Human Papillomaviruses: Biology and Laboratory Testing
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1 For our patients and our population Human Papillomaviruses: Biology and Laboratory Testing Geoffrey Higgins Microbiology and Infectious Diseases
2 For our patients and our population HPV Associated Cancers Number of Cases in 2008 Number Attributable to Infection Percent HPV Positive Cervix uteri 530, , % Anus % Vagina % Penis % Vulva % Oropharynx % De Martel C. et al. Global Burden of cancers attributable to infection in Lancet Oncology :
3 L1 Homology Genera (<60%) Species (60-<70% Types (70-<90%) Doorbar J et al. Rev. Med.Virol 2016; 25:2-23
4 HPV Type in 10,575 Cervical Cancers Paraffin Embedded Tissues From 35 Countries Total HPV Positive (n=8977) Squamous Cell Carcinoma (n=8252) Adeno/ Adenosquamous Carcinoma (n =625) Other* (n=100) HPV (61%) 5090 (62%) 296 (48%) 51 (51%) HPV (10%) 687 (8%) 201 (32%) 30 (30%) HPV (6%) 446 (5%) 74 (12%) 9 (9%) HPV (4%) 338 (4%) 6 (1%) 1 (1%) HPV (4%) 328 (4%) 6 (1%) 1 (1%) HPV (3%) 252 (3%) 1 (<1%) HPV (2%) 199 (2%) 3 (<1%) 1(1%) HPV (2%) 169 (2%) 4 (<1%) 2 (2%) HPV (2%) 134 (2%) 5 (<1%) 3 (3%) HPV (1%) 108 (1%) 4 (<1%) 1 (1%) HPV (1%) 90 (1%) 5 (<1%) HPV (<1%) 72 (<1%) 2 (<1%) 1(1%) HPV (<1%) 57 (<1%) 2 (<1%) HPV (<1%) 43 (<1%) HPV (<1%) 31 (<1%) HPV Other 170 (2%) 163 (2%) 7 (1%) HPV Untyped 52 (<1%) 44 (<1%) 8 (1%) 0 HPV 16/ % HPV % (Gardasil 9) Modified from De Sanjose S. et al. Lancet Oncology 2010; 11:
5 HPV-type from infection to cancer: a meta-analysis of 115,789 women (Guan P. et al. Int. J. Cancer 2012; 131: ) HPV type as a proportion of All HPV Positives HPV
6 HPV HPV type as a proportion of All HPV Positives HPV HPV 35 is not in Gardasil 9 HPV-type from infection to cancer: meta-analysis of 115,789 Women. Guan P. et al. Int. J. Cancer 2012; 131:
7 ORF FUNCTION L1 Major capsid protein L2 Minor capsid protein E1 Control of Viral DNA replication E2 Transcription regulation E4 Virus particle/dna production E5?Transforming: e.g. EGFR E6 Transformation: degrades p53 E7 Transformation: binds prb Doorbar J et al. The Biology and Life Cycle of Human Papillomaviruses
8 HPV Pathogenesis Doorbar J et al. The Biology and Life Cycle of Human Papillomaviruses
9 Cell Cycle Regulation and HPV Doorbar J. et al. Rev. Med. Virol 2016; 25:2-23
10 Differences between HR and LR HPV Doorbar J et al. The Biology and Life Cycle of Human Papillomaviruses
11 The Site of HPV Infection may Determine Outcome Reserve, Cells Cuboidal Cells Stem Cells Egawa N. et al. Viruses 2015; 7: ;
12 HPV DNA integration into a human chromosome Structure of the single copy of HPV-16 DNA integrated into the SiHa cell line derived from a cervical carcinoma. (Fields Virology, (2001) 4th ed.) Integration may decrease E2 mediated repression of the HPV early promoter increasing E6 and E7 expression
13 What is the outcome of HPV Infection? Natural History of HPV Infection 90% of infection clears within 2 years Moscicki A.-B. et al. Vaccine 2012; 30S: F24 F33
14 What is the outcome of HPV Infection? Cumulative Incidence of CIN 3 in Women HPV Positive at Enrolment Less than 30 years Greater than 30 years Mosicki A-B et al Vaccine 2006; 24S3:42-51
15 Fig 2 Cumulative incidence rate for CIN3+ for women (24,000) according to baseline test results Joakim Dillner et al. BMJ 2008;337:bmj.a1754 Assay HC II for 6/7 sites 2008 by British Medical Journal Publishing Group
16 Laboratory Guidelines for HPV Testing National Cervical Screening Program Guidelines 2016 Medical Benefit Schedule National Pathology Accreditation Advisory Council (NPAAC) Requirements for Laboratories reporting tests for the National Cervical Screening Program (First Edition 2017) Working Draft Second Edition, 201?
