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Screening & Early Detection of Cervical Precancers: A Clinical Role for HPV DNA Testing Dinesh Gupta,  PhD  Director, NCRL, India Executive Member, IACR Life Member, AOGIN “ We are what we repeatedly do. EXCELLENCE, then, is a HABIT.”
Natural History of Cervical Carcinogenesis CIN 1 Normal CIN 2 CIN 3 CIS/ Ca LSIL HSIL 40% 20% Spontaneous Regression Rate ASC-US (ASC-H) Moderate Severe Mild dysplasia 66% Co-factors affecting natural history ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
NATURAL HISTORY OF CERVICAL CANCER NORMAL EPITHELIUM HPV-INFECTION PERSISTENT HPV-INFECTION CIN II/III CANCER Sexual exposure! ~80% ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],10-25% 10-30% Integration of viral DNA Genetic predisposition? 5-10% 8-10 yrs CIN I  60-70% Ref: Dr Albert Singer’s presentation at Asian HPV Summit, Blue Mountain 2003 >90%
HPV-mediated progression to cervical cancer
NATURAL HISTORY OF SQUAMOUS INTRAEPITHELIAL LESIONS Oscar, 1992 > 12% 5% 1% PROGRESS Ca Cx 56% 35% 32% PERSIST - 22% 11% PROGRESS CIN 3 32% CIN 3 (SEVEVER DYSPIASIA) 43% CIN 2 (MODERATE DYSPIASIA) 57% CIN 1  (MILD DYSPIASIA) REGRESS Clinical LESION
Ca Cx Screening: Considerations ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Screening by cervical cytology: review ,[object Object],[object Object],[object Object]
Squamous cell maturation
Changes in squamous epithelium Normal Cytology HSIL
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Quality of smear & reporting choose one or more descriptor from each category, and describe
Quality of smear & reporting considerations ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
End Result… ,[object Object],[object Object],[object Object],[object Object]
Gynaecological Cytopathology advanced… Liquid Based Cytology (LBC) ThinPrep 2000 Processor 1 st  to US FDA Approval LiquiSmear Processor
Controlled Membrane Transfer TM  technology controls dispersion, collection and transfer of diagnostic cells from the sample to the slide  ,[object Object],[object Object],[object Object],real-time process control
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Controlled Membrane Transfer Technology New Quantitative Stain
Based on a cohort analysis of 5,671 women above age 30 within a study that included 7932 women ages 15 to 76 (median 34) RE: C Clavel, 2003 LBC: Sensitivity improved … Monolayer cells on 19mm dia circle
Key Elements/ Features: A- Superficial Sq Cells B- Intermediate Cells C- Parabasal Cells D- Metaplastic (Basal) cells
Colposcopy Technique
Usually -directed biopsy ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
5 Considerations ,[object Object],[object Object],[object Object],[object Object],[object Object],Documentation is crucial
The Transformation Zone
Remember… ,[object Object],[object Object],[object Object],[object Object],[object Object]
The HPV TEST   Because of the strong association (>99.7%) between HPV and cervical cancer, this test helps  DETERMINE THE NEXT STEP  for a woman with lower genital abnormality among symptomatic, or identifies latent disease among healthy. This test checks for HPV genetic material, or DNA, within the cervical cells.  A swab of cervical cells is taken and the specimen is sent to a laboratory and examined for HPV . If the HPV test is positive for a high-risk strains, the abnormalities are more likely precancerous. If the test is negative, the cervical changes are more likely benign or regress or maintain.
