Simultaneous Detection of Collagen I Alpha II and Cytokeratin 19 mRNA by Multiplex qPCR in Liquid Biopsy in Diagnosis of Patients with Resectable Solid Tumors
Abstract
:1. Introduction
2. Results
2.1. Preparation of the Standard Curve
2.2. Simplex RT-qPCR Standardization
2.3. Repeatability and Reproducibility of Intra- and Inter-Assay of RT-qPCR
2.4. Absence of Interactions between Real-Time Reactions
2.5. Limit of Detection (LOD) and RT-qPCR Efficiency
2.6. Baseline Characteristics of Patients
2.7. Comparative Analysis of mRNA Expression Level in Extracellular Vesicles of ACs vs. CPs
2.8. Combined Biomarker Prediction
3. Discussion
4. Materials and Methods
4.1. Patients and Human Samples
4.2. Blood Sample Collection and RNA Isolation from Extracellular Vesicles
4.3. Multiplex RT-qPCR Assay
4.4. Amplification Efficiency (E)
4.5. Statistical Analysis
5. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Dilutions | ARN (ng) | Ct Values for the Different Dilutions of Human XpressRef Universal Total ARN | |||||
---|---|---|---|---|---|---|---|
β-Actin Mean Ct ± SD | CV (%) | KRT19 Mean Ct ± SD | CV (%) | COL1A2 Mean Ct ± SD | CV (%) | ||
Intra-assay | 200 | 17.02 ± 0.44 | 2.58 | 19.83 ± 0.23 | 1.18 | 17.91 ± 0.11 | 0.63 |
20 | 19.82 ± 0.44 | 2.21 | 22.50 ± 0.22 | 0.96 | 21.32 ± 0.37 | 1.75 | |
2 | 23.45 ± 0.16 | 0.68 | 26.65 ± 0.26 | 0.99 | 24.80 ± 0.38 | 1.55 | |
0.2 | 27.11 ± 0.29 | 1.07 | 29.61 ± 0.33 | 1.12 | 28.44 ± 0.24 | 0.85 | |
0.02 | 30.19 ± 0.23 | 0.77 | 33.32 ± 0.17 | 0.51 | 31.78 ± 0.27 | 0.85 | |
0.002 | 33.13 ± 0.23 | 0.69 | 36.50 ± 1.15 | 3.16 | 35.31 ± 1.05 | 3.00 | |
0.0002 | 36.61 ± 0.31 | 0.84 | - | - | - | - | |
Inter-assay | 200 | 16.89 ± 0.39 | 2.33 | 19.97 ± 0.29 | 1.45 | 19.22 ± 0.68 | 3.52 |
20 | 20.04 ± 0.44 | 2.17 | 23.30 ± 0.39 | 1.66 | 22.65 ± 0.68 | 3.01 | |
2 | 23.29 ± 0.26 | 1.10 | 26.63 ± 0.31 | 1.15 | 25.70 ± 0.88 | 3.42 | |
0.2 | 27.15 ± 0.35 | 1.30 | 30.16 ± 0.28 | 0.93 | 29.68 ± 0.67 | 2.24 | |
0.02 | 30.89 ± 0.23 | 0.74 | 33.96 ± 0.61 | 1.81 | 33.38 ± 0.81 | 2.44 | |
0.002 | 33.45 ± 0.43 | 1.27 | 36.68 ± 0.92 | 2.50 | 35.77 ± 0.95 | 2.66 | |
0.0002 | 36.63 ± 0.57 | 1.54 | - | - | - | - |
Target | Multiplex | Slope | R2 | Efficiency |
---|---|---|---|---|
COL1A2 | Singleplex | −3.325 | 0.987 | 99.9% |
KRT19 | Singleplex | −3.240 | 0.996 | 103.5% |
ACTB | Singleplex | −3.453 | 0.999 | 94.8% |
COL1A2 | Triplex | −3.043 | 0.957 | 113.1% |
KRT19 | Triplex | −3.594 | 0.979 | 89.8% |
ACTB | Triplex | −3.330 | 0.995 | 99.7% |
Clinical and Pathological Features of Individuals Included in This Study (n = 60) | ||||
---|---|---|---|---|
Clinic Pathological Features | AC (n = 13) | CPs (n = 47) | ||
Age Median (Range) | 41.46 (26–58) | 70.36 (46–90) | ||
Gender | ||||
Female | 9 (69.23%) | 24 (51.06%) | ||
Male | 4 (30.77%) | 23 (48.94%) | ||
Curative Resections | - | 47 (100%) | ||
Clinical and Pathological Features of Patients with Tumor Process (n = 47) | ||||
Colorectal Cancer (CRC) (n = 31) | Prostate Cancer (PC) (n = 3) | |||
