Pathophysiological Implications of Urinary Peptides in Hepatocellular Carcinoma
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Ethics
2.2. Study Design
2.3. Sample Preparation
2.4. CE-MS Analysis
2.5. CE-MS Data Processing
2.6. Support Vector Machine Model Generation and Classification
2.7. Peptide Sequencing
2.8. In Silico Protease Prediction
2.9. Immunohistochemistry
2.10. Statistics
3. Results
3.1. Identification of Urinary Peptides as HCC Progression Markers by CE-MS Analysis
3.2. Development of the 31 HCC Progression Markers to a Multivariate Classification Model
3.3. CE-MS and Peptide Sequence Characteristics of the Peptide Marker Candidates
3.4. Differential Expression of KLK6 and MEP1A in HCC, Cirrhosis and Normal Liver Tissue
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Study Phase | Discovery | Validation | |||||
---|---|---|---|---|---|---|---|
Patient Group | HCC | Non-HCC | p * | HCC | Non-HCC | p * | |
Patients/samples, n | 18/18 | 51/51 | n.a. | 39/39 | 87/87 | n.a. | |
Age, years, mean/range | 58/28–76 | 52/18–82 | 0.08 | 67/38–87 | 56/20–85 | 0.0001 | |
Female/male, n | 3/15 | 20/31 | 0.14 | 9/30 | 35/52 | 0.07 | |
No. detected peptides, mean/range | 1753/623–2965 | 2329/920–4488 | 0.02 | 2872/1073–4617 | 2461/811–4057 | 0.008 | |
Liver cirrhosis, n (%) | 18 (100) | 25 (49) | <0.0001 | 28 (72) | 47 (54) | 0.08 | |
Diabetes mellitus, n (%) | 3 (17) | 13 (25) | 0.53 | 16 (41) | 23 (26) | 0.14 | |
Body mass index, mean/range | 26.8/18.3–32.1 | 27.8/20.3–41.7 | 0.76 | 27.7/20.2–41.0 | 27.5/16.4–46.6 | 0.52 | |
Platelet count, ×109/L, mean/range | 99/26–310 | 162/25–391 | 0.02 | 195/28–595 | 234/44–961 | 0.04 | |
a-Fetoprotein (AFP), µg/L, mean/range | 1821/3–22,826 | 4/1–50 | <0.0001 | 6669/1–107,202 | 39/1–1493 | <0.0001 | |
Alkaline phosphatase, U/L, mean/range | 151/60–380 | 125/45–797 | 0.008 | 291/83–1781 | 196/37–693 | 0.02 | |
Aspartate aminotransferase (AST), U/L, mean/range | 74/24–284 | 49/13–120 | 0.03 | 112/26–457 | 69/13–1606 | <0.0001 | |
Alanine aminotransferase (ALT), U/L, mean/range | 61/14–288 | 53/10–304 | 0.84 | 69/12–242 | 72/7–1038 | 0.37 | |
AST:ALT ratio, mean/range | 1.48/0.89–3.41 | 1.17/0.20–3.20 | 0.05 | 2.11/0.55–7.46 | 1.13/0.18–3.06 | 0.0004 | |
Approx. Ishak Fibrosis Score as per FIB-4 index, n, 0–1/2–3/4–6 | 0/1/17 | 14/12/17 | 0.0004 | 2/9/28 | 36/28/23 | <0.0001 | |
Albumin, g/L, mean/range | 32/19–48 | 40/26–51 | 0.0003 | 34/16–48 | 38/15–70 | 0.01 | |
Bilirubin, µmol/L, mean/range | 27/9–86 | 26/3–163 | 0.05 | 43/6–254 | 34/3–390 | 0.03 | |
ALBI stage, n, 1/2/3 | 2/11/5 | 26/15/4 | 0.003 | 11/20/8 | 47/22/18 | 0.009 | |
Liver Disease Etiology, n | |||||||
Primary HCC | 0 | 0 | 1 | 0 | |||