digene Hybrid Capture 2 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Probe High Risk:  13 HR HPV types  16, 18, 31, 33, 35, 39, 45, 51, 52, 56,  58, 59, and 68 Probe Low Risk  LR HPV types 6, 11, 41, 42, 43
[object Object],[object Object],[object Object],[object Object],Genomic Configuration of HPV URR L1 L2 E1 E2 E3 E4 E5 E6 E7 High-Risk Low-Risk pRb  p53  Epithelial cell DNA  Blocked expression  HPV DNA  Regulatory gene   Transcription into host genome   Transforming into host genome
Specimen Collection  Using the DNA Collection Device 1 2 3 4
digene Hybrid Capture 2  ® Denature  Physiologic specimen Hybridize with  full-length RNA  probe Capture  and detect  hybrids Sensitive  Luminescence
Single Sample Collection Device ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],(STM) ,[object Object],[object Object],[object Object]
Hybrid Capture 2  (US FDA Approved) Manual Version 2 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Moving forward
Sensitivity ,[object Object],[object Object],Sensitivity for High Grade Cervical Disease The digene HPV DNA TM Test vs. Cytology Alone Sensitivity (%) 40 50 60 70 80 90 100 Cytology The digene HPV test TM Median = 73.0 Median = 98.5
[object Object],[object Object],[object Object],[object Object],WHY only - hc2
BROAD  INTERPRETATION OF –ve HPV DNA  TEST  RESULT -regardless age  ,[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],BROAD  INTERPRETATION OF +ve HPV DNA  TEST  RESULT
Viral load cut-off by HC2 is clearly indicative of higher disease stage (likely to be  not missed  at Colposcopy) J Pathol  2003;  201 : 1–6. 5 August 2003  Very low levels of viral DNA may not be clinically meaningful! Clinical sensitivity of hc2 hpv Viral load threshold for CIN development Viral load threshold for no clinical infection
Adapted from Snijders et al. Journal of Pathology 2003; 201:1-6 Analytical versus Clinical Sensitivity The Analytical Sensitivity of PCR methods can be <10 copies of HPV DNA The Analytical Sensitivity of HC2 is 5,000 copies of HPV DNA
“ HC2 was more sensitive…than PCR for detecting 2-year cumulative CIN3 or cancer”* * Schiffman et al, Am J Clin Path 2005; 124: 722-732 Lorincz, HPV Testing by Hybrid Capture. Monsonego J(ed): Emerging Issues of  HPV Infections: From Science to Practice. Basel, Karger, 2006: 54-62 Data from 16 published studies showed a median Clinical Sensitivity of 82% for PCR Analytical versus Clinical Sensitivity
HPV Genome: locations of general primer sets AMPLIMER SIZE Claim:  Utilises sequence variation in the 22-bp interprimer region permitting identification of HPV genotypes. (also claim it does not permit formal classification of HPV genotypes.   Ref: Bernhardt Kleter et al, J Clin Microbiol Aug. 1999, 2508-17 ) 65 bp E6 E7 L1 L2 E4 E5 E2 E1 1 7900 4000 5000 6000 7000 1000 2000 3000 MY 9/11 GP 5+/6+ SPF 1/2 150 bp 450 bp PRIMER SETS
Clinical Efficacy of Screening Options for Cervical Precursors Generally accepted detection range of various diagnostic tests normal cervix HPV  infection HPV persistence high grade  neoplasia invasion low grade  neoplasia Pap Smear (cytology) HC2 HPV DNA Testing VIA/ VILI Histopathology Expert Colposcopy
Emerging paradigm: Ca Cx Screening
“ There is  sufficient evidence  that testing for human papillomavirus infection as the primary screening modality can reduce cervical cancer incidence and mortality rates.” – IARC/WHO, 2005 IARC, WHO. IARC Handbooks of Cancer Prevention: Cervical Cancer Screening. Volume 10. IARC Press; 2005.
