENT2 Database
4.6. Statistical Analyses
4.6.1. Single-Molecule Analysis
4.6.2. Paired-Molecule Analysis
4.6.3. Profile Analysis
4.7. Results Validation
4.7.1. Cross-Validation Procedure
4.7.2. Validation
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Patient Type | Number | Mean Age | Mean Thickness (mm) | Thickness Distribution | |
---|---|---|---|---|---|
<1 mm * Number | ≥1 mm * Number | ||||
Female controls | 72 | 41.3 | 0.00 | 0 | 0 |
Male controls | 64 | 45.3 | 0.00 | 0 | 0 |
Female melanoma | 48 | 54.5 | 1.60 | 23 | 22 |
Male melanoma | 48 | 58.0 | 1.08 | 31 | 14 |
Total | 232 |
Cytokines | Controls Median (n = 136) | Melanoma Median (n = 96) | p-Value Mann–Whitney Controls vs. Melanoma | AUC ± S.E. by ROC Analysis |
---|---|---|---|---|
IL-1b | 0.53 | 0.65 | 0.04 | 0.61 ± 0.05 |
IL-1Ra | 26.77 | 17.83 | 0.14 | 0.56 ± 0.04 |
IL-2 | 3.45 | 2.14 | 0.14 | 0.66 ± 0.10 |
IL-4 | 2.95 | 2.88 | 0.47 | 0.53 ± 0.04 |
IL-5 | 2.77 | 2.34 | 0.22 | 0.60 ± 0.08 |
IL-6 | 5.37 | 3.17 | 0.04 | 0.70 ± 0.09 |
IL-7 | 2.24 | 2.24 | 0.72 | 0.52 ± 0.05 |
IL-8 | 6.63 | 6.4 | 0.55 | 0.52 ± 0.04 |
IL-9 | 45.58 | 42.05 | 0.29 | 0.54 ± 0.04 |
IL-10 | 7.08 | 4.56 | 0.10 | 0.63 ± 0.08 |
IL-12(p70) | 15.74 | 16.07 | 1.00 | 0.50 ± 0.05 |
IL-13 | 2.43 | 2.99 | 0.83 | 0.52 ± 0.09 |
IL-15 | 52.22 | 30.11 | 1.00 | 0.50 ± 0.27 |
IL-17 | 16.72 | 13.1 | 0.37 | 0.54 ± 0.04 |
Eotaxin | 95.28 | 106.28 | 0.38 | 0.53 ± 0.04 |
FGF-2 | 32.68 | 30.01 | 0.26 | 0.55 ± 0.04 |
G-CSF | 4.73 | 5.41 | 0.33 | 0.55 ± 0.05 |
GM-CSF | 10.21 | 10.63 | 0.67 | 0.53 ± 0.06 |
IFN-γ | 19.1 | 23.2 | 0.48 | 0.53 ± 0.04 |
IP-10 (CXCL10) | 438.69 | 501.41 | 0.04 | 0.58 ± 0.04 |
MCP-1(MCAF) | 18.59 | 12.49 | 0.24 | 0.58 ± 0.07 |
MIP-1a (CCL3) | 1.78 | 1.74 | 0.59 | 0.52 ± 0.04 |
MIP-1b (CCL4) | 54.36 | 56.26 | 0.43 | 0.53 ± 0.04 |
PDGF-BB | 1603.74 | 1033.41 | 0.01 | 0.61 ± 0.05 |
RANTES (CCL5) | 11,353.34 | 8735.27 | 0.01 | 0.57 ± 0.06 |
TNF-α | 16.4 | 18.06 | 0.26 | 0.52 ± 0.06 |
VEGF | 59.75 | 57.98 | 0.88 | 0.54 ± 0.06 |
Cytokines | Melanoma Breslow Thickness <1 mm | Melanoma Breslow Thickness ≥1 mm | Mann-Whitney <1 mm vs. ≥1 mm | ||
---|---|---|---|---|---|