Alcoholic cirrhosis (C2) | 7 | 4 | 7 | 6 | |||
Virus-related cirrhosis (HBV/HCV/HDV) | 3/4/0 | 0/4/1 | 3/4/0 | 2/2/0 | |||
Cryptogenic/Biliary cirrhosis | 0/0 | 3/1 | 1/0 | 2/0 | |||
Hereditary (Mucoviscidosis/Hemochromatosis/AATD) | 1/1/0 | 0/0/0 | 1/0/1 | 0/0/0 | |||
Cholestasis (PBC/PSC/SSC/PFIC) | 2/0/0/0 | 2/2/1/1 | 2/5/0/0 | 2/14/1/0 | |||
Autoimmune hepatitis (AIH) | 0 | 2 | 0 | 0 | |||
NAFLD/NASH/NASH-LC | 0/0/0 | 9/8/4 | 0/2/12 | 18/6/15 | |||
GI cancer (CCA/PCA) with LC | 0/0 | 0/0 | 0/0 | 7/2 | |||
Other benign liver diseases (CHP/CDL) | 0/0 | 0/0 | 0/0 | 7/3 | |||
No liver disease | 0 | 9 | 0 | 0 |
Peptide ID † | Group-Wise Comparison § of Peptide Distributions HCC (n = 18) vs. Non-HCC Liver Disease (n = 51) | Rank Correlation of Peptide Amplitudes to a Grading Score 0 = Non-LC #, 1 = LC & 2 = HCC | Peptide Distribution in the Discovery Patient Groups | |||||||
---|---|---|---|---|---|---|---|---|---|---|
Non-LC # (n = 26) | LC (n = 25) | HCC (n = 18) | ||||||||
p-Value ‡ for Group Differences | AUC for Group Differences | Spearman Rho Coef. | p-Value ‡ for Rank Differences | Mean Amp (SD) | Freq. | Mean Amp (SD) | Freq. | Mean Amp (SD) | Freq. | |
54 | 8.34 × 10−03 | 0.70 | 0.353 | 1.43 × 10−02 | 2 (10) | 4 | 7 (28) | 12 | 53 (85) | 56 |
1059 | 4.57 × 10−02 | 0.63 | 0.303 | 4.06 × 10−02 | 4 (18) | 8 | 3 (13) | 12 | 30 (55) | 33 |
1160 | 4.23 × 10−02 | 0.63 | 0.431 | 1.83 × 10−03 | 1 (4) | 8 | 34 (46) | 48 | 61 (81) | 67 |
1778 | 1.50 × 10−02 | 0.65 | 0.407 | 3.66 × 10−03 | 5 (26) | 4 | 18 (34) | 32 | 29 (40) | 50 |
2314 | 2.92 × 10−02 | 0.65 | 0.385 | 6.52 × 10−03 | 1911 (3077) | 42 | 2696 (2346) | 80 | 3017 (2222) | 94 |
3559 | 1.30 × 10−02 | 0.63 | 0.396 | 4.97 × 10−03 | 47 (88) | 31 | 184 (223) | 60 | 201 (224) | 67 |
3662 | 8.27 × 10−04 | 0.76 | 0.489 | 3.08 × 10−04 | 76 (91) | 62 | 161 (188) | 76 | 256 (177) | 89 |
4564 | 6.81 × 10−03 | 0.72 | 0.479 | 4.18 × 10−04 | 270 (130) | 92 | 639 (579) | 96 | 591 (449) | 94 |
4610 | 2.50 × 10−03 | 0.65 | 0.398 | 4.74 × 10−03 | 0 (0) | 0 | 64 (157) | 16 | 166 (272) | 39 |
4829 | 1.54 × 10−02 | 0.66 | 0.409 | 3.49 × 10−03 | 59 (142) | 27 | 277 (358) | 76 | 245 (286) | 67 |
5284 | 5.61 × 10−03 | 0.70 | 0.362 | 1.15 × 10−02 | 63 (102) | 42 | 81 (137) | 44 | 172 (174) | 67 |
6601 | 3.57 × 10−06 | 0.72 | 0.470 | 5.54 × 10−04 | 0 (0) | 0 | 1 (4) | 4 | 199 (286) | 44 |
6607 | 8.67 × 10−04 | 0.65 | 0.380 | 7.39 × 10−03 | 0 (0) | 0 | 19 (83) | 8 | 259 (737) | 33 |
8720 | 6.37 × 10−04 | 0.75 | −0.587 | 8.05 × 10−06 | 658 (664) | 73 | 231 (641) | 48 | 31 (98) | 11 |
9080 | 3.34 × 10−02 | 0.65 | 0.391 | 5.69 × 10−03 | 813 (2125) | 15 | 893 (1236) | 48 | 1618 (1815) | 61 |