digene hc2 HPV: Long-term Predictive Value for HSIL Cervical Cancer incidence rates among screen negative women by study group  Osmanabad RCT of Cervical Screening: India Sankarnarayanan et al, NEJM 2009; 360 (14)  HPV Screening for Cervical Cancer in Rural India ,[object Object],Cancer Deaths No. of women Study Group Arms (2000-2007) ASR HPV (hc2) 34 24,395 12.7 Cytology (Pap) 54 23,797 21.5 VIA 56 23,041 20.9 ASR: Age Standardised Rate Incidence Rate/ 100,000 Control  64 23,041 25.8 0.52 (0.33–0.83) 0.89 (0.62–1.27) 0.86 (0.60–1.25) Hazard Ratio At 95% CI 1.00
Long Term Predictive Values Cytology and HPV: Joint European Cohort Study (7 cohort studies) ,[object Object],hc2 HPV: Long-term Predictive Value for HSIL 381 4,778 7,443 Total 232 40 67 837 Cyto+/ HPV+ 10 46 89 436 Cyto+ / HPV- 107 111 268 1,962 Cyto-/HPV+ 32 4,571 7,019 21,060 Cyto- / HPV-   After 72 months After 60 months     # with CIN3+ # still in follow-up # baseline  No. of Women Baseline group No. of women
OUR LABORATORY MEDICINE SUPPORT EXPERIENCE:  National Clinical Reference Laboratory, New Delhi, India  SYMPTOMATIC PATIENTS:  ANNUAL HPV TESTING V/S %POSITIVITY (2003-2008) ,[object Object],[object Object],[object Object],56 105 93 106 162 341 0 500 1000 1500 2000 2500 2003 2004 2005 2006 2007 2008 Year  # HPV Tests/  Total +ve 0 2 4 6 8 10 12 14 16 18 20 % Positivity Total HPV +ve HPV % vity
5-YR AGE-INCREMENT TREND FOR HPV PREVALENCE & POSITIVITY. PERIOD: 2003-2008 (60-MONTH-DATA) ,[object Object],[object Object],OUR LABORATORY MEDICINE SUPPORT EXPERIENCE National Clinical Reference Laboratory, New Delhi, India
Does Pretreatment Human Papillomavirus (HPV) Titers Predict Radiation Response and Survival Outcomes in Cancer Cervix? – A pilot study.  Gynaecological Oncology: 2006 Niloy R. Datta, MD, DNB  , Piyush Kumar, MD  , Shalini Singh, MD, DNB  ,  Dinesh Gupta, PhD # , Anurita Srivastava, MD * , Tapan N. Dhole, MD,  Departments of Radiotherapy   and Microbiology+, Regional Cancer Centre, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Rae Barelli Road, Lucknow, India and Gentech Laboratories, New Delhi   HPV in Ca Cx Treatment
 
 
Benefits of HPV as a Primary Screen ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Can HPV complement colposcopy? ,[object Object],[object Object],[object Object],[object Object],[object Object]
Who are at risk? ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Why should we take HPV test? ,[object Object],[object Object],[object Object],[object Object],[object Object]
Does HPV mean cancer? ,[object Object],[object Object],[object Object],[object Object]
Lynnette Denny  AORTIC May 2006 “ secondary prevention (cytology, colposcopy, biopsy, histology), has not been successful in any developing countries in the world due to the complexity of the infrastructure required to make this approach work and the substantial human, financial and logistical resources required. Consequently cervical cancer prevention efforts in developing countries either do not exist or they are extremely limited.
Begin a Cervical Cancer Screening Program ,[object Object],[object Object],[object Object],[object Object],[object Object]
“ Dhanyawad ” Thank U! Delhi Moms  are going  HPV-way as  much as  make overs... India Times for a big change Testing for HPV DNA once in a life time reduces the risk of CERVICAL CANCER  almost 100%
What is the incidence of High-Grade CIN in women with abnormal smears? 90% Refer to HPV or Colpo and directed Biopsy CIN 3 (Severe dysplasia) 70% hc2 HPV +ve Refer to Colpo CIN 1/ CIN 2 (Moderate dysplasia) 30% (ASC-H) hc2 HPV / Repeat smear 6 mo/ refer to Colpo when either is positive ASC-US (Mild Dysplasia) Incidence of  HG CIN (CIN2/3) Action?
[object Object],[object Object],[object Object],[object Object],[object Object]
Table 2. Staging System for Cervical Cancer

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Basics To Ca Cx Screening (Eastern Biotech)

  • 1. Screening & Early Detection of Cervical Precancers: A Clinical Role for HPV DNA Testing Dinesh Gupta, PhD Director, NCRL, India Executive Member, IACR Life Member, AOGIN “ We are what we repeatedly do. EXCELLENCE, then, is a HABIT.”
  • 2.
  • 3.
  • 4. HPV-mediated progression to cervical cancer
  • 5. NATURAL HISTORY OF SQUAMOUS INTRAEPITHELIAL LESIONS Oscar, 1992 > 12% 5% 1% PROGRESS Ca Cx 56% 35% 32% PERSIST - 22% 11% PROGRESS CIN 3 32% CIN 3 (SEVEVER DYSPIASIA) 43% CIN 2 (MODERATE DYSPIASIA) 57% CIN 1 (MILD DYSPIASIA) REGRESS Clinical LESION
  • 6.
  • 7.
  • 9. Changes in squamous epithelium Normal Cytology HSIL
  • 10.
  • 11.
  • 12.
  • 13. Gynaecological Cytopathology advanced… Liquid Based Cytology (LBC) ThinPrep 2000 Processor 1 st to US FDA Approval LiquiSmear Processor
  • 14.
  • 15.
  • 16. Based on a cohort analysis of 5,671 women above age 30 within a study that included 7932 women ages 15 to 76 (median 34) RE: C Clavel, 2003 LBC: Sensitivity improved … Monolayer cells on 19mm dia circle
  • 17. Key Elements/ Features: A- Superficial Sq Cells B- Intermediate Cells C- Parabasal Cells D- Metaplastic (Basal) cells
  • 19.
  • 20.
  • 22.
  • 23. The HPV TEST Because of the strong association (>99.7%) between HPV and cervical cancer, this test helps DETERMINE THE NEXT STEP for a woman with lower genital abnormality among symptomatic, or identifies latent disease among healthy. This test checks for HPV genetic material, or DNA, within the cervical cells. A swab of cervical cells is taken and the specimen is sent to a laboratory and examined for HPV . If the HPV test is positive for a high-risk strains, the abnormalities are more likely precancerous. If the test is negative, the cervical changes are more likely benign or regress or maintain.
  • 24.
  • 25.
  • 26. Specimen Collection Using the DNA Collection Device 1 2 3 4
  • 27. digene Hybrid Capture 2 ® Denature Physiologic specimen Hybridize with full-length RNA probe Capture and detect hybrids Sensitive Luminescence
  • 28.
  • 29.
  • 30.
  • 31.
  • 32.
  • 33.
  • 34. Viral load cut-off by HC2 is clearly indicative of higher disease stage (likely to be not missed at Colposcopy) J Pathol 2003; 201 : 1–6. 5 August 2003 Very low levels of viral DNA may not be clinically meaningful! Clinical sensitivity of hc2 hpv Viral load threshold for CIN development Viral load threshold for no clinical infection
  • 35. Adapted from Snijders et al. Journal of Pathology 2003; 201:1-6 Analytical versus Clinical Sensitivity The Analytical Sensitivity of PCR methods can be <10 copies of HPV DNA The Analytical Sensitivity of HC2 is 5,000 copies of HPV DNA
  • 36. “ HC2 was more sensitive…than PCR for detecting 2-year cumulative CIN3 or cancer”* * Schiffman et al, Am J Clin Path 2005; 124: 722-732 Lorincz, HPV Testing by Hybrid Capture. Monsonego J(ed): Emerging Issues of HPV Infections: From Science to Practice. Basel, Karger, 2006: 54-62 Data from 16 published studies showed a median Clinical Sensitivity of 82% for PCR Analytical versus Clinical Sensitivity
