American Society of Cytopathology Core Curriculum in Molecular Biology

Transcription

1 American Society of Cytopathology Core Curriculum in Molecular Biology

2 American Society of Cytopathology Core Curriculum in Molecular Biology Chapter 5 Applications of Molecular Testing Hybrid Capture II Triage Timothy St. John, CT(ASCP) Sandra Giroux, MS, SCT(ASCP)CFIAC Fletcher Allen Health Care Burlington, Vermont

3 Hybrid Capture II Triage Learning Objectives Describe the association of HPV and cervical cancer. List the common high risk subtypes of HPV. Understand the role of persistent HPV infection in the development of cervical cancer. Define when it is appropriate to utilize HPV testing. List the basic steps in the Digene HPV Hybrid Capture test. Define when it is not appropriate to test for HPV DNA.

4 Why Test for HPV? (Human Papilloma Virus) HPV causes cervical cancer o Most women infected with HPV will not develop cervical cancer, or have abnormal Paps o Most infections will be defeated by the host immune system o There is no way to predict which patients will progress Cervical cancer is a preventable disease

5 HPV Causes Cervical Cancer HPV is recognized as the primary causal factor in the development of cervical cancer 1, 2 o HPV DNA is found in 99.8% of cervical carcinomas 3 80% of these are HPV 16, 18, 31, 45 o HPV infection strongly predicts subsequent cervical squamous intraepithelial lesions (SIL) 4 1 National Institutes of Health (NIH). Consensus Development Conference Statement, World Health Organisation/EUROGIN Joint Experts Meeting on Cervical Cancer Screening, Bosch FX, European HPV Clinical Summit Meeting, January Liaw K-L, Glass AG, Manos MM, et al. Detection of Human Papillomavirus DNA in Cytologically Normal Women and Subsequent Cervical Squamous Intraepithelial Lesions. J Nat Cancer Inst 1999; 91:

6 Cervical Cancer is a Preventable Disease Cancer of the cervix: Has a known cause Can be prevented by careful screening and follow-up Can nearly always be cured if detected early 5 5 Pisani P, Parkin DM, Ferlay J. Estimates of the worldwide mortality from eighteen major cancers in Implications for prevention and projections of future burden. Int J Cancer 1993; 55:

7 Persistent HPV Infection o Persistent infection with high-risk human papilloma virus is necessary for the development and maintenance of CIN 3 6 o Women are at risk of progression only as long as the infection with high-risk human papilloma virus persists o Persistent HPV infection increases by over 250 times the relative risk of cervical cancer 4 6 Lenhart S. The U.S. national cervical cancer public education campaign. In: New Approaches to the Detection and Elimination of Cervical Cancer, a report of the European HPV Clinical Summit Meeting, Liaw K-L, Glass AG, Manos MM, et al. Detection of Human Papillomavirus DNA in Cytologically Normal Women and Subsequent Cervical Squamous Intraepithelial Lesions. J Nat Cancer Inst 1999; 91:

8 HPV Prevalence Falls but Cervical Cancer Incidence Rises with Age 7,8 o o o The prevalence of HPV DNA is relatively high in women < 25 years HPV prevalence is much lower among older women (>30 years) Women over 30 years with normal cytology but positive for high-risk HPV have a 116-fold risk for developing high-grade squamous intraepithelial lesions (HSIL) compared with similar women who are HPV negative 9 7 Nobbenhuis MAE, Walboomers JMM, Helmerhorst TJM et al. Relation of human papillomavirus status to cervical lesions and consequences for cervical cancer screening: a prospective study. The Lancet 1999; 354: NCI SEER DATA, Melkert PW, Hopman E, van den Brule AJ, et al. Prevalence of HPV in cytomorphologically normal cervical smears, as determined by the polymerase chain reaction, is aged-dependent. Int J Cancer 1993; 53:

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11 ASC-US/LSIL Triage Study (ALTS) Over 3400 women from 4 U.S. clinical centers, sponsored by National Cancer Institute 10 ALTS compared the sensitivity and specificity of three management strategies to detect CIN 3 in women referred with ASC- US (Atypical Squamous Cells of Undetermined Significance) or Low Grade Squamous Intraepithelial Lesion (LSIL) on their Pap test. 10 In certain settings, the study supported the use of Digene s Hybrid Capture II HPV DNA Test to detect significant cervical cancer precursor lesions Solomon D, et al. J Natl Cancer Inst. 2001;93:

