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

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

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

4. 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: (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

5. 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

6. 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

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

8. 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)

9. Invasive CxCa after initial Screening in RCTs in 2nd Round
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 ( 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

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

11. 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

12. 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

13. >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.

14. 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

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

16. 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

17. Cobas (Roche)
Partial genotyping
FDA approval
automation
Market power

18. PCR basierter Cobas Test: Genotypisierung und Sensitivität
Athena Studie: „Addressing the need for advanced screening“
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 Oct;136(4):

19. 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?

20. 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

21. 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

22. 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

23. 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

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

25. 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

26. 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

27. 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

28. 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

29. 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

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

31. © AMK

32. 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 CIN %
for CervixCa 91.7%
Specificity for CIN %
PPV for CIN %
NPV for CIN %
High specificity for disease
Simple, robust technology
Low resource settings
Zhao et al., 2013

33. Simplicity of Oncoprotein E6 Cervical Test (Workstation Setup)

34. 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

35. 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

36. 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.

37. 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

38. 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