9510 | 5.03 × 10−03 | 0.72 | 0.370 | 9.56 × 10−03 | 2227 (4587) | 31 | 2847 (4477) | 68 | 4563 (3380) | 83 |
9728 | 1.90 × 10−03 | 0.73 | 0.511 | 1.51 × 10−04 | 345 (745) | 23 | 902 (1038) | 56 | 1359 (916) | 89 |
10177 | 3.24 × 10−03 | 0.71 | 0.358 | 1.29 × 10−02 | 162 (288) | 38 | 170 (224) | 48 | 476 (376) | 89 |
11725 | 6.63 × 10−04 | 0.65 | 0.385 | 6.64 × 10−03 | 0 (0) | 0 | 4 (12) | 8 | 28 (50) | 33 |
12459 | 8.13 × 10−06 | 0.86 | −0.526 | 8.68 × 10−05 | 373 (152) | 96 | 335 (236) | 92 | 119 (124) | 61 |
13134 | 7.85 × 10−04 | 0.73 | 0.311 | 3.45 × 10−02 | 32 (73) | 27 | 170 (375) | 24 | 364 (411) | 67 |
13176 | 1.20 × 10−03 | 0.73 | 0.410 | 3.47 × 10−03 | 27 (54) | 27 | 103 (190) | 40 | 171 (155) | 72 |
14389 | 5.32 × 10−04 | 0.74 | −0.400 | 4.53 × 10−03 | 53 (64) | 54 | 20 (37) | 40 | 0 (0) | 0 |
14925 | 7.69 × 10−04 | 0.73 | −0.567 | 1.82 × 10−05 | 689 (1120) | 58 | 237 (627) | 32 | 0 (0) | 0 |
15342 | 1.38 × 10−04 | 0.80 | −0.604 | 3.66 × 10−06 | 766 (308) | 100 | 420 (323) | 76 | 224 (219) | 78 |
17066 | 1.33 × 10−04 | 0.66 | 0.397 | 4.89 × 10−03 | 0 (3) | 4 | 8 (53) | 4 | 257 (693) | 33 |
17805 | 8.75 × 10−04 | 0.75 | −0.514 | 1.37 × 10−04 | 394 (478) | 69 | 235 (349) | 52 | 16 (36) | 22 |
19681 | 5.57 × 10−04 | 0.76 | −0.630 | 1.14 × 10−06 | 127 (129) | 85 | 34 (53) | 40 | 9 (21) | 17 |
20237 | 1.82 × 10−04 | 0.79 | 0.483 | 3.69 × 10−04 | 292 (480) | 35 | 316 (635) | 56 | 940 (1064) | 94 |
24328 | 2.07 × 10−05 | 0.84 | −0.636 | 8.84 × 10−07 | 5557 (3207) | 100 | 2341 (2674) | 100 | 890 (1187) | 72 |
29919 | 1.20 × 10−02 | 0.69 | 0.465 | 6.63 × 10−04 | 199 (505) | 23 | 525 (661) | 60 | 857 (1223) | 78 |
Peptide ID † | Exp. Mass [Da] | CE Time [min] | Protein | AA | Proteases for N-Terminal Cleavage | Peptide Sequence (Black) with Flanking Regions (Grey) ‡ | Proteases for C-Terminal Cleavage |
---|---|---|---|---|---|---|---|
54 | 807.39 | 23.2 | CLU | 366–371 | --- | LNEQ|FNWVSR|LANL | --- |
1059 | 920.34 | 21.2 | --- | --- | --- | --- | --- |
1160 | 928.51 | 24.6 | UMOD | 592–599 | MEP1A, MMP3 | RSGS|VIDQSRVL|NLGP | MEP1A, KLK6, CTS [B,D,E] |
1778 | 981.49 | 24.4 | ACTB | 107–115 | MEP1A, MMP [3,13], CTS [B,D,E] | HPVL|LTEAPLNPK|ANRE | --- |
2314 | 1032.45 | 25.9 | ACTB | 95–103 | --- | YNEL|RVAPEEHPV|LLTE | MEP1A, MMP [3,13], CTS [B,D,E] |
3559 | 1134.59 | 24.0 | COL3A1 | 755–766 | --- | ADGV|PGKDGPRGPTGP|IGPP | --- |
3662 | 1142.55 | 22.0 | ADGRF3 | 56–64 | --- | DKAW|NERIDRPFP|ACPI | --- |
4564 | 1199.55 | 20.8 | TKT | 343–352 | --- | DGDT|KNSTFSEIFK|KEHP | --- |
4610 | 1201.53 | 24.9 | GAGE12H | 58–70 | MEP1A, MMP3, CTSB | AAAQ|KGEDEGASAGQGP|KPEA | MMP3 |
4829 | 1217.48 | 27.7 | COL3A1 | 179–191 | --- | PAGP|pGPpGPpGTSGHp|GSPG | KLK6, CTSB |
5284 | 1250.64 | 20.6 | HBB | 136–147 | --- | QKVV|AGVANALAHKYH | C-terminal end |
6601 | 1352.78 | 22.0 | AHNAK | 772–784 | --- | EVDV|NLPKADVDISGPK|IDVT | MEP1A, KLK6 |
6607 | 1353.53 | 23.8 | FGA | 605–617 | MEP1A | YKMA|DEAGSEADHEGTH|STKR | --- |
8720 | 1513.62 | 29.5 | CDH1 | 397–410 | --- | ITTL|KVTDADAPNTPAWE|AVYT | --- |
9080 | 1539.74 | 40.4 | COL18A1 | 1400–1416 | MEP1A, CTSB | EGRQ|GPpGPpGPPGPPSFPGP|HRQT | MMP3 |
9510 | 1576.68 | 44.9 | --- | --- | --- | --- | --- |
9728 | 1594.77 | 40.3 | COL1A1 | 1177–1194 | MMP3 | DAGP|VGPpGPpGPpGPpGPPSA|GFDF | MMP [3,13], CTSB |
10177 | 1624.73 | 25.1 | COL2A1 | 1150–1167 | --- | GPSG|DQGASGpAGpSGpRGPpG|PVGP | --- |
11725 | 1733.73 | 29.8 | GSN | 605–621 | CTSD | AAYL|WVGTGASEAEKTGAQEL|LRVL | MEP1A, MMP3, CTS [D,E] |
12459 | 1782.85 | 26.0 | --- | --- | --- | --- | --- |
13134 | 1836.79 | 31.1 | COL1A2 | 918–937 | MMP3 | SPGV|NGApGEAGRDGNPGNDGPpG|RDGQ | MEP1A, MMP [3,13], CTSB |
13176 | 1840.83 | 41.9 | COL2A1 | 1193–1213 | MEP1A, KLK6 | PRGR|SGETGPAGppGNPGPPGPpGP|PGPG | KLK6 |
14389 | 1930.89 | 31.6 | COL3A1 | 618–639 | MEP1A, CTSB | TGPQ|GPpGPTGPGGDKGDTGPpGPQG|LQGL | MEP1A, MMP [3,13], CTSB |
14925 | 1972.96 | 25.0 | PCSK1N | 223–241 | MEP1A | RRAA|DHDVGSELPPEGVLGALLR|VKRL | MEP1A, MMP [3,13] |
15342 | 2009.88 | 32.4 | COL5A2 | 136–157 | MEP1A, MMP [3,13], CTSB | GAPG|SKGEAGpTGPMGDpGTVGPPGP|VGER | MMP3 |
17066 | 2169.98 | 33.7 | COL16A1 | 1145–1166 | MEP1A, MMP [3,13], CTSB | GPQG|NSGEKGDQGFQGQPGFpGPPGP|PGFP | --- |
17805 | 2232.00 | 33.6 | COL5A1 | 999–1021 | --- | PPGV|VGpQGpTGETGpMGERGHPGPpG|PPGE | MEP1A, CTSB |
19681 | 2380.08 | 35.8 | COL3A1 | 201–228 | MEP1A, CTSB | PGYQ|GPPGEPGQAGpSGpPGppGAIGPSGPAG|KDGE | MEP1A, MMP [3,13], CTSB |
20237 | 2430.60 | 35.7 | --- | --- | --- | --- | --- |
24328 | 2854.37 | 34.6 | COL3A1 | 616–646 | CTSB | GETG|PQGPpGPTGpGGDKGDTGPpGPQGLQGLpGT|GGPP | MEP1A, CTSB |
29919 | 3524.75 | 32.4 | CLU | 390–423 | --- | VTTV|ASHTSDSDVPSGVTEVVVKLFDSDPITVTVPVEV|SRKN | MMP3 |
ACTB, Actin, cytoplasmic 1; ADGRF3, Adhesion G-protein coupled receptor F3; AHNAK, Neuroblast differentiation-associated protein; CDH1, Cadherin-1; CLU, Clusterin; COL1A1, Collagen α-1(I) chain; COL1A2, Collagen α-2(I) chain; COL2A1, Collagen α-1(II) chain; COL3A1, Collagen α-1(III) chain; COL5A1, Collagen α-1(V) chain COL5A2, Collagen α-2(V) chain; COL16A1, Collagen α-1(XVI) chain; COL18A1, Collagen α-1(XVIII) chain; CTS, Cathepsin; FGA, Fibrinogen α chain; GAGE12H, G antigen 12H; GSN, Gelsolin; HBB, Hemoglobin subunit β; KLK6, Kallikrein-6; MEP1A, Meprin A subunit α; MMP, Matrix metallopeptidase; PCSK1N, ProSAAS; TKT, Transketolase; UMOD, uromodulin. |
Protease | Peptide Substrate Distribution [Avg. Ion Counts ± SD] | Fold-Change Case/Control | p | |
