Clinical Study – Confidence HPV 2013-2016

Cellcall’s Confidence HPV Test [1-3] for Primary HPV Screening Summary of Baseline Results – Ongoing Clinical Study (n>6000)

INTRODUCTION Millions of women are screened for cervical cancer each year in the Western World and via such programs cervical cancer was reduced by fifty percent, but the system lacks efficiency, automation and objectivity [4]. Traditional cervical cancer screening (cytology or Pap smear or Pap test named after Doctor Georgios Papanikolaou who invented it) was introduced over 50 years ago and has not changed much since. Cells scraped from the cervix are fixed on a glass slide and checked for physical signs of dysplasia indicative of malignant transformations (precancer or cancer). The evaluation is ultimately performed by highly experienced professionals, and patients are managed according to very complex algorithms [5]. This system is subjective, very intricate, with many ramifications, not very sensitive for the disease and cannot identify "at-risk individuals"[4]. In the early 80s Professor Harald zur Hausen discovered that a virus, called the Human papillomavirus causes the large majority of cervical cancers [6-9]. Today we are preparing for the HPV test, which detects the presence of the virus in the cervical sample, to take over the place of the Pap smear in screening [10-12]. It is a major step forward being very sensitive (approx. 95% versus 50%), highly objective and finding almost all at risk women [13-15]. However it brings a new major problem to solve: specificity. Only about 10% of the women infected with the virus develop dysplasia which has to be treated [16, 17]. The question is how to find these women (how to "triage" them). The obvious answer to this question is to bring back the Pap smear in lack of anything better. But because of the low sensitivity of Pap smear many repeat tests called follow-ups will be required, and the complexity will not disappear either. The scientific community has been long looking for adequate answers to this question, and numerous attempts for a triage test are known: HPV RNA, HPV typing, p16 staining, mRNA marker panels, methylation marker panels, HPV methylation testing etc. [18]. The latest US guideline for primary HPV screening recommends a genotyping triage for hrHPV positivity only for HPV16 and HPV18 based on their highest positive predictive values combined with a cytology triage for the HPV16/18 negative but hrHPV positive cases [11]. The latest European guideline for cervical cancer screening recommends the primary HPV screening with no genotyping triage, with a cytology triage alone [12]. Nevertheless, the currently recommended guidelines and the potential protocols of the close future worldwide require reliable, sensitive, objective, high throughput, highly automated, contamination safe HPV tests with a genotyping potential. 1

Confidence CE IVD | Cellcall Kft.| Röppentyű u. 48., Budapest, H-1139, Hungary | www.cellcall.hu

Clinical Study – Confidence HPV 2013-2016 METHODS Real-time PCR technology Real-time PCR technology, as a more suitable technology for high-throughput workflow, offers numerous advantages over conventional PCR. The PCR amplification and detection runs in a sealed PCR plate, also the PCR product stays in the PCR plate sealed. There is no post-PCR manipulation of the PCR product, the risk of getting in contact with the PCR product is avoided which is a major source of PCR contamination in the conventional PCR technology. On the basis of these considerations the real-time PCR reaction does not necessarily require any PCR product elimination processes. Confidence real-time HPV test The Cellcall’s Confidence HPV Test is a qualitative in vitro test for the detection of Human Papillomavirus in patient specimens. The test utilizes amplification of target DNA by multiplex real-time polymerase chain reaction (PCR) for the detection of 14 high-risk (hr)HPV types. The test specifically identifies HPV16 and HPV18 while concurrently detects the other high risk types in group (HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Cellcall’s Confidence HPV Test operates with Confidence DNA Prep Kit (Cellcall, ref.: CO11000101) for DNA preparation and Confidence HPV Kit (Cellcall, ref.: CO21004001) for real-time hrHPV type specific PCR reaction. High-throughput application of Confidence HPV Test Cellcall offers quality controlled high-throughput and highly automated protocols for the application of Confidene HPV Test. The system is able to process over 500 samples per day. Aliquoting of samples with barcode identification, DNA extraction was performed by a silica-filter-plate based purification method on Tecan EVO liquid handling robot with contamination safe filter tip pipetting. After the 96-tip robotic PCR setup, HPV real-time PCR was performed on the QuantStudio 6 Flex platform in 384 plate format. The PCR results were automatically analyzed by Lab Assistant Grey software, Cellcall. The whole procedure is standardized, and the contamination risk is monitored by preparation and amplification non template control (PREP NTC and PCR NTC) where both results in a certain PCR plate should be negative for valid run result. The quality of the sample DNA is guaranteed by the amplification of genomic target control (human PCR control) and internal control (IC: artificial DNA added to the sample during DNA preparation). The PCR procedure is monitored by PCR positive control (PC). 2

