Analytical Validation of NavDx+Gyn, a cfDNA-Based Fragmentomic Profiling Assay for HPV-Driven Gynecologic Cancers
Abstract
:1. Introduction
2. Materials and Methods
2.1. Test Characteristics
2.2. Bioinformatics
2.3. Validation Materials
2.4. Determination of Assay Performance Characteristics
2.4.1. Specificity (Limit of Blank)
2.4.2. Sensitivity (Detection Limit)
2.4.3. Accuracy, Precision, and Linearity
3. Results
3.1. Detection Capability
3.1.1. Specificity (Limit of Blank)
3.1.2. Sensitivity (Detection Limit) and Limit of Quantitation
3.2. Analytical Accuracy
3.3. Precision Studies
3.4. Linearity
4. Discussion
5. Patents
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A. Dilution Series for Analytical Validation Parameters: Accuracy, Precision, Linearity, LoD, and LoQ
Dilution Series Used to Determine Accuracy, Precision, and Linearity (copies/µL) | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Concentration | HPV Type | |||||||||||||
Level * | 16 | 18 | 31 | 33 | 35 | 39 | 45 | 51 | 52 | 56 | 58 | 59 | 66 | 68 |
1 | 4696 | 4317 | 4240 | 3822 | 4277 | 3790 | 4611 | 4299 | 3582 | 4431 | 4616 | 3605 | 4026 | 3747 |
2 | 940 | 808 | 862 | 775 | 832 | 973 | 1187 | 1076 | 895 | 1160 | 1122 | 891 | 1009 | 915 |
3 | 468 | 400 | 422 | 393 | 423 | 476 | 589 | 546 | 441 | 574 | 561 | 439 | 486 | 449 |
4 | - | 77 | 81 | 77 | 80 | 91 | 113 | 104 | 86 | 107 | 109 | 86 | 96 | 84 |
5 | 44 | 39 | 41 | 39 | 40 | 43 | 55 | 49 | 42 | 49 | 53 | 40 | 45 | 41 |
6 | - | 7.9 | 8.4 | 8.2 | 8.4 | 9.2 | 11.3 | 9.6 | 9.0 | 10.3 | 11.3 | 8.3 | 9.2 | 7.5 |
7 | 4.7 | 3.5 | 5.0 | 4.2 | 5.0 | 3.9 | 4.3 | 4.5 | 3.6 | 5.0 | 5.4 | 3.3 | 5.0 | 4.2 |
8 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 |
9 | - | 15 | 15 | 15 | 15 | 15 | 15 | 15 | 15 | 15 | 15 | 15 | 15 | 15 |
10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
11 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
12 | - | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
13 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.50 | 2.50 | 2.50 |
14 | - | 1.7 | 1.7 | 1.7 | 1.7 | 1.7 | 1.7 | 1.7 | 1.7 | 1.7 | 1.7 | 1.67 | 1.67 | 1.67 |
15 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.50 | 1.50 | 1.50 |
16 | - | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 |
17 | - | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 |
18 | - | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 |
19 | - | 0.17 | 0.17 | 0.17 | 0.17 | 0.17 | 0.17 | 0.17 | 0.17 | 0.17 | 0.17 | 0.17 | 0.17 | 0.17 |
20 | 0.75 | - | - | - | - | - | - | - | - | - | - | - | - | - |
21 | 0.63 | - | - | - | - | - | - | - | - | - | - | - | - | - |
22 | 0.42 | - | - | - | - | - | - | - | - | - | - | - | - | - |
23 | 0.21 | - | - | - | - | - | - | - | - | - | - | - | - | - |
24 | 0.13 | - | - | - | - | - | - | - | - | - | - | - | - | - |
25 | 0.08 | - | - | - | - | - | - | - | - | - | - | - | - | - |
26 | 0.04 | - | - | - | - | - | - | - | - | - | - | - | - | - |
Appendix B. Linearity Across the Entire Detectable Concentration Range
Concentration Levels in the First Phase of the LoQ/LoD Experiment | |
---|---|
HPV16 | HPV Types 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 |
20 | 20 |
10 | 15 |
5 | 10 |
2.5 | 5 |
1.5 | 2.5 |
1.5 | |
Concentration Levels in the Second Phase of the LoQ/LoD Experiment | |
HPV16 | HPV Types 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 |
0.75 | 3.00 |
0.63 | 2.50 |
0.42 | 1.67 |
0.21 | 0.83 |
0.13 | 0.50 |
0.08 | 0.33 |
0.04 | 0.17 |
HPV Type | Equation | R2 Value |
---|---|---|
HPV16 | Y = 0.8872 × X + 1.262 | 0.9997 |
HPV18 | Y = 0.9880 × X + 9.602 | 0.9967 |
HPV31 | Y = 1.002 × X − 1.407 | 0.9989 |
HPV33 | Y = 1.086 × X − 4.624 | 0.9984 |
HPV35 | Y = 0.9909 × X + 4.416 | 0.9992 |
HPV39 | Y = 0.9368 × X + 0.09014 | 0.9993 |
HPV45 | Y = 0.8977 × X + 0.6558 | 0.9981 |
HPV51 | Y = 0.9950 × X − 0.8600 | 0.9999 |
HPV52 | Y = 0.9919 × X + 0.2239 | 0.9986 |
HPV56 | Y = 0.9318 × X + 1.669 | 0.9977 |
HPV58 | Y = 0.9035 × X + 1.781 | 0.9996 |
HPV59 | Y = 0.9983 × X − 0.01611 | 0.9988 |
HPV66 | Y = 0.9371 × X − 0.8443 | 0.9973 |
HPV68 | Y = 0.9084 × X − 0.4254 | 0.9975 |
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HPV Type | HPV DNA Score (Copies/μL) | ||
---|---|---|---|
LoB | LoD | LoQ | |
HPV-16 | 0.0134 | 0.1009 | 0.1009 |
HPV-18 | 0.0273 | 0.2552 | 0.2552 |
HPV-31 | 0.0621 | 0.2423 | 0.2423 |
HPV-33 | 0 | 0.2963 | 0.2963 |
HPV-35 | 0 | 0.3022 | 0.3040 |
HPV-39 | 0.0779 | 0.2726 | 0.2726 |
HPV-45 | 0.0282 | 0.2520 | 0.2520 |
HPV-51 | 0 | 0.1983 | 0.2699 |
HPV-52 | 0.0581 | 0.2489 | 0.2489 |
HPV-56 | 0 | 0.1255 | 0.1255 |
HPV-58 | 0.0926 | 0.3100 | 0.3100 |
HPV-59 | 0.0315 | 0.1848 | 0.1848 |
HPV-66 | 0.0612 | 0.3147 | 0.3147 |
HPV-68 | 0 | 0.2874 | 0.2874 |