Count | Median | Count | Median | p Value | |
IL-1b | 26 | 0.65 | 16 | 0.72 | 0.66 |
IL-1Ra | 44 | 18.24 | 27 | 17.83 | 0.59 |
IL-2 | 5 | 1.9 | 7 | 2.38 | 0.25 |
IL-4 | 54 | 2.85 | 35 | 3.06 | 0.81 |
IL-5 | 15 | 2.34 | 9 | 1.7 | 0.86 |
IL-6 | 7 | 0.83 | 8 | 3.34 | 0.18 |
IL-7 | 31 | 2.24 | 15 | 2.9 | 0.34 |
IL-8 | 47 | 7.8 | 30 | 5.78 | 0.01 |
IL-9 | 52 | 42.55 | 35 | 41.91 | 0.64 |
IL-10 | 11 | 3.84 | 10 | 5.12 | 0.92 |
IL-12(p70) | 40 | 20.48 | 20 | 14.64 | 0.51 |
IL-13 | 11 | 2.43 | 8 | 3.2 | 0.60 |
IL-15 | 2 | 13.02 | 1 | 42.31 | 0.67 |
IL-17 | 38 | 13.15 | 30 | 11.79 | 0.94 |
Eotaxin | 54 | 108.94 | 35 | 93.24 | 0.71 |
FGF-2 | 49 | 29.73 | 34 | 31.46 | 0.77 |
G-CSF | 27 | 5.45 | 10 | 4.12 | 0.30 |
GM-CSF | 17 | 14.06 | 25 | 9.78 | 0.22 |
IFN-γ | 51 | 23.49 | 31 | 22.13 | 0.51 |
IP-10 (CXCL10) | 53 | 491.81 | 35 | 574.3 | 0.88 |
MCP-1(MCAF) | 18 | 10.56 | 11 | 24.83 | 0.02 |
MIP-1a (CCL3) | 54 | 1.82 | 35 | 1.71 | 0.56 |
MIP-1b (CCL4) | 53 | 55.51 | 35 | 53.52 | 0.73 |
PDGF-BB | 53 | 1048.16 | 35 | 1080.98 | 0.47 |
RANTES (CCL5) | 53 | 10,341.43 | 35 | 7534.18 | 0.03 |
TNF-α | 43 | 16.65 | 19 | 22.78 | 0.06 |
VEGF | 51 | 63.58 | 35 | 52.26 | 0.45 |
Cytokines | No. of Pairs | Spearman R Correlation of Serum Expression with Breslow Thickness | p-Value (2-Tails) |
---|---|---|---|
IL-1b | 42 | 0.04 | 0.80 |
IL-1Ra | 71 | 0.04 | 0.75 |
IL-2 | 12 | 0.01 | 0.97 |
IL-4 | 89 | −0.02 | 0.88 |
IL-5 | 24 | 0.01 | 0.96 |
IL-6 | 15 | 0.24 | 0.40 |
IL-7 | 46 | 0.28 | 0.06 |
IL-8 | 77 | −0.23 | 0.05 |
IL-9 | 87 | −0.09 | 0.42 |
IL-10 | 21 | −0.14 | 0.55 |
IL-12(p70) | 60 | −0.02 | 0.86 |
IL-13 | 19 | −0.09 | 0.72 |
IL-15 | 3 | 1.00 | 0.33 |
IL-17 | 68 | 0.05 | 0.67 |
Eotaxin | 89 | −0.01 | 0.96 |
FGF-2 | 83 | 0.03 | 0.81 |
G-CSF | 37 | 0.04 | 0.83 |
GM-CSF | 42 | −0.40 | 0.01 |
IFN-γ | 82 | 0.07 | 0.53 |
IP-10 (CXCL10) | 88 | 0.04 | 0.72 |
MCP-1(MCAF) | 29 | 0.39 | 0.04 |
MIP-1a (CCL3) | 89 | −0.09 | 0.40 |
MIP-1b (CCL4) | 88 | −0.02 | 0.86 |
PDGF-BB | 88 | −0.10 | 0.35 |
RANTES (CCL5) | 88 | −0.20 | 0.06 |
TNF-α | 62 | 0.31 | 0.01 |
VEGF | 86 | −0.02 | 0.87 |
Cytokines | All Controls (Male + Female) | All Melanoma (Male + Female) | ||||
