Internal study data for the validation of AmpFire HPV 16/18/HR assay

according to Meijer criteria1

AmpFire is a registered trademark of Atila Biosystems Ltd.

Distributed by Fujirebio Europe N.V.

___________________________________________________________________

Ghent, Belgium, 19. December 2019

Content

Criteria 1: Relative sensitivity and specificity data____________________ page 2

Criteria 2: Detection of high-risk types_____________________________ page 5

Criteria 3: CE marking_________________________________________ page 5

Criteria 4: Inter- and intra-laboratory reproducibility data_______________ page 6

Publications_________________________________________________ page 9

1 C.J.L.M. Meijer. Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening
in women of 30 years and older. Int J Cancer. 2009; 124(3): 516–520.
Criteria 1
At least 90% of the sensitivity for CIN2 + of an established and validated HPV test and at
least 98% of the specificity for CIN2 + of an established and validated HPV test in women
over the age of 30 years or older. Non-inferiority vs comparator assay (p-value for non-
inferiority test should be lower than 0,05).

Results for the AmpFire HPV 16/18/HR

Cervical specimens were collected in PreservCyt solution from 4715 healthy women aged
between 30-64 years. From each specimen collected 2 mL was aliquoted into a new tube for
HPV DNA testing and the remainder of the specimen was used for cytology study by Thin-
Prep 2000 system (Hologic Inc.). LINEAR ARRAY® HPV Genotyping (Roche Diagnostics)
results were available for most of the specimens.
All specimens were tested by AmpFire HPV Screening 16/18/HR Test and Cobas® 4800 HPV
Test (Roche Diagnostics) in accordance with their respective assay package insert and user’s
manual. The clinical sensitivity and specificity for detection of disease, which is defined as (1)
Cervical Intraepithelial Neoplasia (CIN2) or greater histology result or (2) Cervical
Intraepithelial Neoplasia (CIN3) or greater histology result, was calculated.

Relative sensitivity for CIN2+ cases

Table 1: Comparison data

Cobas 4800 HPV Cobas 4800 HPV

positive negative
AmpFire HPV 16/18/HR positive 69 0
AmpFire HPV 16/18/HR negative 2 6

Table 2: Results according to criteria

Results Meijer criteria

Test-statistic (according to Meijer et al.)1 2,02
p-value non-inferiority 0,02 < 0,05
Clinical sensitivity AmpFire HPV 16/18/HR 89,6 %
Clinical sensitivity Cobas HPV 92,2 %
Relative clinical sensitivity 0,97 ≥ 0,90

Conclusion
P-value is lower than 0,05 meaning non-inferior vs comparator assay Cobas4800 HPV.

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Relative specificity for < CIN2 cases (HPV53 included)

Table 3: Comparison data

Cobas 4800 HPV Cobas 4800 HPV

positive negative
AmpFire HPV 16/18/HR positive 456 139*
AmpFire HPV 16/18/HR negative 74 3969

*62/139 samples: HPV53

Table 4: Results according to criteria

Results Meijer criteria

Test-statistic (according to Meijer et al.)1 1,17
p-value non-inferiority 0,12 < 0,05
Clinical specificity ATILA AmpFire HPV 87,2 %
Clinical specificity Cobas HPV 88,6 %
Relative clinical specificity 0,98 ≥ 0,98

Conclusion

P-value is higher than 0,05 meaning inferior vs comparator assay Cobas 4800 HPV. This is
related to the fact that the AmpFire HPV 16/18/HR assay detects also HPV53 which is not
detected by the Cobas 4800 HPV assay. The group of samples detected as positive with
AmpFire HPV 16/18/HR but negative with Cobas 4800 HPV (n=139) includes 62 HPV53
samples.

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Relative specificity for < CIN2 cases (HPV53 excluded)

Table 5: Comparison data

Cobas 4800 HPV Cobas 4800 HPV

positive negative

AmpFire HPV 16/18/HR positive 456 77

AmpFire HPV 16/18/HR negative 74 3969

Table 6: Results according to criteria

Results Meijer criteria

Test-statistic (according to Meijer et al.)1 5,79
p-value non-inferiority 4 x 10-9 < 0,05
Clinical specificity AmpFire HPV 16/18/HR 88,4 %
Clinical specificity Cobas 4800 HPV 88,4 %
Relative clinical specificity 0,999 >= 0,98

Conclusion

When excluding the HPV53 samples, relative clinical specificity equals 1 meaning that
AmpFire HPV 16/18/HR is perfect aligned with the comparator assay Cobas 4800 HPV. P-
value is quasi 0 meaning perfect, non-inferior vs comparator assay Cobas 4800 HPV.

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Criteria 2
At least detection of high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68

Results for AmpFire HPV16/18/HR

AmpFire HPV 16/18/HR detects HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58,
59, 66, 68

Criteria 3
CE marking

Results for AmpFire HPV16/18/HR

AmpFire HPV16/18/HR is a CE-marked IVD product.

