Monitoring and Ordering Practices for Human Papilloma Virus in Cervical Cytology

1
Monitoring and Ordering Practices for Human
Papilloma Virus in Cervical Cytology

• Christine Noga Booth, MD
• Cytopathology Fellowship Program Director
• RJT-Pathology Laboratory Medicine Institute
• Cleveland Clinic

2
HPV and Cervical Cytology

• Early 1980s
• Link between HPV and cervical carcinoma
  discovered
• Mid-1990s
• First test to detect HPV made available
• Current roles
• Patient screening, triage and management

3
Anogenital HPV Infections
Spectrum of clinical expression

• Latent infection - no identifiable lesion
• Exophytic condylomas
• Low-grade and high-grade neoplasia
• Invasive cancers Cervix, vulva, anus, penis,
• head neck, esophagus, conjunctiva

4
Natural History of HPV Infections
Wright and Schiffman (2003) NEJM
5
HPV Integration

• Not part of normal viral life cycle
• Random sites in human genome
• Loss of 50 HPV DNA including E2 leading to
  increased expression of E6/E7
• Integration detected even in LSIL lesions but
  more common in HSIL

5
6
Mechanism for Transformation

• Requires integration of viral genome into host
  genome
• HPV genes E6 E7 are always conserved with loss
  of E2 which normally regulates transcription of
  E6 E7
• E6
• Inhibits p53
• Allows cell to enter S phase without normal DNA
  repair function
• E7
• Binds retinoblastoma tumor suppressor gene
  product (pRb)
• Allows cells to proceed uninhibited through S
  phase

7
E7 leads to increased expression of p16INK4a
MTM labs
8
Anogenital HPV Types

• High-risk types 16, 18, 31, 33, 35, 39, 45, 51,
  52, 56, 58, 59, 66, 68, 73, 82
• Possible high-risk 23, 53
• Low-risk types 6, 11, 40, 42, 43, 44, 54, 61, 70,
  72, 81

Munoz et al. (2003) NEJM
9
HPV DNA Testing Methods
Methods currently in use

• FDA Approved
• Hybrid Capture 2 (Qiagen) 1995
• Cervista (Hologic) March 2009
• Cobas (Roche) April 2011
• APTIMA (GenProbe) Oct 2011
• In situ hybridization (ISH)
• Home Brew Polymerase chain reaction
  (PCR) "consensus" primers - all anogenital
  HPV "type specific" - single type of HPV

10
HPV DNA Testing Methods

• Hybrid Capture 2 (Qiagen) separate high-risk
  and low-risk probe mixtures
• Invader (Cervista) separate tests for high
  risk mixture and HPV 16/18
• Cobas 4800 (Roche) concurrent testing for HPV
  16/18 and 12 other hrHPV
• APTIMA (GenProbe) E6/E7 mRNA from 14 hrHPV

11
HPV DNA Testing Methods hc2

• Hybrid Capture 2 High-Risk HPV DNA Test
• Commercially available (Qiagen), FDA-approved
• High-risk Probe mixture
• 16/18/31/33/35/39/45/51/52/56/58/59/68
• Sensitivity is about 5,000 copies of HPV
• May cross react with low-risk HPV DNA

12
HPV DNA Testing Methods Invader

• Commercially available (Cervista), FDA-approved
• Probe mixture
• High-risk 16,18, 31, 33, 35, 39, 45,
• 51,52, 56, 58, 59, 66, 68
• Isothermal signal amplification procedure with
  detectable fluorescence
• Does not cross react with low-risk HPV DNA
• Contains internal control for sample DNA
  sufficiency

13
HPV DNA Testing Methods Cobas

• Commercially available (Roche), FDA-approved
• Probe mixture
• Individual results for 16 and 18
• Pooled results for 31, 33, 35, 39, 45, 51,52,
  56, 58, 59, 66, 68
• Qualitative PCR for L1
• Contains internal control for sample DNA
  sufficiency

14
HPV DNA Testing Methods Aptima

• Commercially available (Gen-Probe), FDA-approved
• Probe mixture
• Pooled results for 16,18, 31, 33, 35, 39, 45,
  51,52, 56, 58, 59, 66, 68
• Qualitative nucleic amplification that detects
  E6/E7 mRNA
• Similar sensitivity but possible increased
  specificity to HC2

15
Cervista vs. Hc2

• Independent study (SHENCCAST II) in China
  (presented at AOGIN 2010)
• Biopsy confirmed CIN 2
• Cervista
• sensitivity 90.7, specificity 90.2
• Hc2
• sensitivity 94.7, specificity 87.9
• Clinically equivalent (area under ROC curve)

16
HPV Testing Quality Assurance

• Test Validation
• Analytic validation/clinical validation
• Laboratory evaluation designed to insure that
  the test is operating in your hands as expected
• 2) Internal Quality Control
• Internal Standards known positives and
  negatives in each run, Active review of results
  looking for patterns/trends, Rerunning of
  equivocal results
• 3) External Peer Comparison
• Required by CLIA 88 for all analytes
• CAP LAP or self-developed program

17
CAP Interlaboratory Comparison for hrHPV

• Started in 2008 (piloted in 2007)
• Designed for labs doing only hrHPV testing
• ThinPrep, SurePath, Standard Transport Media
  modules or mixed specimen module
• Hc2, PCR, Third Wave
• 3 mailings per year, 5 specimens each mailing
• 2008 data
• 3,296 laboratory responses
• 98.3 concordance with reference result

18
HPV DNA Testing
Potential clinical uses

• Management of ASC - US
• Secondary follow-up - abnormal Pap
• Follow-up post treatment
• Primary screening
• QC

19
HPV DNA Testing for ASCUS Triage
Kaiser Permanente study

• 46,009 women with ThinPrep Paps
• ASCUS rate of 3.5 973 women
• 82 participation median age 37 yrs
• Used Hybrid Capture II for high-risk HPV

Manos et al. JAMA (1999)
20
For CIN 2 ()Triage Method
Sens Refer
Kaiser ASCUS Study

• HPV DNA testing 89 40
• Repeat Pap test 76 39

• repeat conventional Pap smear gt ASCUS

Manos et al. (1999) JAMA
21
HPV DNA Testing for Triage
NCI-sponsored ALTS trial

• Multicenter, randomized, prospective trial
• 3 arms Immediate colposcopy HPV DNA
  liquid-based Pap Repeat Pap test
• 2,324 women with ASCUS published

Solomon et al. JAMA (2001)
22
For CIN 2 ()Triage Method
Sens Refer
ALTS Trial - ASCUS

• HPV DNA testing 96 56
• Repeat Pap test
• gt ASCUS 85 58
• gt LSIL 60 26

23
Meta-analysis of ASCUS-HPV Triage Studies
(1999-2005)Arbyn et al GynGynecol Oncol 99
(2005) S7-S11

• 16 studies met criteria for inclusion
• Using HC2 with a disease threshold of CIN2
• Sensitivity 94 (CI 92-96) (range 80 - 100)
• Specificity 62.4 (CI 56-68) (range 37 - 80)
• 6 studies included repeat cytology
• Sensitivity of HC2 was 14 higher than repeat
  cytology with essentially equal specificity

24
Consensus Guidelines for the use of HPV in
Cervical Specimens

• ASCCP 2012 updated consensus guidelines for the
  management of abnormal cervical cancer screening
  tests and cancer precursors
• Representatives from 23 professional societies
  including ACS, AAFP, ACOG, ASCCP, CDC and CAP
• J Lower Genital Tract Dis. 2013 17(4)S1-S27.
• CETC statement on HPV test utilization
• Endorsed by ACS, ASCP, ASCCP, ASC, CAP, IAC and
  PSC
• Diagn Cytopathol. 2009 Jul37(7)542-3 as well as
  Arch Pathol Lab Med and Am J Clin Pathol

25
Consensus Guidelines for the use of HPV in
Cervical Specimens

• ASC/ASCCP/ASCP 2012 Screening Guidelines for
  Cervical Cancer
• Representatives from 25 professional
  organizations
• Am J Clin Pathol. 2012137516-542
• Systematic evidence based review and consensus
  symposium
• Screening recommendations address age appropriate
  screening strategies and use of HPV testing
• Age 30 HPV testing should not be used as part
  of the primary screen.
• Age 30-65 HPV and cytology co-testing is the
  preferred screening test.
• Age 65 No screening following adequate
  negative prior screening

26
2012 ASCCP Guidelines

• Testing for low-risk HPV types has NO role in
  routine cervical cancer screening or for the
  evaluation of women with abnormal cervical
  cytology.

27
Role of HPV Testing in ASC-HLiman et al Cancer
Cytopathol 105 (2005) 457-460

• 48 samples of ASC-H with HPV and histo/cyto
  follow-up (2001-2003)
• All proven HSIL (22 cases) were HR HPV
• 80 of LSIL (5 total) were HR HPV
• 50 of patients with negative follow-up were HPV

28
HPV Testing for Screening
Questions to address

• Who gets screened
• Testing method and with or without Pap
• Screening frequency
• Management of screen positives and negatives
• These questions were addressed by the
  ASC/ASCCP/ASCP 2012 Screening Guidelines for
  Cervical Cancer

29
HPV Testing for Screening
Summary of clinical data

• Consistently more sensitive than Pap
• Specificity is probably less than Pap
• Combination of Pap and HPV increases sensitivity,
  but reduces specificity - very high negative
  predictive value

30
HPV Testing for Screening
FDA approval HPV-Pap Co-test

• The various FDA approved HPV tests are also
  approved as an adjunct to cervical cytology
  screening in women 30 years and older.

31
HPV Testing for Screening
Key advantage of using

• Negativity for high-risk HPV identifies which
  women are at very low risk for having or
  developing CIN 2,3 over next 5 yrs
• Allows targeted screening

32
HPV Testing for Screening
Management of HPV () / Pap (-)

• Risk for having CIN 2,3 is about 5 in
  well-screened population
• About half have transient infections and become
  HPV negative by 6 mos
• Almost all CIN 2,3 occurs in women with
  persistent HPV

33
Negative Cytology - HPV DNA Positive
HPV 16/18
() - Colposcopy
(-)
Repeat co-test in 12 months
High-risk HPV ()
Both Negative
Pap gt ASC-US
J Lower Genital Tract Dis. 2013, 17(4)S1-S27.
34
HPV positive rates and ASC-US

• Most recent HPV Q-Probe data conclude that an
  HR-HPV positive rate of 43.7 is an appropriate
  quality metric
• Q-Probes 2005 HPV Testing (QP053). Northfield,
  Ill College of American Pathologists 2005.
• In other studies the median rate is reported as
  34.1 to 50.6, depending on the age of the
  population studied.

35
HPV Triage CAP Survey 2006Arch Pathol Lab Med
2008 132 1290-1294

• 679 labs responded
• 73 send to reference lab, 9 perform in house
• 45 offer low risk HPV testing
• Digene HC is most commonly used test
• Median test volume 55/month
• 24.5 doing primary screening in women gt30yr
• Median rates for positivity ASCUS 36.6, ASC-H
  50, 4 for HPV screening in conjunction with Pap

36
ASC-US Cases versus HPV

• HPV () labs labs
• 2003 2006
• lt10 18.2 8.1 10-24 12.9 11.3
• 25-40 28.3 38.7 41-60
  32.0 36.9 gt60 8.6 5.0
• Median rate 2006 36.6
• From the 2003 and 2006 College of American
  Pathologists PAP questionnaire Arch Pathol Lab
  Med. 2008 Aug132(8)1290-4.

37
HPV percentiles for ASC-US, ASC-H and HPV30-NIL

• percentile ASC-US ASC-H NIL
• Labs 157 73 29
• 5th 4.0 0 0
• 10th 15.2 0 0
• 25th 26.0 1.8 1.9
• 50th 36.6 50.0 4.0
• 75th 47.8 71.0 11.0
• 90th 53.2 89.0 24.5
• 95th 62.2 98.7 25.9
• From the 2006 College of American Pathologists
  PAP questionnaire Arch Pathol Lab Med. 2008
  Aug132(8)1290-4.

38
Gynecologic Cytopathology Quality Consensus
Conference (GCQC2) 2011

• College of American Pathologists laboratory-based
  survey funded by the Centers for Disease Control
  and Prevention
• Paper-based survey
• Follow-up Web-based survey
• National Consensus Conference

39
GCQC2 2011

• Goal of Survey and Results To identify what
  metrics are collected, how metrics are analyzed
  and what benchmarks are used to determine
  variance in performance and what actions are
  taken to address performance issues.

40
GCQC2 Working Group 5Monitoring of HPV Rates

• Christine Booth MD, FCAP, Chair
• Michael Henry MD, FCAP, Senior Author
• Carol Filomena MD, FCAP
• Marilee Means, PhD, SCT(ASCP)
• Patricia Wasserman MD, FCAP
• Christine Bashleben, CAP staff

41
CAP Survey

• 1245 Laboratories received survey
• 546 laboratory responses (525 regarding HPV
  practices) to paper survey
• 51 additional questions posed from working group
• Up to 34 responses received
• Voting at the GCQC2 in June, 2011

42
Original Survey Results HPV testing practices

• HPV results obtained at the time of the Pap test
  are routinely incorporated into the Pap report
  (n525)
• Yes 61.9
• No 38.1

43
Original Survey Results HPV testing practices

• How are HR-HPV tests for ASC-US ordered? (N518)
  
• Ordered as a "reflex test" by providers
• Ordered reflexively by the laboratory independent
  of the primary provider initial order
• Offered for reflex testing for women under 21
  years of age

87.6 23.4 7.9
44
Original Survey Results HPV testing practices

• Laboratory limits ASC-US reflex testing to women
  over the age of 20 (n512)
• Yes 15.6
• No 84.4

If no, why? (from online survey) Clinician
driven Patient demands Resolve diagnostic
dilemmas Ordered out of habit
45
Online Question Results

• 71 of respondents will perform reflex HR-HPV in
  ASC-US in women under 20 at clinicians
  insistence
• 51 of respondents will call clinician to educate
  about published guidelines before performing
  reflex HR-HPV in ASC-US in women under 20
• 6 of respondents will not perform reflex HR-HPV
  in ASC-US in women under 20 despite clinicians
  insistence
• Some respondents will perform reflex HR-HPV
  testing in women under 20 at clinicians
  insistence and will include a comment in the
  report indicating that modern guidelines do not
  recommend HPV tests for women equal and less than
  20 years of age and if obtained, the results
  should be ignored for management.
• Some respondents require that clinicians who want
  reflex HR-HPV testing in women under 20 submit a
  separately collected test directly to virology

46
Original Survey Results HPV testing practices

• Which HR-HPV tests are reflexively offered from a
  cytology specimen? (n520 multiple responses
  allowed)
• ASC-US reflex 90.6
• ASC-H reflex 47.1
• AGC or other glandular abnormalities 28.3
• Pap test with any squamous epithelial abnormality
  23.5
• LSIL with a Pap test regardless of age 20.8
• LSIL reflex in postmenopausal women 14.8

47
Online Question Results

• Does your lab offer HPV reflex testing on
• ASC-H?
• 50 offer reflex HR-HPV testing for ASC-H results
• 50 do not offer HR-HPV testing for ASC-H results
• Some respondents state that knowledge of HPV in
  ASC-H is beneficial in the following cases
• No colposcopic follow up if HPV is negative
• If HPV negative, patient returns to routine
  testing
• Useful in resolving diagnostic dilemmas with
  confidence
• Useful in older women with negative history
• Useful in pregnant women
• Helps PCP decide to go to LEEP or not

48
Original Survey Results HPV testing practices

• If an HR-HPV test is not ordered reflexively on a
  Pap test by the submitting clinician, does the
  pathologist have the discretion to order an
  HR-HPV test?
• Yes 201 38.7
• No 319 61.3

49
Original Survey Results HPV testing practices

• Laboratory finds it useful to order HR-HPV
  testing independently of the clinician to resolve
  diagnostic discrepancies between the
  cytotechnologist and pathologist in Pap test
  diagnosed as the following (n129 multiple
  responses allowed)
• ASC-US 97 75.2
• ASC-H 76 58.9
• AGC 40 31.0
• HSIL 25 19.4
• LSIL 24 18.6
• SCC 10 7.8
• ADC 7 5.4

50
Online Question Results

• If you do use HR-HPV results to resolve
  diagnostic discrepancies, please explain how
• To fine tune ASC-US criteria
• To aid in the CT-P disagreements for ASC-US
  cases
• In borderline Pap interpretations
• HPV negative cases results in re-evaluations
• HPV positive cases results in re-evaluations
• To resolve diagnostic dilemmas at any age

51
Survey results Lab Volume Analysis for HPV
Testing Practices

1. Smaller labs are more likely to reflexively order
   HPV testing independent of the initial order
   (P0.001)
2. Larger labs are more likely to reflexively order
   HPV testing with any squamous abnormality
   (P0.001)

52
Original Survey Results HPV testing practices

• Laboratory offers low-risk HPV testing (n520)
• Never 45.6
• Only on request 39.2
• Routinely bundled with HR-HPV 11.9
• Other 3.3

53
Use of HPV Test Results for Quality Assessment
HR-HPV rates are monitored to determine potential trends in accuracy of diagnoses HR-HPV rates are monitored to determine potential trends in accuracy of diagnoses
ASC-US reflex HR-HPV results ASC-US reflex HR-HPV results
Yes 221 (53.9)
No 189 (46.1)
HPV DNA Results HPV DNA Results
Yes 119 (32.7)
No 245 (67.3)
54
HPV Monitoring by Pap Test Result and Laboratory
or Individual
Monitoring of HPV rates (n392) Monitoring of HPV rates (n392) Monitoring of HPV rates (n392) Monitoring of HPV rates (n392)
Pap test Result Laboratory Cytotechnologist Pathologist
ASC-US 53.3 13.8 21.4
NILM 21.4 5.9 5.9
LSIL 17.9 4.3 5.9
HSIL 14.8 3.3 4.6
Multiple responses allowed
55
Original Survey Results HPV for Quality
Assessment

• HPV results are compared to ASC-US/SIL ratios for
  pathologists to determine potential trends in
  over or under diagnosis? (n513)
• Yes 28.8
• No 71.2

• If no, why not? (from online survey)
• Too complex to get data
• Pap and HPV results in separate systems
• Does not affect pathologist sign-out tendencies

56
CAP Checklist CYP.07653

• HR-HPV Records
• If available, records are maintained for
  high-risk human papillomavirus (HR-HPV) tests
  performed on ASC-US including
• Total number of HR-HPV tests performed on ASC-US
  cases
• Total number of POSITIVE HR-HPV ASC-US cases
• NOTE The percentage of ASC-US cases with a
  positive HR-HPV result may be a helpful quality
  metric for both overall laboratory performance
  and individual performance of pathologists,
  especially when combined with an individual's
  ASC-SIL ratio. Data for other HR-HPV testing
  results (e.g. co-testing with a Pap test in women
  gt 30 years of age) may also be helpful quality
  metrics but should be kept separately.

57
Consensus Good Laboratory Practice Statements

• Laboratories should only offer HR-HPV testing for
  gynecologic cytology specimens.
• Not appropriate to offer low-risk HPV testing for
  any clinical circumstance.
• 81 agreement

58
Consensus Good Laboratory Practice Statements

• Laboratories should encourage clinicians to
  consider the latest consensus guidelines in
  ordering HR-HPV tests on gynecologic specimens.

59
Consensus Good Laboratory Practice Statements

• Laboratories should be cautious in using HPV test
  results to change or influence cytologic
  interpretations.
• 83.9 of conference participants voted that
  HR-HPV test results should not be used to either
  downgrade or upgrade Pap test interpretations.

60
Consensus Good Laboratory Practice Statements

• While there is significant variability in
  interinstitutional HPV-positive rates in ASC-US
  Pap tests, monitoring the HPV-positive rate in
  ASC-US Pap tests is a valuable broad measure of
  quality.
• Performance beyond 2 SDs of the mean should
  prompt reassessment of diagnostic criteria used
  in the evaluation of Pap tests and/or
  investigation of the prevalence of HPV positivity
  in the population from which the Pap tests are
  obtained.

Tworek et al, Arch Pathol Lab Med.
200713115251531
61
Consensus Good Laboratory Practice Statements

• Monitoring the HPV-positive rate in other
  diagnostic categories such as LSIL and the
  comparison of these HR-HPV rates to published
  benchmarks is also a valuable broad measure of
  quality for a laboratory and possibly for
  individuals.

62
Consensus Good Laboratory Practice Statement

• When possible, individual ASC-US/HR-HPV results
  should be compared to ASC-US/SIL ratios for
  pathologists to determine potential trends in
  over- and under-diagnosis.
• No consensus agreement
• If laboratory is able to extract information from
  LIS, still a beneficial practice

63
Indicator Indicator Explanation Explanation Explanation
ASC/SIL HPV NIL ASC-US SIL
Normal


Normal
Normal
Normal A B C


Cibas et al. Am J Clin Pathol 200812997-101
64
(No Transcript)
65
Additional Statements

• Laboratories should routinely document all
  available HPV test results performed over the
  last five years preceding histopathologic
  diagnoses of cervical carcinoma including
  laboratory site and date where each HPV test was
  performed.
• 70 agreement

66
Additional Statements

• Laboratories should routinely document all
  available HPV test results performed over the
  last five years preceding histopathologic
  diagnosis of cervical carcinoma including both
  specific HPV test and platform, and FDA-approved
  versus laboratory-developed test.
• 81 agreement

67
Additional Statements

• Is it appropriate for a lab to order a HR-HPV
  test as a diagnostic test independent of the
  clinician?
• 84 conference participants responded no

68
Questions?