---|---|---|---|---|---|---|
No. of Pairs | Spearman R Correlation of Serum Expression with Age | p Value (2 Tails) | No. of Pairs | Spearman R Correlation of Serum Expression with Age | p-Value (2 Tails) | |
IL-1b | 85 | 0.14 | 0.21 | 44 | −0.13 | 0.40 |
IL-1Ra | 121 | 0.15 | 0.10 | 75 | −0.08 | 0.50 |
IL-2 | 19 | −0.21 | 0.38 | 13 | 0.23 | 0.46 |
IL-4 | 135 | 0.08 | 0.36 | 95 | −0.05 | 0.66 |
IL-5 | 30 | 0.34 | 0.07 | 25 | −0.20 | 0.33 |
IL-6 | 21 | 0.00 | 0.99 | 16 | −0.01 | 0.96 |
IL-7 | 81 | 0.35 | 0.001 | 48 | −0.09 | 0.53 |
IL-8 | 128 | 0.12 | 0.17 | 83 | −0.20 | 0.07 |
IL-9 | 135 | 0.12 | 0.17 | 93 | 0.05 | 0.61 |
IL-10 | 39 | 0.05 | 0.77 | 22 | −0.09 | 0.69 |
IL-12(p70) | 110 | 0.30 | 0.002 | 62 | −0.08 | 0.55 |
IL-13 | 27 | 0.39 | 0.04 | 19 | 0.21 | 0.38 |
IL-15 | 2 | - | - | 4 | - | - |
IL-17 | 108 | 0.12 | 0.23 | 73 | −0.01 | 0.93 |
Eotaxin | 132 | 0.13 | 0.13 | 95 | −0.01 | 0.97 |
FGF-2 | 129 | 0.02 | 0.85 | 88 | 0.01 | 0.94 |
G-CSF | 92 | −0.03 | 0.76 | 40 | −0.38 | 0.02 |
GM-CSF | 48 | −0.05 | 0.72 | 46 | −0.10 | 0.53 |
IFN-γ | 136 | 0.09 | 0.31 | 86 | −0.12 | 0.26 |
IP-10 (CXCL10) | 136 | 0.22 | 0.01 | 94 | 0.20 | 0.05 |
MCP-1(MCAF) | 47 | 0.11 | 0.45 | 30 | −0.17 | 0.38 |
MIP-1a (CCL3) | 133 | 0.23 | 0.01 | 95 | −0.16 | 0.13 |
MIP-1b (CCL4) | 136 | 0.30 | 0.0005 | 94 | −0.15 | 0.15 |
PDGF-BB | 135 | 0.02 | 0.86 | 94 | −0.11 | 0.29 |
RANTES (CCL5) | 136 | −0.02 | 0.84 | 94 | −0.08 | 0.42 |
TNF-α | 120 | 0.17 | 0.06 | 65 | −0.14 | 0.28 |
VEGF | 135 | 0.17 | 0.05 | 92 | 0.09 | 0.41 |
Cytokines | Male Melanoma | Female Melanoma | Mann–Whitney |
---|---|---|---|
Median Value | Median Value | p-Value | |
IL-1b | 0.63 | 0.785 | 0.07 |
IL-1Ra | 15.17 | 24.125 | 0.25 |
IL-2 | 1.9 | 3.09 | 0.14 |
IL-4 | 3.05 | 2.75 | 0.06 |
IL-5 | 2.34 | 2.02 | 0.36 |
IL-6 | 3.34 | 3.0 | 0.73 |
IL-7 | 2.24 | 2.31 | 0.49 |
IL-8 | 6.57 | 6.32 | 0.64 |
IL-9 | 44.35 | 38.84 | 0.14 |
IL-10 | 3.84 | 6.31 | 0.13 |
IL-12(p70) | 16.94 | 14.57 | 0.98 |
IL-13 | 2.19 | 3.23 | 0.60 |
IL-15 | - | 30.11 | - |
IL-17 | 13.1 | 12.96 | 0.67 |
Eotaxin | 135.15 | 91.8 | 0.002 |
FGF-2 | 32.55 | 29.73 | 0.25 |
G-CSF | 5.09 | 5.45 | 0.60 |
GM-CSF | 11.71 | 9.97 | 0.47 |
IFN-γ | 20.83 | 27.33 | 0.26 |
IP-10 (CXCL10) | 567.19 | 468.93 | 0.13 |
MCP-1(MCAF) | 10.995 | 25.6 | 0.05 |
MIP-1a (CCL3) | 1.86 | 1.7 | 0.49 |
MIP-1b (CCL4) | 56.89 | 51.09 | 0.30 |
PDGF-BB | 1145.64 | 900.76 | 0.06 |
RANTES (CCL5) | 9536.67 | 7879.5 | 0.19 |
TNF-α | 17.355 | 20.11 | 0.34 |
VEGF | 65.345 | 50.515 | 0.49 |
Missing Values | Num. Melanoma | Num. Controls | Training Set Size | Testing Set Size | Predictors: Sex or Age | AUC (ROC) | Accuracy | No Info Rate | p-Value |
---|---|---|---|---|---|---|---|---|---|
Removed * | 72 | 124 | 138 | 58 | Sex, Age | 0.674 | 0.621 | 0.64 | 0.66 |
Sex | 0.658 | 0.638 | 0.64 | 0.56 | |||||
Age | 0.761 | 0.724 | 0.64 | 0.11 | |||||
None | 0.615 | 0.586 | 0.64 | 0.83 | |||||
Set to 0 ** | 96 | 136 | 164 | 68 | Sex, Age | 0.621 | 0.588 | 0.59 | 0.55 |
Sex | 0.510 | 0.588 | 0.59 | 0.55 | |||||
Age | 0.704 | 0.662 | 0.59 | 0.13 | |||||
None | 0.619 | 0.588 | 0.59 | 0.55 |
Cytokines | Median | p-Value (Mann–Whitney) | ||||||
---|---|---|---|---|---|---|---|---|
Ctrls (201) | Melanoma | Ctrls vs. all | (with Bonferroni Correction) | |||||
All (310) | Prim. (83) | Metast. (227) | Ctrls vs. Prim. | Ctrls vs. Metast. | Prim. vs. Metast. | |||
IL-1b | 6.66 | 7.04 | 6.73 | 7.15 | <0.0001 | 0.15 | <0.0001 | 0.71 |
IL-1Ra | 9.38 | 7.02 | 7.03 | 7.01 | <0.0001 | <0.0001 | <0.0001 | 1.31 |
IL-2 | 2.58 | 2.81 | 2.58 | 3.00 | 0.59 | 0.96 | 1.00 | 1.00 |
IL-4 | 3.46 | 3.46 | 3.17 | 3.58 | 0.96 | 0.45 | 1.00 | 0.15 |
IL-5 | 2.81 | 3.00 | 3.00 | 3.00 | 0.48 | 1.00 | 1.00 | 1.00 |
IL-6 | 5.61 | 6.39 | 5.98 | 6.64 | <0.0001 | 0.06 | <0.0001 | 0.06 |
IL-7 | 7.22 | 5.49 | 5.17 | 5.73 | <0.0001 | <0.0001 | <0.0001 | 0.01 |
IL-8 | 3.32 | 3.32 | 3.17 | 3.32 | 0.99 | 1.00 | 1.00 | 1.00 |
IL-9 | 2.58 | 2.81 | 2.58 | 2.81 | 0.76 | 1.00 | 1.00 | 0.48 |
IL-10 | 3.70 | 4.88 | 4.75 | 5.04 | <0.0001 | <0.0001 | <0.0001 | 0.66 |
IL-12(p70) | 4.25 | 2.32 | 2.81 | 2.00 | <0.0001 | <0.0001 | <0.0001 | 0.12 |
IL-13 | 5.64 | 5.52 | 5.55 | 5.49 | 0.96 | 1.00 | 1.00 | 1.00 |
IL-15 | 6.95 | 6.83 | 6.30 | 6.97 | 0.44 | 0.001 | 1.00 | 0.01 |
IL-17 | 5.67 | 6.83 | 6.07 | 7.37 | <0.0001 | 0.08 | <0.0001 | 0.05 |
Eotaxin | 4.75 | 5.29 | 4.95 | 5.39 | <0.0001 | 0.21 | <0.0001 | 0.06 |
FGF-2 | 7.11 | 7.03 | 6.25 | 7.24 | 0.51 | <0.0001 | 0.53 | <0.0001 |
G-CSF | 11.83 | 11.55 | 12.25 | 11.37 | 0.95 | 0.001 | 0.30 | 0.006 |
GM-CSF | 4.81 | 4.64 | 4.52 | 4.64 | 0.83 | 1.00 | 1.00 | 0.99 |
IFN-γ | 4.70 | 5.58 | 4.91 | 5.83 | <0.0001 | 0.03 | <0.0001 | 0.01 |
IP-10 (CXCL10) | 2.81 | 3.86 | 3.17 | 4.25 | <0.0001 | 0.36 | <0.0001 | 0.01 |
MCP-1(MCAF) | 3.32 | 3.46 | 3.00 | 3.58 | 0.03 | 1.00 | 0.03 | 0.01 |
MIP-1a (CCL3) | 5.13 | 8.11 | 7.76 | 8.24 | <0.0001 | <0.0001 | <0.0001 | 0.07 |
MIP-1b (CCL4) | 5.29 | 7.90 | 7.35 | 8.13 | <0.0001 | <0.0001 | <0.0001 | 0.12 |
PDGF-BB | 13.21 | 13.01 | 13.21 | 12.83 | 0.02 | 0.60 | 0.001 | 0.003 |
RANTES (CCL5) | 6.86 | 8.16 | 7.81 | 8.22 | <0.0001 | <0.0001 | <0.0001 | 1.00 |
TNF-α | 6.13 | 7.54 | 7.27 | 7.57 | <0.0001 | <0.0001 | <0.0001 | 0.003 |
VEGF | 8.95 | 8.97 | 8.67 | 9.03 | 0.37 | 0.001 | 1.00 | 0.002 |
Cytokines | AUC ± S.E. of ROC Analysis | |||
---|---|---|---|---|
Ctrls vs. all Melanoma | Ctrls vs. Primary | Ctrls vs. Metastatic | Primary vs. Metastatic | |
IL-1b | 0.62 ± 0.03 | 0.57 ± 0.04 | 0.63 ± 0.03 | 0.54 ± 0.04 |
IL-1Ra | 0.88 ± 0.02 | 0.88 ± 0.03 | 0.88 ± 0.02 | 0.53 ± 0.04 |
IL-2 | 0.51 ± 0.03 | 0.54 ± 0.04 | 0.51 ± 0.03 | 0.53 ± 0.04 |
IL-4 | 0.50 ± 0.03 | 0.55 ± 0.04 | 0.52 ± 0.03 | 0.57 ± 0.04 |
IL-5 | 0.52 ± 0.03 | 0.51 ± 0.04 | 0.52 ± 0.03 | 0.51 ± 0.04 |
IL-6 | 0.64 ± 0.03 | 0.59 ± 0.04 | 0.66 ± 0.03 | 0.59 ± 0.04 |
IL-7 | 0.86 ± 0.02 | 0.91 ± 0.03 | 0.85 ± 0.02 | 0.61 ± 0.04 |
IL-8 | 0.50 ± 0.03 | 0.52 ± 0.04 | 0.51 ± 0.03 | 0.52 ± 0.04 |
IL-9 | 0.51 ± 0.03 | 0.53 ± 0.04 | 0.52 ± 0.03 | 0.55 ± 0.04 |
IL-10 | 0.68 ± 0.03 | 0.65 ± 0.03 | 0.69 ± 0.03 | 0.55 ± 0.04 |
IL-12(p70) | 0.78 ± 0.02 | 0.77 ± 0.03 | 0.79 ± 0.02 | 0.58 ± 0.04 |
IL-13 | 0.50 ± 0.03 | 0.50 ± 0.04 | 0.50 ± 0.03 | 0.50 ± 0.04 |
IL-15 | 0.52 ± 0.03 | 0.64 ± 0.01 | 0.52 ± 0.03 | 0.61 ± 0.04 |
IL-17 | 0.61 ± 0.03 | 0.54 ± 0.04 | 0.63 ± 0.03 | 0.59 ± 0.04 |
Eotaxin | 0.63 ± 0.03 | 0.57 ± 0.04 | 0.65 ± 0.03 | 0.59 ± 0.04 |
