Screening Methods for HPV and Dysplasia Detection Prevention of Cervical Cancer: THE ROLE OF HPV INFECTION Vilnius, 26.4.2017 Screening Methods for HPV and Dysplasia Detection Andreas M. Kaufmann, PhD andreas.kaufmann@charite.de Clinic for Gynecology, Charité-Universitätsmedizin Berlin, Germany

The most important measure to prevent cervical cancer in future is HPV VACCINATION

where screening established Cervical cancer – Prevention in Germany Trends in Incidence and Mortality Most effective cancer prevention programme where screening established 3

Progression from Infection to Cervical Cancer 7-30 years Infection CIN 1 CIN 2/3 Invasive CxCa Ca. 50% 8-18 months Virus production transformation CxCa invasion Aus Nature Reviews Cancer: Vol. 7: 11-22 (Januar 2007) Basal cells in the cervical epithelium rest on the basement membrane, which is supported by the dermis. Human papillomavirus (HPV) is thought to access the basal cells through micro-abrasions in the cervical epithelium. Following infection, the early HPV genes E1, E2, E4, E5, E6 and E7 are expressed and the viral DNA replicates from episomal DNA (purple nuclei). In the upper layers of epithelium (the midzone and superficial zone) the viral genome is replicated further, and the late genes L1 and L2, and E4 are expressed. L1 and L2 encapsidate the viral genomes to form progeny virions in the nucleus. The shed virus can then initiate a new infection. Low-grade intraepithelial lesions support productive viral replication. An unknown number of high-risk HPV infections progress to high-grade cervical intraepithelial neoplasia (HGCIN). The progression of untreated lesions to microinvasive and invasive cancer is associated with the integration of the HPV genome into the host chromosomes (red nuclei), with associated loss or disruption of E2, and subsequent upregulation of E6 and E7 oncogene expression. LCR, long control region. infection DNA Detection E6/E7 RNA/Protein detection! Cytology Screening Methods: © AMK

CxCa Prevention (primary screening) abnormal cells in micropscope 1) PAP smear 2) HPV Test Cervical samples (liquid-based cytology) Pictured here is the cervical sample collection system using liquid-based cytology (LBC). A brush-like instrument is used to collect cell samples from just inside the cervix, which are then placed in liquid and sent to a central laboratory for analysis. The histopathologist examines the stained cells to see if any abnormal cells are present. If there are abnormal cells present, the investigator will ask that the subject return to the clinic for a visual examination of the cervix using a colposcope. This is the normal sequence of events in a cervical cancer screening programme. High-risk HPV16,18,31,33,… Low-risk HPV6, 11, 42,….. When positive triage, diagnosis, therapy 5

Primary Screening: HPV Detection vs Cytology HPV Test: high Sensitivity Internationally shift from cytology to HPV Test 2) WHO: „countries that do not yet have cytology established should start directly with HPV Testing“ 3) Maybe use cytology as triage method 4) Potentially better molecular triage tests possible But Cytology: low Sensitivity / high Specificity Cuzick et al., Int J Cancer 2008

Parameter of Test Quality Depending on frame work conditions: Age, Interval, Test characteristics… Cytology HPV DNA Sensitivity (CIN2+) 50% (80%) >95% Specificity 98% 80-90% Positive Predictive Value >20-40% <10% Negative Predictive Value 50% >98% Post Test Probability ??? >10% Which conditions adequate for the program? © AMK

Comparison of 6 HPV Tests in a Screening Population @ six HPV tests from residual liquid-based screening cytology specimens, all tests except for NorChip showed high sensitivity for high-grade lesions that were positive by cytology, suggesting that they are suitable for primary screening and that dual co-testing with cytology as well is unnecessary. Positivity rates in cytology-negative specimens were similar for the DNA-based tests, but were lower for the APTIMA test, suggesting it can maintain the high sensitivity of the DNA tests, but with a better specificity, so that fewer women would need triage tests or short-term follow-up. However, a long-term low-risk period after a negative test has yet to be demonstrated for APTIMA or any RNA-based test, as has been shown for some of the DNA-based tests, especially Hybrid Capture 2 (Dillner et al, 2008, Cuzick et al, 2008b, Mesher et al, 2010, Rijkaart et al, 2012). Direct demonstration of this is desirable to support its use in primary screening, The NorChip test had lower sensitivity but higher specificity, suggesting its role may be more in triage than primary screening. © AMK Cuzick et al., BJC 108 (2013)