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Criteria 4
Inter- and intra-laboratory reproducibility should be not less than 87%.

Results for the AmpFire HPV16/18/HR

The inter- and intra-laboratory reproducibility of the AmpFire HPV Assay has been
evaluated using Thin-Prep cervical brush samples. All tests (intra-lab and inter-lab tests)
were performed from the original Thin-Prep samples stored at -20°C. Samples were
previously test with Cobas HPV assay giving 122/500 positive samples. Cobas test gave
fewer positive numbers because it doesn’t detect HPV53. Intra-lab tests were performed
with a time difference of 3 weeks.

Inter-laboratory results

Table 7: Comparison data

AmpFire HPV16/18/HR 1st test

positive negative total
positive 135 1 136
AmpFire HPV16/18/HR negative 3 361 364
2nd test total 138 362 500

Table 8: Results according to criteria

Results Meijer criteria

Lower confidence bound for agreement 98 % ≥ 87 %
Kappa 0,98 ≥ 0,5

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Intra-laboratory results

Table 9: Comparison data

AmpFire HPV16/18/HR 1st test

positive negative total
positive 136 1 137
AmpFire HPV16/18/HR negative 2 358 360
2nd test total 138 359 497*

Table 10: Results according to criteria

Results Meijer criteria

Lower confidence bound for agreement 98 % ≥ 87 %
Kappa 0,98 ≥ 0,5

Conclusion
*Three samples failed on one of the tests in the intra-lab study. These samples were excluded
from the data analysis.
Even if these 3 samples are included in worst case scenario as discrepant results, the
AmpFire HPV16/18/HR assay still applies to the requirement of at least 87% reproducibility.

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Additional reproducibility data (included also in the instructions for use)

The inter- and intra-run reproducibility of the AmpFire HPV16/18/HR Assay has been
evaluated using plasmid DNA of each of the 15 HPV genotypes at 200 copies/reaction.
The assay was tested across an equal distribution of three lots of reagents over 6 days.
Data are summarized in table below.

Table 11

Inter-Run Intra-Run Total

HPV Conc % Correct 95% Confidence Mean SD %CV SD %CV SD %CV
Genotype Interval Ct

HPV16 200 100% (90/90) (97.29% – 100%) 18.15 0.15 0.82 0.17 0.94 0.35 1.93%
HPV18 200 100% (90/90) (97.29% – 100%) 21.84 0.35 1.60 0.43 1.97 0.48 2.20
HPV31 200 100% (90/90) (97.29% – 100%) 40.17 1.02 2.54 0.49 1.22 1.12 2.79
HPV33 200 100% (90/90) (97.29% – 100%) 34.40 0.62 1.80 0.85 2.76 0.84 2.44
HPV35 200 100% (90/90) (97.29% – 100%) 32.36 0.85 2.62 0.83 2.56 0.30 0.93
HPV39 200 100% (90/90) (97.29% – 100%) 31.94 0.51 1.60 0.43 1.34 0.53 1.66
HPV45 200 100% (90/90) (97.29% – 100%) 38.8 1.67 4.30 1.30 3.35 1.63 4.2
HPV51 200 100% (90/90) (97.29% – 100%) 26.38 0.26 0.99 0.20 0.76 0.46 1.74
HPV52 200 100% (90/90) (97.29% – 100%) 30.85 0.50 1.62 0.55 1.78 0.83 2.69
HPV53 200 100% (90/90) (97.29% – 100%) 37.94 0.93 2.45 1.02 2.69 1.40 3.69
HPV56 200 100% (90/90) (97.29% – 100%) 29.86 0.40 1.34 0.53 1.77 0.56 1.88
HPV58 200 100% (90/90) (97.29% – 100%) 39.70 1.23 3.10 1.44 3.63 1.24 3.12
HPV59 200 100% (90/90) (97.29% – 100%) 30.47 0.20 0.66 0.27 0.88 0.26 0.85
HPV66 200 100% (90/90) (97.29% – 100%) 30.88 0.40 1.30 0.55 1.78 0.54 1.75
HPV68 200 100% (90/90) (97.29% – 100%) 28.59 0.48 1.68 0.47 1.64 0.41 1.43

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Publications and Posters

Zhang W et al. Evaluation of an isothermal amplification HPV detection assay (AmpFire) for
primary cervical cancer screening. Poster submitted for IPV 2020.
Tang YW et al. An Isothermal, Multiplex Amplification Assay for Detection and Genotyping of
Human Papillomaviruses in Formalin-Fixed, Paraffin-Embedded Tissues. J Mol Diagn. 2020
Mar;22(3):419-428.
Jang D et al. Performance of AmpFire HPV assay on neck cervical lymp node aspirate and
oropharyngeal samples. Journal of Virological Methods 279 (2020) 113840
Kleemann M et al. Evaluation of a Novel Isothermal Amplification Assay for rapid HPV
Detection and Genotyping. Poster P19-03 Eurogin 2018 Lisbon