Conc. Level a | Accuracy—Mean % Recovery b | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
HPV Type | ||||||||||||||
16 | 18 | 31 | 33 | 35 | 39 | 45 | 51 | 52 | 56 | 58 | 59 | 66 | 68 | |
1 | 88.7 | 98.7 | 100.3 | 108.7 | 99.0 | 93.7 | 89.8 | 100.5 | 99.2 | 93.1 | 90.3 | 99.7 | 93.8 | 90.8 |
2 | 88.8 | 107.0 | 97.7 | 104.3 | 104.3 | 92.8 | 90.3 | 100.8 | 99.1 | 94.5 | 91.2 | 101.8 | 92.0 | 91.0 |
3 | 90.0 | 113.1 | 99.0 | 101.1 | 101.4 | 94.3 | 89.4 | 101.9 | 100.3 | 93.5 | 91.6 | 97.5 | 93.7 | 89.5 |
4 | n/a | 114.7 | 105.6 | 105.4 | 112.0 | 94.9 | 90.8 | 100.2 | 98.4 | 94.6 | 92.8 | 94.9 | 92.3 | 89.3 |
5 | 96.2 | 111.1 | 100.8 | 101.6 | 102.8 | 97.7 | 89.3 | 101.3 | 99.3 | 98.7 | 94.1 | 100.5 | 98.2 | 90.5 |
6 | n/a | 107.8 | 100.3 | 102.5 | 101.2 | 94.7 | 83.4 | 96.4 | 97.1 | 97.4 | 90.4 | 99.0 | 91.6 | 98.5 |
7 | 87.6 | 125.3 | 91.4 | 102.2 | 89.4 | 103.1 | 103.1 | 101.0 | 100.1 | 97.2 | 89.2 | 105.7 | 96.6 | 89.9 |
Conc. Level a | Intra-Assay/Method Precision—%CVs of Mean Effective Concentration (copies/μL) b | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
HPV Type | ||||||||||||||
16 | 18 | 31 | 33 | 35 | 39 | 45 | 51 | 52 | 56 | 58 | 59 | 66 | 68 | |
1 | 1.1 | 5.5 | 2.5 | 2.4 | 1.6 | 1.6 | 4.2 | 1.6 | 2.8 | 4.5 | 0.9 | 2.0 | 4.9 | 2.1 |
2 | 1.2 | 1.9 | 2.2 | 4.7 | 1.6 | 3.3 | 3.9 | 2.1 | 1.0 | 4.9 | 2.7 | 3.3 | 2.3 | 2.4 |
3 | 1.2 | 4.2 | 4.9 | 3.6 | 3.7 | 1.2 | 5.2 | 3.2 | 4.7 | 2.5 | 5.0 | 2.9 | 3.4 | 2.9 |
4 | n/a | 4.0 | 4.0 | 3.5 | 2.3 | 2.2 | 7.6 | 1.3 | 3.1 | 6.8 | 7.3 | 4.8 | 2.3 | 1.9 |
5 | 1.4 | 6.2 | 1.5 | 3.3 | 5.2 | 2.8 | 4.0 | 3.8 | 3.5 | 5.0 | 6.0 | 2.7 | 5.5 | 5.0 |
6 | n/a | 5.3 | 4.4 | 7.6 | 7.8 | 1.3 | 10.9 | 6.9 | 6.1 | 7.2 | 8.3 | 1.2 | 13.8 | 6.8 |
7 | 3.9 | 7.4 | 2.7 | 18.2 | 9.4 | 13.7 | 13.5 | 3.0 | 7.5 | 12.5 | 12.5 | 3.8 | 3.7 | 0.3 |
Conc. Level a | Inter-Assay/Intermediate Precision—%CVs of Mean Effective Concentration (copies/μL) b | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
HPV Type | ||||||||||||||
16 | 18 | 31 | 33 | 35 | 39 | 45 | 51 | 52 | 56 | 58 | 59 | 66 | 68 | |
1 | 1.1 | 4.7 | 2.4 | 3.7 | 1.4 | 1.9 | 3.7 | 1.4 | 2.5 | 3.8 | 1.0 | 2.1 | 4.0 | 2.4 |
2 | 1.6 | 1.8 | 3.4 | 3.8 | 1.3 | 2.8 | 3.2 | 2.6 | 1.2 | 4.1 | 2.2 | 3.1 | 1.9 | 2.0 |
3 | 1.9 | 6.9 | 4.4 | 5.5 | 3.4 | 1.0 | 4.3 | 4.4 | 3.9 | 2.0 | 4.2 | 2.5 | 2.9 | 2.5 |
4 | n/a | 6.4 | 3.3 | 6.0 | 2.0 | 2.0 | 6.8 | 3.9 | 3.1 | 6.0 | 6.8 | 4.1 | 2.1 | 2.3 |
