Quantification of HPV16 E6/E7 mRNA Spliced Isoforms Viral Load as a Novel Diagnostic Tool for Improving Cervical Cancer Screening
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Lines
2.2. Patients
2.3. Uterine Cervical Samples Analysis and Criteria of Selection
2.4. HR-HPV DNA Genotyping
2.5. HR-HPV E6/E7 mRNA Detection
2.6. Nucleic Acid Extractions
2.7. HPV16 DNA VL Quantification Assay
2.8. HPV16 E6/E7 mRNA VLs Quantification Assay
2.9. Statistical Analysis
3. Results
3.1. Validation of HPV16 DNA and E6/E7 mRNA VLs Quantification Assays
3.2. Quantification of HPV16 DNA and E6/E7 mRNA VLs in UCS
3.3. HPV16 E6/E7 mRNA VLs Are Increased in High Grade Cervical Lesions
3.4. Comparison of the Pap Test, HPV16 DNA VLs, and HPV16 E6/E7 mRNA VLs Sets Diagnostic Performances for Detection of High Grade Cervical Lesions
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
HPVs | human papillomaviruses |
FL | full length E6/E7 mRNA |
SP | + spliced E6/E7 mRNA containing intact E7 ORF |
T | total E6/E7 mRNA corresponding to SP + E6^E7 mRNA |
VL | viral loads |
E6^E7 | E6/E7 mRNA containing disrupted E6 and E7 ORFs calculated by the following subtraction T-SP |
ASC-US | atypical squamous cells of unknown significance |
LSIL | low-grade squamous intraepithelial lesion |
CC | cervical cancer |
CIN | cervical intraepithelial neoplasia |
CIN2+ | CIN of grade 2 or more: CIN2, CIN3, cancer |
CIN3+ | CIN of grade 3 or more: CIN3, cancer |
UCS | uterine cervical smears |
ASC-H | atypical squamous cells- cannot exclude high grade |
HSIL | high grade squamous intraepithelial lesion |
ROC | receiver operating curves |
AUC | area under roc curve |
NPV | negative predictive value |
PPV | positive predictive value |
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Primer/Probe Set | Sequences | |
---|---|---|
HPV16 E6 | Forward | 5′-GAGAACTGCAATGTTTCAGGACC-3′ |
Reverse | 5′- TGTATAGTTGTTTGCAGCTCTGTGC-3′ | |
Probe | JOE-5′-CAGGAGCGACCCAGAAAGTTACCACAGTT-3′-TAMRA | |
HPV16 E2 | Forward | 5′-AACGAAGTATCCTCTCCTGAAATTATTAG-3′ |
Reverse | 5′-CCAAGGCGACGGCTTTG-3′ | |
Probe | FAM-5′-CACCCCGCCGCGACCCATA-3′-TAMRA | |
β-globin | Forward | 5′-TGCATCTGACTCCTGAGGAGAA-3′ |
Reverse | 5′-GGGCCTCACCACCAACTTC-3′ | |
Probe | TET-5′-CTGCCGTTACTGCCCT-3′-TAMRA |
Primer/Probe Set | Sequences | |
---|---|---|
FL-E6/E7 | Forward | 5′-GTGTACTGCAAGCAACAGTTA-3′ |
Reverse | 5′-CCCATCTCTATATACTATGCATAAATCC-3′ | |
Probe | FAM-5′-CTGCGACGTGAGGTATATGACTTTGCT-3′-TAMRA | |
SP-E6/E7 | Forward | 5′-GATTTGCAACCAGAGACAACTG-3′ |
Reverse | 5′-GCTGGACCATCTATTTCATCCT-3′ | |
Probe | FAM-5′-TGAGCAATTAAATGACAGCTCAGAGGAGG-3′-TAMRA | |
T-E6/E7 | Forward | 5′-GACTCTACGCTTCGGTTGTG-3′ |
Reverse | 5′-TGTGCCCATTAACAGGTCTT-3′ | |
Probe | FAM-5′-CGTACAAAGCACACACGTAGACATTCG-3′-TAMRA | |
β-actin | Forward | 5′-GACCCAGATCATGTTTGAGACC-3′ |
Reverse | 5′-CCAGAGGCGTACAGGGATA-3′ | |
Probe | FAM-5′-TGTACGTTGCTATCCAGGCTGTGC-3′-TAMRA |