Confidence CE IVD | Cellcall Kft.| Röppentyű u. 48., Budapest, H-1139, Hungary | www.cellcall.hu

Clinical Study – Confidence HPV 2013-2016 Study Design Since mid-2013 Cellcall has been conducting a prospective study with outpatient and colposcopy clinics in Hungary enrolling over 6,000 women to assess the performance of the Confidence HPV Test. Cervical samples were collected by Cervex Brush Combi (Rovers) in PreservCyt® Solution (Thinprep Hologic Corp). As a clinically validated comparator test cobas HPV test (Roche, CE) [19-21] was used. We also compared the results of the Confidence HPV to the Full Spectrum HPV detection test (Genoid/Synlab, CE) [22, 23] and GenoID in-house genotyping test (Genoid/Synlab in house). RESULTS Clinical sensitivity and specificity The performance of the HPV test was in practical terms identical to the Roche cobas HPV test regarding the relative sensitivity and specificity and negative predictive values. The Confidence HPV test showed comparable PPV to the cobas HPV test. Cellcall Confidence HPV Test n=4532 women, baseline sample, 30+, CIN2+

se sp ppv npv

Total 4532 Test + Test -

Diesase + 72 3

Diesase - 839 3618

est

lower 0,887523178 0,7999655 0,062350328 0,997580698

upper 0,991673834 0,823138082 0,098497243 0,999829111

0,96 0,811756787 0,079034029 0,9991715



Cellcall Confidence HPV Test n=5717 women, baseline sample, 25+, CIN2+

se sp ppv npv

Total 5717 Test + Test -

Diesase + 86 3

est lower 0,966292135 0,904638434 0,775764037 0,76463719 0,06379822 0,051343665 0,999313344 0,997994624

Diesase - 1262 4366 upper 0,992993943 0,786604268 0,078193289 0,999858373



Table 1. Sensitivity, specificity, positive and negative predictive value of Confidence HPV Test 3

Confidence CE IVD | Cellcall Kft.| Röppentyű u. 48., Budapest, H-1139, Hungary | www.cellcall.hu

Clinical Study – Confidence HPV 2013-2016 Histology was considered to be the gold standard confirmation method, i.e. cases with no histology were presumed gold standard negatives. Cellcall Confidence HPV Test Roche cobas HPV Test n=2547 women, baseline sample, 30+, CIN2+ n=2547 women, baseline sample, 30+, CIN2+

se sp ppv npv

Total 2547

Diesase +

Diesase -

448

Test +

64

393

3

2032

Test -

3

2087

lower 0,874672964 0,803637511 0,097611077 0,995697859

upper 0,990669157 0,834314122 0,156803859 0,999695881

est

lower

upper

0,955223881 0,841532258 0,140043764 0,998564593

0,874672964 0,826556533 0,109549308 0,995810917

0,990669157 0,855696555 0,175286159 0,999703887

Total 2547

Diesase +

Diesase -

Test +

64

Test - est 0,955223881 0,819354839 0,125 0,998525799



se sp ppv npv

Cellcall Confidence HPV Test GenoID Full Spectrum HPV Test n=1879 women, baseline sample, 30+, CIN2+ n=1879 women, baseline sample, 30+, CIN2+



Total 1879

Diesase +

Diesase -

377

Test +

8

401

1494

Test -

0

1470

est

lower

upper

Total 1879

Diesase +

Diesase -

Test +

8

Test -

0

est

lower upper 0,517503485 1 0,779599204 0,81646707 0,009012662 0,04053136 0,996278842 1

se sp ppv npv

1 0,798503474 0,020779221 1





se 1 0,517503485 1 sp 0,785676109 0,766374447 0,8040779 ppv 0,019559902 0,008481481 0,038175707 npv 1 0,996218224 1

Table 2. Sensitivity, specificity, positive and negative predictive value of Confidence HPV Test compared to Roche cobas and GenoID Full Spektrum among 30+ 4

Confidence CE IVD | Cellcall Kft.| Röppentyű u. 48., Budapest, H-1139, Hungary | www.cellcall.hu

Clinical Study – Confidence HPV 2013-2016 Cellcall Confidence HPV Test Roche cobas HPV Test n=3310 women, baseline sample, 25+, CIN2+ n=3310 women, baseline sample, 25+, CIN2+

se sp ppv npv

Total 3310

Diesase +

Diesase -

718

Test +

74

636

3

2515

Test -

3

2597

lower 0,890334633 0,763185068 0,074077897 0,996522145

upper 0,991892083 0,792140968 0,115877144 0,999754233

est

lower

upper

Total 3310

Diesase +

Diesase -

Test +

74

Test - est 0,961038961 0,777915249 0,093434343 0,998808578



se sp ppv npv

0,961038961 0,890334633 0,991892083 0,803278689 0,789149641 0,816856885 0,104225352 0,082728423 0,12907377 0,998846154 0,99663171 0,999761986

Cellcall Confidence HPV Test GenoID Full Spectrum HPV Test n=2274 women, baseline sample, 25+, CIN2+ n=2274 women, baseline sample, 25+, CIN2+



Total 2274

Diesase +

Diesase -

523

Test +

12

560

1739

Test -

0

1702

est

lower

upper

Total 2274

Diesase +

Diesase -

Test +

12

Test -

0

est

lower upper 0,639702565 1 0,750851324 0,786027107 0,011642311 0,038852504 0,996802109 1

se sp ppv npv

1 0,768788683 0,022429907 1





se 1 0,639702565 1 sp 0,752431477 0,734104218 0,770102583 ppv 0,020979021 0,010886024 0,036359157 npv 1 0,996732724 1



Table 3. Sensitivity, specificity, positive and negative predictive value of Confidence HPV Test compared to Roche cobas and GenoID Full Spectrum among 25+ In the HORIZON study the goal was to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Positive agreement between the assays was measured as the conditional probability that the results of all compared assays were positive given that at least one assay returned a positive result. Among primary screening samples from women aged 30–

5

Confidence CE IVD | Cellcall Kft.| Röppentyű u. 48., Budapest, H-1139, Hungary | www.cellcall.hu

Clinical Study – Confidence HPV 2013-2016 65 years (n = 2,881), 23% tested positive on at least one assay, and 42 to 58% of these showed positive agreement on any compared pair of the assays [24]. In our study, Confidence HPV test was compared to Roche cobas HPV and GenoID Full Spectrum HPV. In the case of cobas HPV we found close to 70 % agreement in the high risk HPV positive cases. The overall kappa value was more than 0.75, which is a very good agreement considering the recent findings in this field. Confidence HPV VS cobas HPV Confidence HPV VS Full Spectrum HPV n=2547 women, baseline sample, 30+ n=1879 women, baseline sample, 30+ ANY hrHPV+

Positive agreement

68 %

ANY hrHPV+

Positive agreement

47,6%

est kappa 0.7516768 Total 2547 Confidence + Confidence -

lower 0.7129363 Roche + 387 70

upper 0.7904174 Roche - 125 1965

kappa

lower 0.5046227 FS + 256 153

upper 0.5949868 FS - 129 1341

est 0.5498048 Total 1879 Confidence + Confidence -



Confidence HPV VS cobas HPV Confidence HPV VS Full Spectrum HPV n=3310 women, baseline sample, 25+ n=2274 women, baseline sample, 25+ ANY hrHPV+

Positive agreement

70,5 %

ANY hrHPV+

est kappa 0.7762921 Total 3310 Confidence + Confidence -

lower 0.7423099 Roche + 621 89

upper 0.8102742 Roche - 171 2429

kappa

est 0.5930074 Total 2274 Confidence + Confidence -

Positive agreement

52,9 %

lower 0.5519465 FS + 383 189

upper 0.6340684 FS - 152 1550

Table 4. Agreement of HPV positivity among 30+ and 25+

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Confidence CE IVD | Cellcall Kft.| Röppentyű u. 48., Budapest, H-1139, Hungary | www.cellcall.hu