17 HPV Assays: A selection Assay Qiagen Digene HR HC II Assay Type Hybrid Capture Signal Amplific n Target Whole Genome Cell Control No Analytical Control No HPVs Detected 16,18,31,33,35,39,45 51,52,56,58,59,68 Reports HR HPV present Separate 16, 18, 45 typing assay.
18 Digene Hybrid Capture II HPV Assay RNA probes against 13 HPV types Monoclonal Ab against RNA-DNA hybrids Chemiluminesent Signal Amplification
19 Clinical versus Analytical Sensitivity Lower Sensitivity Improves Specificity Digene Hybrid Capture II Product Information
20 HPV (HC2), Pap or Cotest? 1,011,000 women followed for 5 years Gage J. et al. JNCI 2014; 106: dju153 doi: /jnci/dju153
21 NPAAC Guideline (1 st Edition 2017) S4.1 Laboratories must use only commercially supplied HPV NAT (ARTG listed) that are validated for primary population based screening. S4.2 The HPV NAT method must test for HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, and separately identify HPV 16 and HPV18 (+/-45). S4.4 HPV NAT assays used in primary screening as part of the National Cervical Screening Program must be demonstrated by the manufacturer, or in published studies to fulfil the following criteria: Proven non-inferiority to validated reference assays (e.g. Hybrid Capture 2 (HC2) in cross-sectional equivalence studies using guidelines for test requirements which were developed by an international consortium 3. HSIL sensitivity >90% of HC2 in women of at least 25 years of age. HSIL specificity >98% of HC2 in women of at least 25 years of age. Reproducibility lower confidence bound of 87% Contain a control to monitor inhibition and/or assay failure. Contain a control for cellularity to detect inadequate or empty cervical samples. There is no requirement that the inhibition and cellularity controls are independent
22 HPV Assays: A selection Assay Qiagen Digene HR HC II Assay Type Hybrid Capture Signal Amplific n Target Whole Genome Cell Control No Inhibition Control No HPVs Detected 16,18,45,31,33,52,58 35,39,51,56,59, 68 Reports HR HPV present Separate typing assay 16, 18, 45. Abbott Molecular HPV/m2000rt PCR L1 DNA (~150 bases) Human b-globin No 16,18,45,31,33,52,58 35,39,51,56,59,66,68 16, 18, Other HR HPV, IC BD Onclarity HPV Assay PCR E6/E7 DNA Human b-globin No 16,18,45,31,33,52,58 35,39,51,56,59,66,68 G1: 16, 18, 45, IC G2: 31, 33/58, 56/59/66, IC G3: 51, 52, 35/39/68, IC Cepheid Xpert HPV PCR E6/E7 DNA Human gene No 16,18,45,31,33,52,58 35,39,51,56,59,66,68 16, 18/45, Other HR HPV, IC Hologic Aptima HPV Assay TMA E6/E7 mrna No (Separate) Yes RNA IC 16,18,45,31,33,52,58 35,39,51,56,59,66,68 HR HPV present Separate typing assay 16, 18/45. Roche cobas 4800/6800 PCR L1 DNA (~200 bases) Human b-globin No 16,18,45,31,33,52,58 35,39,51,56,59,66,68 16, 18, Other HR HPV, IC
23 Athena (Roche) Trial: 3 years follow-up data CIN 2 CIN 3 HPV 16 HPV 18 Other Negative
24 CIN 3 Detection in ASCUS Populations Age >21 years Examples of Product Information Data Estimate 95% CI Estimate 95% CI Cobas 4800 Comparator (DNA, CE) Sensitivity (%) , , 96.6 Specificity (%) , , 72.3 PPV (%) , , 9.4 NPV (%) , , 99.9 Prevalence (%) , , 3.9 BD Onclarity Comparator (DNA, FDA) Sensitivity (%) , , 93.7 Specificity (%) , , 61.9 PPV (%) , , 5.0 NPV (%) , , 99.8 Prevalence (%) % Aptima HPV Comparator (DNA) Sensitivity (%) , , 97.3 Specificity (%) , , 56.7 PPV (%) , , 9.4 NPV (%) , , 99.8 Prevalence (%)
25 Do HPV Tests Agree? Differential Detection of Human Papillomavirus Genotypes and Cervical Intraepithelial Neoplasia by Four Commercial Assays Rebolj M. et al J Clin Micro 2016; 54: (All, yrs (n = 651) Normal Cytology (n =558) Abnormal Cytology (n = 93) 29% 22% 68%