5 | 1.7 | 5.6 | 2.6 | 2.8 | 5.5 | 2.9 | 5.1 | 3.1 | 3.4 | 5.3 | 5.5 | 4.4 | 5.7 | 4.4 |
6 | n/a | 7.2 | 3.9 | 8.1 | 6.5 | 5.8 | 13.5 | 5.7 | 5.3 | 6.4 | 8.4 | 9.5 | 11.6 | 5.8 |
7 | 4.2 | 11.6 | 6.6 | 16.7 | 7.9 | 11.3 | 11.0 | 2.5 | 8.4 | 10.2 | 10.2 | 6.5 | 3.9 | 3.4 |
HPV Type | Equation | R2 Value |
---|---|---|
HPV16 | Y = 0.8868 × X + 2.518 | 0.9996 |
HPV18 | Y = 0.9843 × X + 22.50 | 0.9962 |
HPV31 | Y = 1.003 × X − 3.387 | 0.9987 |
HPV33 | Y = 1.088 × X − 10.92 | 0.9982 |
HPV35 | Y = 0.9892 × X + 10.03 | 0.9991 |
HPV39 | Y = 0.9368 × X − 0.05201 | 0.9991 |
HPV45 | Y = 0.8978 × X + 0.5441 | 0.9978 |
HPV51 | Y = 0.9955 × X − 2.500 | 0.9998 |
HPV52 | Y = 0.9918 × X + 0.5093 | 0.9984 |
HPV56 | Y = 0.9314 × X + 3.252 | 0.9973 |
HPV58 | Y = 0.9030 × X + 3.403 | 0.9996 |
HPV59 | Y = 0.9982 × X + 0.09592 | 0.9986 |
HPV66 | Y = 0.9376 × X − 2.244 | 0.9968 |
HPV68 | Y = 0.9086 × X − 0.9594 | 0.9971 |
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Hutcheson, J.; Conway, D.; Kumar, S.; Wiseman, C.; Chakraborty, S.; Skrypkin, E.; Horan, M.; Gunning, A.; Williams, C.K.; Kuperwasser, C.; et al. Analytical Validation of NavDx+Gyn, a cfDNA-Based Fragmentomic Profiling Assay for HPV-Driven Gynecologic Cancers. Diagnostics 2025, 15, 825. https://doi.org/10.3390/diagnostics15070825
Hutcheson J, Conway D, Kumar S, Wiseman C, Chakraborty S, Skrypkin E, Horan M, Gunning A, Williams CK, Kuperwasser C, et al. Analytical Validation of NavDx+Gyn, a cfDNA-Based Fragmentomic Profiling Assay for HPV-Driven Gynecologic Cancers. Diagnostics. 2025; 15(7):825. https://doi.org/10.3390/diagnostics15070825
Chicago/Turabian StyleHutcheson, Joshua, David Conway, Sunil Kumar, Chloe Wiseman, Syandan Chakraborty, Evgeny Skrypkin, Michael Horan, Alicia Gunning, Cassin Kimmel Williams, Charlotte Kuperwasser, and et al. 2025. "Analytical Validation of NavDx+Gyn, a cfDNA-Based Fragmentomic Profiling Assay for HPV-Driven Gynecologic Cancers" Diagnostics 15, no. 7: 825. https://doi.org/10.3390/diagnostics15070825
APA StyleHutcheson, J., Conway, D., Kumar, S., Wiseman, C., Chakraborty, S., Skrypkin, E., Horan, M., Gunning, A., Williams, C. K., Kuperwasser, C., Naber, S. P., & Gupta, P. B. (2025). Analytical Validation of NavDx+Gyn, a cfDNA-Based Fragmentomic Profiling Assay for HPV-Driven Gynecologic Cancers. Diagnostics, 15(7), 825. https://doi.org/10.3390/diagnostics15070825