Clinical Study – Confidence HPV 2013-2016 Confidence HPV VS cobas HPV Confidence HPV VS Full Spectrum HPV n=2547 women, baseline sample, 30+ n=1879 women, baseline sample, 30+ HPV 16+

Positive agreement

63,2 %

est kappa 0.7611736 Total 2547 Confidence + Confidence -

lower 0.7224386 Roche + 108 22

upper 0.7999087 Roche - 41 2376

HPV 16+

Positive agreement

45,3%

kappa

lower 0.5659544 FS + 53 2

upper 0.6500571 FS - 62 1762

est 0.6080057 Total 1879 Confidence + Confidence -

Confidence HPV VS cobas HPV Confidence HPV VS Full Spectrum HPV n=2547 women, baseline sample, 30+ n=1879 women, baseline sample, 30+ HPV 18+

Positive agreement

68,9 %

HPV 18+

est kappa 0.8130074 Total 2547 Confidence + Confidence -

lower 0.7742269 Roche + 31 5

upper 0.8517878 Roche - 9 2502

kappa

est 0.5958705 Total 1879 Confidence + Confidence -

Positive agreement

42,9 %

lower 0.551588 FS + 12 4

upper 0.6401531 FS - 12 1851



Confidence HPV VS cobas HPV Confidence HPV VS Full Spectrum HPV n=2547 women, baseline sample, 30+ n=1879 women, baseline sample, 30+ HPV other HR+

Positive agreement

64,7 %

est kappa 0.7498007 Total 2547 Confidence + Confidence -

lower 0.711068 Roche + 290 56

upper 0.7885335 Roche - 102 2099



HPV other HR+

est kappa 0.4972433 Total 1879 Confidence + Confidence - Table 5. Agreement among 30+

Positive agreement

40,9 %

lower 0.4522503 FS + 183 158

upper 0.5422362 FS - 106 1432

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Confidence CE IVD | Cellcall Kft.| Röppentyű u. 48., Budapest, H-1139, Hungary | www.cellcall.hu

Clinical Study – Confidence HPV 2013-2016 Intended use of Cellcall’s Confidence Test Confidence HPV Test shows similar sensitivity and specificity results as the cobas HPV test (Roche, CE) with high agreement in genotyping. We recommend the following intended use for Confidence HPV: • Test is indicated for use as a first-line primary screening test to identify women at increased risk for the development of cervical cancer or presence of high-grade disease. • Test is indicated for use as a first-line primary screening test to assess the presence or absence of hrHPV types • Test is indicated for use as a first-line primary screening test to assess the presence or absence of hrHPV genotypes 16 and 18.



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Confidence CE IVD | Cellcall Kft.| Röppentyű u. 48., Budapest, H-1139, Hungary | www.cellcall.hu

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Heideman, D.A., et al., Clinical validation of the cobas 4800 HPV test for cervical screening purposes. J Clin Microbiol, 2011. 49(11): p. 3983-5. Wright, T.C., et al., Primary cervical cancer screening with human papillomavirus: end of study results from the ATHENA study using HPV as the first-line screening test. Gynecol Oncol, 2015. 136(2): p. 189-97. Wright, T.C., Jr., et al., Evaluation of HPV-16 and HPV-18 genotyping for the triage of women with high-risk HPV+ cytology-negative results. Am J Clin Pathol, 2011. 136(4): p. 578-86. Jeney, C., et al., Detection and typing of 46 genital human papillomaviruses by the L1F/L1R primer system based multiplex PCR and hybridization. J Virol Methods, 2007. 140(1-2): p. 32-42. Keegan, H., et al., Human papillomavirus detection and genotyping, by HC2, fullspectrum HPV and molecular beacon real-time HPV assay in an Irish colposcopy clinic. J Virol Methods, 2014. 201: p. 93-100. Rebolj, M., et al., Disagreement between human papillomavirus assays: an unexpected challenge for the choice of an assay in primary cervical screening. PLoS One, 2014. 9(1): p. e86835.



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Confidence CE IVD | Cellcall Kft.| Röppentyű u. 48., Budapest, H-1139, Hungary | www.cellcall.hu