  • 37. HPV Genome: locations of general primer sets AMPLIMER SIZE Claim: Utilises sequence variation in the 22-bp interprimer region permitting identification of HPV genotypes. (also claim it does not permit formal classification of HPV genotypes. Ref: Bernhardt Kleter et al, J Clin Microbiol Aug. 1999, 2508-17 ) 65 bp E6 E7 L1 L2 E4 E5 E2 E1 1 7900 4000 5000 6000 7000 1000 2000 3000 MY 9/11 GP 5+/6+ SPF 1/2 150 bp 450 bp PRIMER SETS
  • 38. Clinical Efficacy of Screening Options for Cervical Precursors Generally accepted detection range of various diagnostic tests normal cervix HPV infection HPV persistence high grade neoplasia invasion low grade neoplasia Pap Smear (cytology) HC2 HPV DNA Testing VIA/ VILI Histopathology Expert Colposcopy
  • 39. Emerging paradigm: Ca Cx Screening
  • 40. “ There is sufficient evidence that testing for human papillomavirus infection as the primary screening modality can reduce cervical cancer incidence and mortality rates.” – IARC/WHO, 2005 IARC, WHO. IARC Handbooks of Cancer Prevention: Cervical Cancer Screening. Volume 10. IARC Press; 2005.
  • 41.
  • 42.
  • 43.
  • 44.
  • 45. Does Pretreatment Human Papillomavirus (HPV) Titers Predict Radiation Response and Survival Outcomes in Cancer Cervix? – A pilot study. Gynaecological Oncology: 2006 Niloy R. Datta, MD, DNB  , Piyush Kumar, MD  , Shalini Singh, MD, DNB  , Dinesh Gupta, PhD # , Anurita Srivastava, MD * , Tapan N. Dhole, MD, Departments of Radiotherapy  and Microbiology+, Regional Cancer Centre, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Rae Barelli Road, Lucknow, India and Gentech Laboratories, New Delhi HPV in Ca Cx Treatment
  • 46.  
  • 47.  
  • 48.
  • 49.
  • 50.
  • 51.
  • 52.
  • 53. Lynnette Denny AORTIC May 2006 “ secondary prevention (cytology, colposcopy, biopsy, histology), has not been successful in any developing countries in the world due to the complexity of the infrastructure required to make this approach work and the substantial human, financial and logistical resources required. Consequently cervical cancer prevention efforts in developing countries either do not exist or they are extremely limited.
  • 54.
  • 55. “ Dhanyawad ” Thank U! Delhi Moms are going HPV-way as much as make overs... India Times for a big change Testing for HPV DNA once in a life time reduces the risk of CERVICAL CANCER almost 100%
  • 56. What is the incidence of High-Grade CIN in women with abnormal smears? 90% Refer to HPV or Colpo and directed Biopsy CIN 3 (Severe dysplasia) 70% hc2 HPV +ve Refer to Colpo CIN 1/ CIN 2 (Moderate dysplasia) 30% (ASC-H) hc2 HPV / Repeat smear 6 mo/ refer to Colpo when either is positive ASC-US (Mild Dysplasia) Incidence of HG CIN (CIN2/3) Action?
  • 57.
  • 58. Table 2. Staging System for Cervical Cancer

Hinweis der Redaktion

  1. ## * * 07/16/96 Preparation: Remove excess mucus from the cervical os and surrounding ectocervix using a cotton or Dacron® swab. Discard the swab. Step 1. Insert brush 1-1.5 centimeters into the cervical os until the largest outer bristles of the brush touch the ectocervix. Rotate three (3) full turns in a counterclockwise direction. Do not insert brush completely into cervical canal. Step 2. Remove brush from the canal. Avoid touching the bristles to the outside of the tube or to any other object. (Not pictured.) Step 3. Insert brush to bottom of transport tube. Then, snap off sampler shaft at score line, leaving brush-end inside tube. Step 4. Re-cap tube securely by snapping it in place. For storage and/or shipping instructions, please see the package insert.