Invasive CxCa after initial Screening in RCTs in 2nd Round 176 464 women aged 20–64 years were randomly assigned to HPV-based (experimental arm) or cytology-based (control arm) screening in Sweden (Swedescreen), The Netherlands (POBASCAM), England (ARTISTIC), and Italy (NTCC). Followed up for a median of 6.5 years (1 214 415 person-years) and identified 107 invasive cervical carcinomas HPV Cytology -60-70% -45% missed CIN3/CxCa in 1st round new CxCa in 2nd round The rate ratio for invasive cervical carcinoma among all women from recruitment to end of follow-up was 0・60 (95% CI 0・40–0・89), with no heterogeneity between studies (p=0・52). Detection of invasive cervical carcinoma was similar between screening methods during the first 2・5 years of follow-up (0・79, 0・46–1・36) but was significantly lower in the experimental arm thereafter (0・45, 0・25–0・81). In women with a negative screening test at entry, the rate ratio was 0・30 (0・15–0・60). The cumulative incidence of invasive cervical carcinoma in women with negative entry tests was 4・6 per 10⁵ (1・1–12・1) and 8・7 per 10⁵ (3・3–18・6) at 3・5 and 5・5 years, respectively, in the experimental arm, and 15・4 per 10⁵ (7・9–27・0) and 36・0 per 10⁵ (23・2–53・5), respectively, in the control arm. Rate ratios did not differ by cancer stage, but were lower for adenocarcinoma (0・31, 0・14–0・69) than for squamous-cell carcinoma (0・78, 0・49–1・25). The rate ratio was lowest in women aged 30–34 years (0・36, 0・14–0・94). Interpretation HPV-based screening provides 60–70% greater protection against invasive cervical carcinomas compared with cytology. Data of large-scale randomised trials support initiation of HPV-based screening from age 30 years and extension of screening intervals to at least 5 years. Higher sensitivity for CIN3 and therapy => less CxCa incidence in follow up © AMK Ronco et al., Lancet 2014; 383: 524–32

CIN2+ Development after HPV Persistence Genotype-specific persistence unknown persistence HPV negative / genotype change Persistence >12 month Elfgren et al., 2016

Quality Control for HPV Testing Smear quality (sampling during colposcopy?) Lab Infrastructure (contamination risk, PCR problem) validated Test Internal control for cellular DNA! Monitoring of processes Intra/inter Lab reproducibility Proficiency Testing of reference laboratory (e.g., Equalis) © AMK

Targets for HPV Detection Generic primer amplify all HPV types L1 is most heterogenous gene (selection pressure) GP-PCR Systems: MY09/11 GP5+/6+ SPF10 450 bp E6-E7: Oncogenes L1 L2 E4 E6 E7 URR E5 E1 E2 150 bp Multiplex PCR mRNA Oncoproteins 65 bp E6/E7 are never deleted! HPV 16 7905 bp L1-L2: Capsid Multiplex PCR Probe-based Systems E1-E5: regulatory genes

>220 commercially available HPV test systems probes DNA, PCR RNA Protein § Guidelines recommend only HR-HPV testing as LR-HPV testing has no clinical use in CxCa screening or triage of positive cytology.

Guideline criteria for validation of HPV Test Formates 14 HR-HPV genotypes included clinically validated versus Hybrid Capture 2 (HC2) and/or GP5+/GP6+ PCR-(EIA) (Diassay) HC2 und Diassay validated in large prospective clinical studies (women >30 years) Shows clin. Sensitivity for CIN2+ of 95% Shows clin. Specificity for CIN2+ of 90.7 to 94.1% New HPV test formats should have relative: 90% of HC2 sensitivity 98% of HC2 specificity © AMK Meijer et al., J Clin Virol. 2009

Parameters for Choice Validation, Approval Target sequence Type spectrum Genotyping Practicability Cost Triage potential Adequacy for a given system

Commercial PCR-based HPV tests Time to first result Released** Target HPV types* Reported QIA hc2 4.5-5 hours (6-7 hrs. total) March 2000 Whole genome 13 HR HR screen Abbott rtHPV 4.5 hours Jan 2009*** L1 + HPV66 16/18 included Cervista ~3 hours March 2009 (HR and 16/18) 16/18 separate Cobas 4800 April 2011 DNA APTIMA 3.5 hrs October 2011 E6/E7 mRNA 16,18/45 separate Anyplex II HPV HR – Seegene 4.5 – 5 hrs June 2012*** All genotyped BD Onclarity HPV Feb 2014*** E6/E7 DNA 16, 18, 31, 45, 51, 52 included Xpert® HPV < 60 minutes April 2014*** 16, 18/45 included * 13 HR types: 16, 18,45, 31, 33, 35, 39, 51, 52, 56, 58, 59 and 68 ** USA FDA approval date unless otherwise indicated *** CE-IVD

Cobas (Roche) Partial genotyping FDA approval automation Market power

PCR basierter Cobas Test: Genotypisierung und Sensitivität Athena Studie: „Addressing the need for advanced screening“ 47.000 Frauen, US multi-center Studie, doppel-verblindet Cobas HPV Test (Roche) 12 hrHPV und HPV16/18 Genotypisierung 93,5% Sensitivität, 69,3% Spezifität für CIN3, 0,3% falsch negativ Identifizierung der Frauen mit höchstem absoluten/relativen Risiko einer CIN3+ 10% HPV16 und/oder 18 positive bei unauffälliger Zytologie hatten eine CIN3+ 16% HPV16 und/oder 18 positive mit ASC-US Zytologie hatten eine CIN3+ Wright TC et al., Am J Clin Pathol. 2011 Oct;136(4):578-586

All HPV 16/18 positives (as of age 25) immediate colposcopy…? CIN 3 Risk when HPV16/18+ Cobas Roche HPV-Test All HPV 16/18 positives (as of age 25) immediate colposcopy…? Clinically and ethically adequate?

Onclarity (BD): Extended Genotyping Genotyping more than 16 and 18 indicator for future disease: Genotyping HPV16/18 recomended by guidelines. Genotype 31 and 33 higher progression risk! >90% of Adenocarcinoma is 16, 18 and 45 associated. Typ 45 to be added to16/18, Detection type-specific persistence in Screening and follow up for risk assessment. Vaccinated populations: less genotypes 16/18 => new needs for Screening

Extended genotyping: OnclARITY (BD) tEST BD Onclarity™ HPV Assay 1 step for: 6 individual genotypes: 16, 18, 31, 45, 51, 52 3 distinct groups: 33, 58 56, 59, 66 35, 39, 68 IC 16 18 45 33,58 31 56,59,66 51 52 35,39,68 Lower risk for disease Basis for 77% invasive SCC and 94% of AdCa Regionale Variance & Importance

Cepheid: Xpert HPV – Test characteristics 3 simple steps: Thinprep sample Routine LBC cytology 1 ml into Cartouche Insert Cartouche start automated PCR analysis Result in 60 min

Result – Xpert HPV (Cepheid) Der GeneXpert® HPV (Human Papillomavirus) Assay qualitative in vitro-Test from ThinPrep Pap-Test PreservCyt-LBC. Real-Time-PCR (RT-PCR) on target DNA (On^cogene E6/E7) of 14 high-risk HPV-Types in a single reaction Differentiation of : HPV 16 HPV 18 and 45 in combination 11 other high-risk Types in a group result (31, 33, 35, 39, 51, 52, 56, 58, 59, 66 und 68) internal validity control  represents by 3 different curves P3, P4 and P5

Result presentation – Xpert HPV Xpert HPV Test uses 6 Fluorochrome channels No high throughput, simple, expensive => adequate? HMBS = Hydroxymethylbilan-Synthase (Housekeeping-Gen)

Advantage by full genotyping Multiple Types differentiated HR-HPV differentiated Persistence identical HPV type vs change in type Test of cure – dysplasia-causing type eliminiert, reinfection with other type? HPV vaccine types identified (loss of protection?) HR-HPV: 16, 18 (31, 45) 10-15% 70-85% HR-HPV: 33, 35, 39, 51, 52, 56, 58, 59, 68, 73 20-35% Umgang mit Ergebnis Infection CINIII/CxCa

Full HPV genotyping testing „HPV Array“ (AID/GenID) Multiplex PCR E1 gene 18 HR-HPV 11 LR-HPV GAPDH Read out by ELISA type staining ELISpot reader 96 samples, high throughput Automatic objective reading, evaluation and reporting in 10 min Simple handling Simple PCR/reader equipment CS PP 02.32