---|---|---|---|---|
HCC Case Group (n = 18) | Non-HCC Liver Disease Control Group (n = 51) | |||
MEP1A | 196.23 ± 93.19 | 365.65 ± 231.12 | 0.54 | 0.003 |
MMP3 | 632.99 ± 317.56 | 393.63 ± 331.29 | 1.60 | 0.007 |
MMP13 | 729.36 ± 402.76 | 495.03 ± 539.78 | 1.47 | 0.012 |
KLK6 | 166.40 ± 79.87 | 67.09 ± 88.51 | 2.47 | <0.0001 |
CTSB | 347.63 ± 173.67 | 643.77 ± 399.10 | 0.53 | 0.004 |
CTSD | 32.90 ± 33.44 | 17.35 ± 31.21 | 1.89 | 0.015 |
CTSE | 34.41 ± 31.99 | 22.56 ± 41.25 | 1.52 | 0.031 |
Tissue Type | Allred IHC Score | |
---|---|---|
KLK6 | MEP1A | |
HCC 1 | 3 | 0 |
HCC 2 | 3 | 1 |
HCC 3 | 3 | 0 |
HCC 4 | 3 | 0 |
HCC 5 | 3 | 0 |
Cirrhosis 1 | 2 | 0 |
Cirrhosis 2 | 2 | 0 |
Cirrhosis 3 | 3 | 0 |
Cirrhosis 4 | 3 | 0 |
Normal liver 1 | 1 | 1 |
Normal liver 2 | 1 | 0 |
Normal liver 3 | 1 | 1 |
Normal liver 4 | 1 | 1 |
Normal liver 5 | 1 | 1 |
HCC-31 Peptide Marker ID | Amino Acid Sequence | Protein Name | Protein Symbol | Biological Source of Identification (Other than Urine) | Reference |
---|---|---|---|---|---|
11354 | 107-LTEAPLNPK-115 | Actin; α skeletal muscle | ACTA1 | Cerebellum tissue ‡ | Marcu et al. [33] |
HCC tumor tissue ‡ | Liang-Qing et al. [34] | ||||
14071 | 95-RVAPEEHPV-103 | Actin, cytoplasmic 1 | ACTB | Lung tissue ‡ | Marcu et al. [33] |
25411 | 179-PGPPGPPGTSGHP-191 | Collagen α-1(III) chain | COL3A1 | Plasma | Zakharova et al. [35] |
33901 | 605-DEAGSEADHEGTH-617 | Fibrinogen α chain | FGA | Serum | Ueda et al. [36] |
Plasma | Koomen et al. [37] | ||||
135817 | 390-ASHTSDSDVPSGVTEV VVKLFDSDPITVTVPVEV-423 | Clusterin | CLU | Cerebrospinal fluid | Belogurov et al. [38] |
57312 | 605-WVGTGASEAEK TGAQEL-621 | Gelsolin | GSN | Plasma | Modzdiak et al. [39] |
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Bannaga, A.; Metzger, J.; Voigtländer, T.; Pejchinovski, M.; Frantzi, M.; Book, T.; James, S.; Gopalakrishnan, K.; Mischak, H.; Manns, M.P.; et al. Pathophysiological Implications of Urinary Peptides in Hepatocellular Carcinoma. Cancers 2021, 13, 3786. https://doi.org/10.3390/cancers13153786
Bannaga A, Metzger J, Voigtländer T, Pejchinovski M, Frantzi M, Book T, James S, Gopalakrishnan K, Mischak H, Manns MP, et al. Pathophysiological Implications of Urinary Peptides in Hepatocellular Carcinoma. Cancers. 2021; 13(15):3786. https://doi.org/10.3390/cancers13153786
Chicago/Turabian StyleBannaga, Ayman, Jochen Metzger, Torsten Voigtländer, Martin Pejchinovski, Maria Frantzi, Thorsten Book, Sean James, Kishore Gopalakrishnan, Harald Mischak, Michael P. Manns, and et al. 2021. "Pathophysiological Implications of Urinary Peptides in Hepatocellular Carcinoma" Cancers 13, no. 15: 3786. https://doi.org/10.3390/cancers13153786