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Clinical Study – Confidence HPV 2013-2016 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 Cellcall Confidence HPV Test n=4532 women, baseline sample, 30+, CIN2+ n=5717 women, baseline sample, 25+, CIN2+ Total 4532 Diesase + Diesase - Total 5717 Diesase + Diesase - Test + 72 839 Test + 86 1262 Test - 3 3618 Test - 3 4366 est lower upper est lower upper se 0,96 0,887523178 0,991673834 se 0,966292135 0,904638434 0,992993943 sp 0,811756787 0,7999655 0,823138082 sp 0,775764037 0,76463719 0,786604268 ppv 0,079034029 0,062350328 0,098497243 ppv 0,06379822 0,051343665 0,078193289 npv 0,9991715 0,997580698 0,999829111 npv 0,999313344 0,997994624 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+ Total 2547 Diesase + Diesase - Total 2547 Diesase + Diesase - Test + 64 448 Test + 64 393 Test - 3 2032 Test - 3 2087 est lower upper est lower upper se 0,955223881 0,874672964 0,990669157 se 0,955223881 0,874672964 0,990669157 sp 0,819354839 0,803637511 0,834314122 sp 0,841532258 0,826556533 0,855696555 ppv 0,125 0,097611077 0,156803859 ppv 0,140043764 0,109549308 0,175286159 npv 0,998525799 0,995697859 0,999695881 npv 0,998564593 0,995810917 0,999703887 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 - Total 1879 Diesase + Diesase - Test + 8 377 Test + 8 401 Test - 0 1494 Test - 0 1470 est lower upper est lower upper se 1 0,517503485 1 se 1 0,517503485 1 sp 0,798503474 0,779599204 0,81646707 sp 0,785676109 0,766374447 0,8040779 ppv 0,020779221 0,009012662 0,04053136 ppv 0,019559902 0,008481481 0,038175707 npv 1 0,996278842 1 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+ Total 3310 Diesase + Diesase - Total 3310 Diesase + Diesase - Test + 74 718 Test + 74 636 Test - 3 2515 Test - 3 2597 est lower upper est lower upper se 0,961038961 0,890334633 0,991892083 se 0,961038961 0,890334633 0,991892083 sp 0,777915249 0,763185068 0,792140968 sp 0,803278689 0,789149641 0,816856885 ppv 0,093434343 0,074077897 0,115877144 ppv 0,104225352 0,082728423 0,12907377 npv 0,998808578 0,996522145 0,999754233 npv 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 - Total 2274 Diesase + Diesase - Test + 12 523 Test + 12 560 Test - 0 1739 Test - 0 1702 est lower upper est lower upper se 1 0,639702565 1 se 1 0,639702565 1 sp 0,768788683 0,750851324 0,786027107 sp 0,752431477 0,734104218 0,770102583 ppv 0,022429907 0,011642311 0,038852504 ppv 0,020979021 0,010886024 0,036359157 npv 1 0,996802109 1 npv 1 0,996732724 1 Table 3.
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