EliSpot Reader and AiDot analysis software used for HPV Easy readout Available in many african countries for CD4 characterization in HIV+ 10 min readout for 96 well plate Evaluation of HPV types Related to DNA content GAPDH internal control

Full Genotyping: HPV Multiplexed Genotyping (WHO Reference test) high analytical sensitivity => Epidemiology GP5+/6+- L1 PCR Biotinylated primer PCR product bonds on beads Schmitt M. et al., JOURNAL OF CLINICAL MICROBIOLOGY, 2006 und 2008

Result Multiplexed Genotyping Gyn Lab Charite Nr. HPV 6 HPV 11 HPV 16 HPV 18 HPV 26 HPV 31 HPV 33 HPV 35 HPV 39 HPV 42 HPV 43 HPV 45 HPV 51 HPV 52 HPV 53 HPV 54 HPV 56 HPV 57 HPV 58 HPV 59 HPV 66 HPV 68 HPV 70 HPV 72 HPV 73 HPV 82 HPV result ß-Control PK mix 75 154 1174 28 24 2 4 11 219 10 71.5 5 121 19 6 310 4.5 365 754 424 9 ok   NK MM 1 3 5.5 6.5 1.5 2.5 NN 1234 1775 16 602 3.5 13 73 1782 8.5 7 763 908 8 45 804 31, 52 845 92 1835 948 0.5 1487 2739 1204 630 12 DNA EXTR HPV Typ Number MFI value relative to ß-Globin internal control Ca. Virus load Multiple Types Full Genotypising Non-sufficient DNA amount

New Test Systems and Molecular Triage Reflex cytology? (co-testing!) All colposcopy? additional biomarker? Molecular Triage? Biomarker Methylation marker HPV oncoprotein expression strength

© AMK

Arbor Vita E6 Cervical Test (Biomarker for disease) Triage test! Simple lateral flow Immunoassay Test Detection of HPV16 and 18 (typing), 5 additional HPV types in development 9 samples in <3 h (hands on time appx. 2 h) Sensitivity for CIN3+ 53.5% for CervixCa 91.7% Specificity for CIN3+ 98.9% PPV for CIN3+ 40.8% NPV for CIN3+ 99.37% High specificity for disease Simple, robust technology Low resource settings Zhao et al., 2013

Simplicity of Oncoprotein E6 Cervical Test (Workstation Setup)

Positivity according to disease stage The AVE6 (blue) detects less lowgrade disease (true progressors) than HC2 HPV test (all infections). AVE6 detects all cancers. Red circle: situation HPV positive but E6 negative / E6 positive: see next slide © AMK

OncoE6TM positive test indicates high risk for future disease Risk prediction: Follow-Up Study, 1 year, 5 years 1 year risk for CIN2+/CIN3+ after positive Test in NILM Pathology patients 30 faches Risiko für CIN3 wenn E6 positiv 30 fold risk after 12 months 88 fold risk after 5 years. 44% after 5 years! © AMK

Screening results and disease burden   AVE6 HR-HPV screening positive 2.1% (41/1999) 37.6% (752/2000) triaging 85% (35/41) colpo 38.4% (298/752) cyto treatment referral 28.5% (10/35) w/changes 5.5% (16/298) ASCUS disease found 100% (10/10) 6.25% (1/16) Comparison of result and follow up depending on screening method AVE6: has only 2.1% positives needed to invite for follow up; 85% follow invitation due to high disease risk; but these are ONLY 35 women who have to travel!!!; of those one third have to receive treatment; all 10 have some degree of disease In contrast: HPV tested 37% are positive = 752 women who need some triage / follow up! Only 38% accepted or reached for cytology swab, no invasive sample give, no will to travel Only 5.5% have some cytology signs of disease (282 triaged in vain) Only 1 case found..15 colposcopy in vain! We need a test with high sensitivity and even more specificity! Also AVE6 allows risk assessment.

Biomarker for high-grade and progressive dysplasia E6 Oncoprotein Expression CIN1 E6 neg. 0.5% E6 pos. 14,5% CIN2/3 E6 neg. 50%? E6 pos. 50%? CxCa E6 pos. 100% Only truely progressive dysplasia expresses E6 in detectable amount © AMK

Summary Primary HPV testing can improve CxCa screening due to higher sensitivity Specificity problem to be solved by biomarkers Adequacy of the test for a given situation Genotyping can be important New assay formats can improve existing possibilities and add a prognostic information