Human Papillomavirus E7 and p16INK4a mRNA Multiplexed Quantification by a QuantiGeneTM Proof-of-Concept Assay Sensitively Detects Infection and Cervical Dysplasia Severity
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Clinical Study Samples
2.3. HPV DNA Testing
2.4. QuantiGeneTM 2.0 Plex Assay Design and Method Adaptation for QG-POC Assay
2.5. Statistical Analysis
3. Results
3.1. QG-POC Assay Detects mRNA Targets Specifically with Good Sensitivity in Cell-Line Samples
3.2. QG-POC Assay Detects HPV-E7 mRNA in Clinical Cervical Smear Samples
3.3. QG-POC Assay-Reported mRNA Expression Strength Indicates Dysplasia Presence and Severity
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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Target mRNA | Gene Accession Number |
---|---|
HPV6-E7 | L41216 |
HPV16-E7 | K02718 |
HPV18-E7 | X05015 |
HPV31-E7 | J04353 |
HPV45-E7 | X74479 |
HPV59-E7 | EU918767 |
HPV68-E7 | Y14591 |
ACTB | NM_001101 |
HPRT1 | NM_000077 |
p16INK4a | NM_000194 |
ASSIST Score 1 | Clinical Group | HPV Genotype 2 (n (%)) | No HPV Detectable | |||||
---|---|---|---|---|---|---|---|---|
16 | 18 | 31 | 59 | 68 | 6 | |||
0 | Normal (n = 177) | 113 (52.6) | 15 (6.9) | 6 (2.8) | 26 (12.1) | 2 (0.9) | 5 (2.3) | 41 (23.2) |
1 | LCIN (n = 58) | 48 (63.2) | 6 (7.9) | 8 (10.5) | 5 (8.6) | 4 (5.3) | 2 (2.6) | 0 (0) |
2 | HCIN (n = 195) | 151 (62.7) | 25 (10.4) | 24 (10.0) | 22 (9.1) | 4 (1.7) | 5 (2.1) | 0 (0) |
3 | CxCa (n = 11) | 10 (90.9) | 1 (9.1) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
total | 441 | 322 (73.0) | 47 (10.7) | 38 (8.6) | 53 (12.0) | 8 (1.8) | 12 (2.7) | 41 (9.1) |
ASSIST Score 1 | Clinical Group | HPV Genotype * (n (%)) | No HPV Detectable | |||||
---|---|---|---|---|---|---|---|---|
16 | 18 | 31 | 59 | 68 | 6 | |||
0 | Normal (n = 177) | 46 (26.0) | 8 (4.5) | 11 (6.2) | 16 (9.0) | 3 (1.7) | 5 (2.8) | 106 (60.0) |
1 | LCIN (n = 58) | 27 (46.6) | 4 (6.9) | 5 (8.6) | 12 (20.7) | 2 (3.4) | 3 (5.1) | 21 (36.2) |
2 | HCIN (n = 195) | 113 (57.9) | 20 (10.3) | 26 (13.3) | 23 (11.8) | 4 (2.1) | 6 (3.1) | 44 (22.6) |
3 | CxCa (n = 11) | 8 (72.7) | 2 (18.2) | 0 (0.0) | 3 (27.3) | 1 (9.1) | 0 (0.0) | 1 (9.1) |
total | 441 | 194 (44.0) | 34 (7.8) | 42 (9.5) | 54 (12.2) | 10 (2.3) | 14 (3.2) | 172 (39.0) |
Cohen’s Kappa | ||||
---|---|---|---|---|
HPV Type | Concordance (%) | Κ | CI (95%) | Agreement + |
6 | 98.41 | 0.66 *** | 0.44–0.87 | Substantial |
16 | 65.53 | 0.35 *** | 0.26–0.43 | Fair |
18 | 95.46 | 0.73 *** | 0.61–0.84 | Substantial |
31 | 93.42 | 0.60 *** | 0.46–0.74 | Moderate |
59 | 79.37 | 0.03 | −0.14–0.20 | Slight |
68 | 95.92 | −0.02 | −0.48–0.44 | No |
QG-POC HPV genotypes # | 70.29 | 0.27 *** | 0.17–0.38 | Fair |
Biomarker | AUC (CI 95%) | Cut-off | Sensitivity (%) | Specificity (%) | Univariate Regression OR (CI 95%) | Multivariate Regression OR (CI 95%) |
---|---|---|---|---|---|---|
HPV16-E7 | 0.68 (0.63–0.72) | 0.025 | 47.6 | 84.7 | 6.03 *** (2.55–18.37) | - |
HPV18-E7 | 0.54 (0.50–0.57) | 0.015 | 16.9 | 90.2 | 3.13 (1.29–18.89) | - |
HPV31-E7 | 0.53 (0.49–0.57) | 0.005 | 17.5 | 88.9 | 1.30 (0.22–9.57) | - |
HPV59-E7 | 0.50 (0.48–0.53) | 0.005 | 1.9 | 100 | 3.88 (0.85–140.64) | - |
Leading HPV-E7 | 0.74 (0.70–0.79) | 0.490 | 63.6 | 75.5 | 4.53 *** (2.44–9.78) | 2.78 ** (1.55–5.97) |
p16INK4a | 0.71 (0.66–0.76) | 0.175 | 64.0 | 68.9 | 5.81 *** (3.04–12.05) | 3.31 ** (1.64–7.13) |
Biomarker | AUC (CI 95%) | Cut-off | Sensitivity (%) | Specificity (%) | Univariate Regression OR (CI 95%) | Multivariate Regression OR (CI 95%) |
---|---|---|---|---|---|---|
Leading HPV-E7 | 0.79 (0.62–0.97) | 0.185 | 81.8 | 78.6 | 1.51 *** (1.21–1.88) | 1.49 * (1.06–2.01) |
p16INK4a | 0.69 (0.48–0.91) | 0.725 | 54.5 | 90.2 | 2.08 ** (1.19–3.56) | 1.06 (0.48–2.31) |
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Skof, A.S.; Rotenberg, L.; Hannemann, P.V.F.; Thies, S.; Boschetti-Grützmacher, E.; Kaufmann, A.M. Human Papillomavirus E7 and p16INK4a mRNA Multiplexed Quantification by a QuantiGeneTM Proof-of-Concept Assay Sensitively Detects Infection and Cervical Dysplasia Severity. Diagnostics 2023, 13, 1135. https://doi.org/10.3390/diagnostics13061135
Skof AS, Rotenberg L, Hannemann PVF, Thies S, Boschetti-Grützmacher E, Kaufmann AM. Human Papillomavirus E7 and p16INK4a mRNA Multiplexed Quantification by a QuantiGeneTM Proof-of-Concept Assay Sensitively Detects Infection and Cervical Dysplasia Severity. Diagnostics. 2023; 13(6):1135. https://doi.org/10.3390/diagnostics13061135
Chicago/Turabian StyleSkof, Anna Sophie, Lina Rotenberg, Paul Viktor Felix Hannemann, Sarah Thies, Eleonora Boschetti-Grützmacher, and Andreas M. Kaufmann. 2023. "Human Papillomavirus E7 and p16INK4a mRNA Multiplexed Quantification by a QuantiGeneTM Proof-of-Concept Assay Sensitively Detects Infection and Cervical Dysplasia Severity" Diagnostics 13, no. 6: 1135. https://doi.org/10.3390/diagnostics13061135