Viral Load Type | DNA VL (Copies/Cell ± SD (Range)) | E6/E7 mRNA VL (log10 (Copies/106 β-actin mRNA Copies) ±SD (Range)) | |
---|---|---|---|
DNA | Total | 0.93 ± 0.2 (0.7–1.06) | |
Episomal | 0 | ||
Integrated | 0.93 ± 0.2 (0.7–1.06) | ||
mRNA | FL-E6/E7 | 4.47 ± 0.18 (4.27–4.59) | |
SP-E6/E7 | 4.86 ± 0.21 (4.63–5.00) | ||
T-E6/E7 | 4.93 ± 0.23 (4.68-5.12) | ||
E6^E7 | 4.09 ± 0.37 (3.73-4.48) |
Patient ID | Cytology Grade | Histology Grade | HPV16 DNA VL (log10 (Copies/106 Cells)) | HPV16 mRNA VL (log10 (Copies/106 β-actin mRNA Copies)) | |||||
---|---|---|---|---|---|---|---|---|---|
Total | Episomal | Integrated | FL-E6/E7 | SP-E6/E7 | T-E6/E7 | E6^E7 | |||
31 | Normal | Unlesional | 5.92 | 5.62 | 5.61 | 2.02 | 2.34 | 2.86 | 2.71 |
44 | Normal | Unlesional | 6.23 | 5.77 | 6.04 | 0.00 | 0.00 | 2.90 | 2.90 |
15 | LSIL | CIN1 | 6.80 | 6.01 | 6.73 | 2.15 | 2.73 | 3.99 | 3.97 |
18 | LSIL | CIN1 | 7.56 | 6.86 | 7.46 | 1.78 | 2.15 | 3.50 | 3.48 |
34 | Normal | CIN1 | 6.08 | 6.06 | 4.81 | 3.45 | 4.06 | 4.09 | 2.94 |
42 | Normal | CIN1 | 5.35 | 5.06 | 5.03 | 1.76 | 0.00 | 3.31 | 3.31 |
43 | Normal | CIN1 | 6.59 | 6.35 | 6.22 | 0.00 | 1.59 | 2.95 | 2.93 |
45 | ASC-US | CIN1 | 7.61 | 6.76 | 7.55 | 1.80 | 2.49 | 4.34 | 4.34 |
51 | LSIL | CIN1 | 6.95 | 6.36 | 6.83 | 2.97 | 0.00 | 4.30 | 4.30 |
55 | ASC-US | CIN1 | 5.89 | 5.79 | 5.19 | 2.91 | 3.04 | 3.27 | 2.87 |
2 | ASC-H | CIN2 | 6.97 | 6.17 | 6.89 | 2.80 | 3.16 | 3.88 | 3.79 |
4 | ASC-US | CIN2 | 5.37 | 4.71 | 5.27 | 0.00 | 0.00 | 1.91 | 1.91 |
9 | LSIL | CIN2 | 7.83 | 7.09 | 7.75 | 3.05 | 3.79 | 5.01 | 4.99 |
47 | ASC-H | CIN2 | 6.96 | 6.47 | 6.79 | 3.53 | 3.94 | 4.45 | 4.29 |
1 | HSIL | CIN3 | 8.09 | 7.48 | 7.97 | 3.52 | 4.19 | 4.72 | 4.56 |
5 | ASC-H | CIN3 | 7.19 | 6.55 | 7.08 | 4.25 | 4.81 | 5.10 | 4.79 |
41 | HSIL | CIN3 | 5.51 | 5.12 | 5.28 | 2.54 | 3.17 | 3.51 | 3.23 |
50 | ASC-H | CIN3 | 7.63 | 6.94 | 7.52 | 4.12 | 5.16 | 5.62 | 5.44 |
3 | HSIL | Invasive Cancer | 7.88 | 7.67 | 7.47 | 3.91 | 4.49 | 5.67 | 5.64 |
6 | HSIL | Invasive Cancer | 7.97 | 7.62 | 7.72 | 4.11 | 4.81 | 5.26 | 5.07 |
HPV16 Viral Load Type | CIN2+ Histology Threshold | CIN3+ Histology Threshold | ||||||
---|---|---|---|---|---|---|---|---|
Histology <CIN2 | Histology CIN2+ | p-Value (Wilcoxon) | Histology <CIN3 | Histology CIN3+ | p-Value (Wilcoxon) | |||
DNA VL (log10 (copies/106 cells)) | Total | Mean ± SD | 6.5 ± 0.7 | 7.1 ± 1.0 | 0.0690 | 6.6±0.8 | 7.4 ± 1.0 | 0.0490 |
Integrated | Mean ± SD | 6.1 ± 1 | 7.0 ± 1.0 | 0.0596 | 6.3±1.0 | 7.2 ± 1.0 | 0.0676 | |
E6/E7 mRNA VL (log10 (copies/106 β-actin mRNA copies)) | FL-E6/E7 | Mean ± SD | 1.9 ± 1.2 | 3.2 ± 1.3 | 0.0201 | 2.0±1.2 | 3.7 ± 0.6 | 0.0102 |
SP-E6/E7 | Mean ± SD | 1.8 ± 1.4 | 3.8 ± 1.5 | 0.0112 | 2.1±1.5 | 4.4 ± 0.7 | 0.0048 | |
T-E6/E7 | Mean ± SD | 3.6 ± 0.6 | 4.5 ± 1.2 | 0.0279 | 3.6±0.8 | 5.0 ± 0.8 | 0.0124 | |
E6^E7 | Mean ± SD | 3.4 ± 0.6 | 4.4 ± 1.1 | 0.0380 | 3.5±0.8 | 4.8 ± 0.9 | 0.0177 |
HPV16 Viral Load Type | CIN2+ Histology Threshold | CIN3+ Histology Threshold | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