Age mean (range) | 71.45 (40–90) | Age mean (range) | 71.66 (64–86) | |
Histological Type | Histological Type | |||
Adenocarcinoma | 31 | Adenocarcinoma | 3 | |
Clinical stage | Clinical stage | |||
Tis */I/II | 20 | II | 2 | |
III | 11 | III ** | 1 | |
Renal Cancer (RC) (n = 6) | Bladder Cancer (BC) (n = 4) | |||
Age mean (range) | 61.66 (44–79) | Age mean (range) | 72.00 (58–86) | |
Histological Type | Histological Type | |||
CCRCC | 6 | Urothelial | 4 | |
Clinical stage | Clinical stage | |||
I/II | 3 | 0a | 1 | |
III | 3 | II | 3 | |
Others (n = 3) | ||||
Age | Sex | Histological Type | Clinical Stage | |
Leiomyosarcoma | 74 | female | Renal leiomyosarcoma | III |
Breast cancer | 74 | female | Ductal invasive | I |
Penile cancer | 66 | male | Squamous | II |
Groups | N | Amplification | Mean ± SD | p-Value | ||||
---|---|---|---|---|---|---|---|---|
COL1A2 | KRT19 | COL1A2 and KRT19 | COL1A2 | KRT19 | COL1A2 | KRT19 | ||
AC | 13 | 12 (92.30%) | 13 (100%) | 12 (92.30%) | 1.21 ± 0.78 | 1.40 ± 1.25 | - | - |
CP | 47 | 30 (63.83%) | 38 (80.85%) | 21 (44.68%) | 26.61 ± 45.46 | 9.69 ± 15.07 | 0.0001 | 0.0031 |
CRC | 31 | 22 (70.96%) | 24 (77.42%) | 15 (48.39%) | 32.11 ± 51.92 | 12.06 ± 18.35 | 0.0001 | 0.0054 |
RC | 6 | 3 (50%) | 5 (83.33%) | 1 (16.67%) | 9.82 ± 6.50 | 8.50 ± 4.75 | 0.0088 | 0.0008 |
BC | 4 | 1 (25%) | 4 (100%) | 1 (25%) | nd | 6.74 ± 4.89 | nd | 0.1567 |
PC | 3 | 1 (33.33%) | 3 (100%) | 1 (33.33%) | nd | 1.84 ± 1.71 | nd | 0.5054 |
Others | 3 | 3 (100%) | 2 (66.67%) | 2 (66.67%) | 16.58 ± 17.14 | nd | 0.0044 | nd |
Total | 60 | 42 (70%) | 51 (85%) | 33 (55%) | nd | nd | nd | nd |
Sensitivity | 0.830 | (0.699–0.911) |
Specificity | 0.846 | (0.578–0.957) |
Positive Predictive Value | 0.951 | (0.839–0.986) |
Negative Predictive Value | 0.579 | (0.363–0.769) |
Diagnostic Accuracy | 0.833 | (0.720–0.907) |
Positive Likelihood Ratio | 5.394 | (2.004–14.520) |
Negative Likelihood Ratio | 0.201 | (0.152–0.265) |
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Estévez Pérez, L.S.; Alén, B.O.; Otero Alén, M.; Hormaetxe, S.D.; Simón, L.; Concha, Á. Simultaneous Detection of Collagen I Alpha II and Cytokeratin 19 mRNA by Multiplex qPCR in Liquid Biopsy in Diagnosis of Patients with Resectable Solid Tumors. Int. J. Mol. Sci. 2024, 25, 9567. https://doi.org/10.3390/ijms25179567
Estévez Pérez LS, Alén BO, Otero Alén M, Hormaetxe SD, Simón L, Concha Á. Simultaneous Detection of Collagen I Alpha II and Cytokeratin 19 mRNA by Multiplex qPCR in Liquid Biopsy in Diagnosis of Patients with Resectable Solid Tumors. International Journal of Molecular Sciences. 2024; 25(17):9567. https://doi.org/10.3390/ijms25179567
Chicago/Turabian StyleEstévez Pérez, Lara Sofía, Begoña O. Alén, María Otero Alén, Saioa Domínguez Hormaetxe, Laureano Simón, and Ángel Concha. 2024. "Simultaneous Detection of Collagen I Alpha II and Cytokeratin 19 mRNA by Multiplex qPCR in Liquid Biopsy in Diagnosis of Patients with Resectable Solid Tumors" International Journal of Molecular Sciences 25, no. 17: 9567. https://doi.org/10.3390/ijms25179567