26 National Cervical Screening Program Guidelines 2016
27 HPV MBS Items: Asymptomatic Screening Five yearly screening for asymptomatic persons, sexually active, vaccinated or unvaccinated aged > 25 years. Cease testing between if no high risk HPV (can test > 75 years if no tests in last 5 years) 73070: A test, including partial genotyping, for oncogenic human papillomavirus that may be associated with cervical pre-cancer or cancer: a) a liquid based cervical specimen; and b) asymptomatic patient aged > 24 years 9 months c) Once in a 57 month period (Fee: 100% = $35.00) Test must identify HPV 16, HPV 18/45 separately from other high risk types Reflex LBC if HR HPV Positive HPV result must be combined with the reflex cytology result (if performed) into a single combined report
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29 HPV MBS Items: Additional/Follow-Up HPV Testing 73072: A test, including partial genotyping, for oncogenic human papillomavirus, performed on a liquid based cervical specimen: a) Patients in population with higher risk of CIN or cancer; i. Immunosuppressed (e.g. HIV, immunosuppressant drugs) HPV and reflex LBC, every 3 years. Colposcopy for any HPV positive i. Early onset sexual activity (unvaccinated and <14 years), aged years: HPV and reflex LBC (one only) b) follow-up patients with i. previously HR HPV but negative/low grade LBC: HPV and reflex LBC, ii. CIN or cancer: Co-test: HPV and LBC
30 HPV MBS Items: Additional/Follow-Up HPV Testing 73072: Continued c) Patient with symptoms suggestive of cervical cancer: Co-test, further assessment d) Follow-up of patient after treatment of high grade CIN Co-test, annually until 2 consecutive negatives e) Follow-up of patient with glandular abnormalities Co-test, annually, indefinitely f) Follow-up of patient exposed to DES in utero Co-test, annually, indefinitely 73075: Repeat HPV testing for failed test 73076: Liquid based cytology ( Fee = $46.00)
31 HPV MBS Item for Hysterectomy Patients 73074: A test, including partial genotyping, for oncogenic human papillomavirus: a) a liquid based vaginal vault specimen; and b) for patients following a total hysterectomy WHY: Secondary VAIN occurs in % of treated CIN patients with elevated rates of invasive disease for at least 20 years. Frequency of testing 1. No evidence or unknown history of cervical disease: HPV yearly until two negative consecutive tests, then cease. 2. LSIL or HSIL found in cervix at operation, or HSIL treated without previous test of cure : Co-test yearly until two consecutive tests negative 3. If hysterectomy was for AIS : Indefinite annual Co-tests
32 NPAAC Guideline 1 st Edition Quality assessment Quality Measures for HPV NAT (applies to all testing settings, including screening, test of cure and self-collected specimens) S5.1 External QAP obligatory S5.2 All external QAP discordances must be investigated. S5.3 Laboratories must monitor detection rates of oncogenic HPV, and unsatisfactory specimens. C5.3(i) HPV results must be stratified for HPV16/18, and other oncogenic HPV. C5.3(ii) The rate of unsatisfactory specimens by testing reason and type. S5.4 Daily testing of an externally sourced non-manufacturer supplied control material
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35 Simulated Plot Provided by Dr Lahn Straney NCSR