TV | YI | NPV. % (95%CI) | PPV. % (95%CI) | Se.% (95%CI) | Spe. % (95%CI) | AUC (95%CI) | TV | YI | NPV. % (95%CI) | PPV. % (95%CI) | Se.% (95%CI) | Spe. % (95%CI) | AUC (95%CI) | ||
DNA | Total | 6.96 | 0.60 | 80 (44–97) | 80 (44–97) | 80 (44–97) | 80 (44–97) | 0.76 (0.52–1.00) | 7.19 | 0.62 | 92 (36–100) | 63 (24–91) | 83 (36–100) | 79 (49–95) | 0.81 (0.53–1.00) |
Integrated | 6.79 | 0.50 | 78 (40–97) | 73 (39–94) | 80 (44–97) | 70 (35–93) | 0.77 (0.55–0.99) | 7.08 | 0.62 | 92 (62–100) | 63 (24–91) | 83 (36–100) | 79 (49–95) | 0.79 (0.54–1.00) | |
mRNA | FL-E6/E7 | 2.54 | 0.60 | 88 (47–100) | 75 (43–95) | 90 (56–100) | 70 (35–99) | 0.84 (0.65–1.00) | 2.54 | 0.57 | 100 (63–100) | 50 (21–79) | 100 (54–100) | 57 (29–82) | 0.92 (0.77–1.00) |
SP-E6/E7 | 3.16 | 0.80 | 90 (56–100) | 90 (56–100) | 90 (56–100) | 90 (56–100) | 0.88 (0.69–1.00) | 3.17 | 0.79 | 100 (72–100) | 67 (30–93) | 100 (54–100) | 79 (49–95) | 0.97 (0.89–1.00) | |
T-E6/E7 | 3.51 | 0.50 | 86 (42–100) | 69 (39–91) | 90 (56–100) | 60 (26–88) | 0.82 (0.60–1.00) | 4.72 | 0.76 | 93 (66–100) | 83 (36–100) | 83 (36–100) | 93 (66–100) | 0.91 (0.73–1.00) | |
E6^E7 | 3.23 | 0.40 | 83 (36–100) | 64 (35–87) | 90 (56–100) | 50 (19–81) | 0.80 (0.58–1.00) | 3.23 | 0.43 | 100 (54–100) | 43 (18–71) | 100 (54–100) | 43 (18–71) | 0.88 (0.69–1.00) | |
Pap test | NA | NA | 83 (52–98) | 100 (63–100) | 80 (44–97) | 100 (69–100) | NA | NA | NA | 100 (74–100) | 75 (35–97) | 100 (54–100) | 86 (57–98) | NA |
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Camus, C.; Vitale, S.; Loubatier, C.; Pénaranda, G.; Khiri, H.; Plauzolles, A.; Carcopino, X.; Halfon, P.; Giordanengo, V. Quantification of HPV16 E6/E7 mRNA Spliced Isoforms Viral Load as a Novel Diagnostic Tool for Improving Cervical Cancer Screening. J. Clin. Med. 2018, 7, 530. https://doi.org/10.3390/jcm7120530
Camus C, Vitale S, Loubatier C, Pénaranda G, Khiri H, Plauzolles A, Carcopino X, Halfon P, Giordanengo V. Quantification of HPV16 E6/E7 mRNA Spliced Isoforms Viral Load as a Novel Diagnostic Tool for Improving Cervical Cancer Screening. Journal of Clinical Medicine. 2018; 7(12):530. https://doi.org/10.3390/jcm7120530
Chicago/Turabian StyleCamus, Claire, Sébastien Vitale, Céline Loubatier, Guillaume Pénaranda, Hacène Khiri, Anne Plauzolles, Xavier Carcopino, Philippe Halfon, and Valérie Giordanengo. 2018. "Quantification of HPV16 E6/E7 mRNA Spliced Isoforms Viral Load as a Novel Diagnostic Tool for Improving Cervical Cancer Screening" Journal of Clinical Medicine 7, no. 12: 530. https://doi.org/10.3390/jcm7120530
APA StyleCamus, C., Vitale, S., Loubatier, C., Pénaranda, G., Khiri, H., Plauzolles, A., Carcopino, X., Halfon, P., & Giordanengo, V. (2018). Quantification of HPV16 E6/E7 mRNA Spliced Isoforms Viral Load as a Novel Diagnostic Tool for Improving Cervical Cancer Screening. Journal of Clinical Medicine, 7(12), 530. https://doi.org/10.3390/jcm7120530