FGF-2 | 0.52 ± 0.03 | 0.67 ± 0.04 | 0.54 ± 0.03 | 0.66 ± 0.04 |
G-CSF | 0.50 ± 0.03 | 0.63 ± 0.04 | 0.55 ± 0.03 | 0.61 ± 0.04 |
GM-CSF | 0.51 ± 0.03 | 0.52 ± 0.04 | 0.52 ± 0.03 | 0.54 ± 0.04 |
IFN-γ | 0.69 ± 0.03 | 0.60 ± 0.04 | 0.72 ± 0.03 | 0.61 ± 0.04 |
IP-10 (CXCL10) | 0.64 ± 0.03 | 0.56 ± 0.04 | 0.67 ± 0.03 | 0.61 ± 0.04 |
MCP-1(MCAF) | 0.56 ± 0.03 | 0.54 ± 0.04 | 0.59 ± 0.03 | 0.62 ± 0.04 |
MIP-1a (CCL3) | 0.93 ± 0.01 | 0.91 ± 0.02 | 0.93 ± 0.02 | 0.58 ± 0.04 |
MIP-1b (CCL4) | 0.87 ± 0.02 | 0.87 ± 0.02 | 0.86 ± 0.02 | 0.58 ± 0.04 |
PDGF-BB | 0.56 ± 0.03 | 0.55 ± 0.04 | 0.60 ± 0.03 | 0.62 ± 0.04 |
RANTES (CCL5) | 0.73 ± 0.03 | 0.73 ± 0.03 | 0.72 ± 0.02 | 0.53 ± 0.04 |
TNF-α | 0.77 ± 0.03 | 0.68 ± 0.03 | 0.80 ± 0.02 | 0.62 ± 0.04 |
VEGF | 0.52 ± 0.03 | 0.63 ± 0.04 | 0.52 ± 0.03 | 0.63 ± 0.04 |
Num. Melanoma | Num. Controls | Training Set Size | Testing Set Size | AUC (ROC) | Accuracy | No Info Rate | p-Value |
---|---|---|---|---|---|---|---|
310 | 201 | 358 | 153 | 0.99 | 0.95 | 0.61 | <0.00001 |
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Cesati, M.; Scatozza, F.; D’Arcangelo, D.; Antonini-Cappellini, G.C.; Rossi, S.; Tabolacci, C.; Nudo, M.; Palese, E.; Lembo, L.; Di Lella, G.; et al. Investigating Serum and Tissue Expression Identified a Cytokine/Chemokine Signature as a Highly Effective Melanoma Marker. Cancers 2020, 12, 3680. https://doi.org/10.3390/cancers12123680
Cesati M, Scatozza F, D’Arcangelo D, Antonini-Cappellini GC, Rossi S, Tabolacci C, Nudo M, Palese E, Lembo L, Di Lella G, et al. Investigating Serum and Tissue Expression Identified a Cytokine/Chemokine Signature as a Highly Effective Melanoma Marker. Cancers. 2020; 12(12):3680. https://doi.org/10.3390/cancers12123680
Chicago/Turabian StyleCesati, Marco, Francesca Scatozza, Daniela D’Arcangelo, Gian Carlo Antonini-Cappellini, Stefania Rossi, Claudio Tabolacci, Maurizio Nudo, Enzo Palese, Luigi Lembo, Giovanni Di Lella, and et al. 2020. "Investigating Serum and Tissue Expression Identified a Cytokine/Chemokine Signature as a Highly Effective Melanoma Marker" Cancers 12, no. 12: 3680. https://doi.org/10.3390/cancers12123680