36 NPAAC Guideline 1 st Edition Quality Measures for HPV NAT, Screening specimens only S5.5 Laboratories must compare their rates of HPV detection in screening tests with the rates most recently reported by the NCSR. C5.5(iii) At least 2000 specimens tested to determine HPV rates. C5.5(iv) The 2000 specimens must be stored and available/eligible for retesting for HPV and LBC according to manufacturer s guideline. C5.5(v) NCSR will produce a quarterly age stratified data set (mean ± 2 SD) C5.5(vi) If the HPV positivity rate in screening tests is not within the two standard deviations, the laboratory must follow the procedures in Appendix A. C5.5(vii) If within 2 SD specimens can be may be discarded C5.5(viii) Compare rates with the NCSR at least quarterly. Laboratories must process 2000 specimens/ 4-6 weeks
37 Specimen Storage Limitations Assay Assay Type Target Cell Control Collection Medium Specimen Stability Qiagen Digene HR HC II Signal Amp Whole Genome No PreserveCyt Digene Cx Kit oC 14 RT, freeze Abbott Molecular HPV/m2000rt PCR L1 DNA (~150 bases) Human b-globin PreserveCyt SurePath Cervi-Collect 4 months@ 15-30oC oC oC BD Onclarity HPV Assay PCR E6/E7 DNA Human b-globin PreserveCyt SurePath BD Cx Kit oC oC oC Cepheid Xpert HPV PCR E6/E7 DNA Human gene PreserveCyt oC Hologic Aptima HPV Assay TMA E6/E7 mrna No PreserveCyt SurePath Aptima Cx kit oC oC 60 Roche cobas 4800/6800 PCR L1 DNA (~200 bases) Human b-globin PreserveCyt SurePath cobas Cx kit oC oC 6
38 NPAAC Guidelines 1 St Edition: Appendix A Protocol to be followed if a batch of results of at least 2000 tests falls outside 2SD (95%) of the mean of the current NCSR data Investigate: a) Pool data with previous batch and check if within range. If out of range b) Compare with ranges of users of the same assay c) Compare age ranges (vaccinated, no vaccinated) positivity rates with NSCR age rates d) If neither, retest the batch of 2000 samples 2. Inform the NCSP Quality and Safety Monitoring Committee including the following information (assay, device, batch and lot numbers of reagents, number of specimens, HPV positivity rates. 3. Report the results of the investigation plus include a) HPV positivity data from the past 10 batches or three months, whichever is greater b) Previous 12 months of RCPAQAP results c) Previous 12 months of non-manufacturer control testing.
39 Simulated Plot Provided by Dr Lahn Straney NCSR
40 Working Draft NPAAC Guidelines 2 nd Edition Quality assessment Quality Measures for HPV NAT (applies to all testing settings, including screening, test of cure and self-collected specimens) S5.1 Participate in external quality assurance program. S5.2 Investigate all discordances in external HPV QAP and document corrective actions. S5.3 Monitor HR HPV detection rates, and unsatisfactory specimens. C5.3(i) Stratified for HPV16/18 (+/- 45), and other oncogenic HPV. S5.4 Externally sourced control material for HPV16/18. S5.5 Reagent batch failure must be notifed to the NCSP Quality Measures for HPV NAT, Screening specimens only S5.6 Monitor HR HPV detection rates and compare with the rates most recently reported by the NCSR. C5.6(i) The NCSR will produce a periodic age stratified data set (including mean and 95% confidence interval). C5.6(ii) If the laboratory s HPV positivity rate in screening tests is not within the 95% confidence interval, the laboratory must investigate the cause (Appendix A) C5.6(iii) Both done quarterly Appendix A Compare age cohorts with NCSR data Compare device specific ranges Investigate further, Consult NCSR No requirement for retrospective testing if laboratory falls outside of range
41 For our patients and our population Human Papillomaviruses: Biology and Laboratory Testing Thank You
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