12 ALTS Conclusions HPV testing is a viable option in the management of ASC-US patients o HPV testing of ASC-US results from the ThinPrep Pap Test vial is 96% sensitive10 o 99% Negative Predictive Value confirms absence of disease HPV testing improves patient management by directing at-risk patients to colposcopy 10 Solomon D, et al. J Natl Cancer Inst. 2001;93:

13 How Can You Test for HPV? Polymerase Chain Reaction (PCR) o time consuming, expensive, not FDA approved o can test for individual HPV subtypes In situ Hybridization (FISH) o time consuming, expensive, not FDA approved o can test for individual HPV subtypes Digene Hybrid Capture II o time consuming, less expensive, FDA approved o can not test for individual HPV subtypes Cervista o recently FDA approved, Invader Technology

14 Hybrid Capture II Test for HPV Consists of an RNA probe that detects 13 High Risk (oncogenic) HPV subtypes o 16,18,31,33,35,39,45,51,52,56,58,59,68 Cut off for assay is 5000 copies

15 Hybrid Capture II The Low Risk probe, which detects non-oncogenic HPV subtypes is no longer in widespread use, and is contraindicated for the purpose of patient management In essence, a positive for Low Risk HPV DNA in the absence of High Risk HPV DNA, is considered as a negative result

16 Specimen Collection Out of Vial Testing One pap sample can be collected and rinsed in the ThinPrep Pap Test vial and used for both the cytology Pap test and the HPV DNA Test when appropriate Clinicians can request automatic reflex testing for HPV DNA, if the Pap result is ASC-US

17 Patient Management and HPV Testing Women with an ASC-US test result are advised to have an HPV test. Those whose HPV result is negative can be returned to routine screening with a high degree of confidence. Their risk of cancer has been shown to be negligible. Only women with a positive HPV result (oncogenic subtypes) require immediate referral for colposcopy.

18 Hybrid Capture II FDA approved procedure to test for HPV. Reimbursed by insurance Current standard of care o That may change with the new FDA approved Cervista HPV DNA test (Hologic Corporation)

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20 Hybrid Capture II Step One Release nucleic acids Clinical specimens are combined with a base solution which disrupts the virus and releases target DNA. No special specimen preparation is necessary.

21 Hybrid Capture II Step One (cont.) DNA denaturation Pipette 4 ml specimen into tube Add specimen transport media (STM) Centrifuge down Pour off supernatant and blot

22 Blotting Remaining Liquid from Tube Resulting in a Cell Pellet

23 Denaturing reagent added with eppendorf pipettor (pipettes different volumes based on the tip syringe being used)

24 Denaturing Specimen and Reagent Mixture Vortexed

25 Denaturing specimen tubes placed in 65 degree water bath for 45 minutes

26 Hybrid Capture II Step Two Hybridize RNA Probe with Target DNA Target DNA combines with specific RNA probes creating RNA:DNA hybrids.

27 RNA Probe added to Denatured Specimen

28 Hybrid Capture II Step Three Capture Hybrids Multiple RNA:DNA hybrids are captured onto a solid phase coated with universal capture antibodies specific for RNA:DNA hybrids.

29 Capture Plate

30 Hybrid Capture II Step Four Label for Detection Captured RNA:DNA hybrids are detected with multiple antibodies conjugated to alkaline phosphatase. Resulting signal can be amplified to at least 3000-fold.

31 Hybrid Capture II Step Five Detect, Read and Interpret Results

32 Hybrid Capture II - Step 5 (cont.) The bound alkaline phosphatase is detected with a chemiluminescent dioxetane substrate. Upon cleavage by alkaline phosphatase, the substrate produces light that is measured on a luminometer in Relative Light Units (RLUs).

33 Luminometer

34 Print Out of Results

35 Remember.. High risk HPV does not mean that HSIL will be present on the pap Most women with high risk HPV have normal paps A negative HPV test is highly predictive that no squamous intraepithelial lesion or cancer exists

36 When Should You Test for HPV? Women >30 years in conjunction with a Pap test o If both are negative the screening interval can be increased up to 3 years. Initial triage for patients >21 years with a Pap result of ASC-US o If HPV is positive then proceed to Colposcopy o If HPV is negative then return to routine screening interval Am J Clin Pathol 2009;131:

37 When Should You Not Test for HPV? Initial triage for patients <21 years with ANY abnormal cytologic finding. Initial triage for the cytologic diagnosis of LSIL and above (ASC-H, HSIL, Atypical Glandular Cells, AIS) Testing for any non-oncogenic HPV types has NO role in cervical cancer screening o Am J Clin Pathol 2009;131: