HPV
Human Papillomavirus
What is it and what are the tes6ng methodologies Presented by Jacqueline Brooks
10-11-2016
1
More than 30 to 40 Types are TransmiHed Sexually Over 120 HPV types have been identified and are referred to by number:
Disease
HPV type
Common Warts Plantar Warts Flat Warts Anogential Warts Anal Warts Genital Cancers
2,7 1,2,4,63 3,10,8 6,11,42,44 and others 6,16,18,31,53,58 High Risk: 16,18,31,45 Other high risk: 33,35,39,51,52,56,58,59 Probably High risk: 26,53,66,68,73,82 More than 15 types
Epidermodysplasia verruciformis Focal Epithelial Hyperplasia (oral) Oral papillomas Oropharyngeal cancer Verrucous cyst Laryngeal papillomatosis
10-11-2016
13,32 6,7,11,16,32 16 60 6,11
2
High Risk Types (16,18,31, and 45
10-11-2016
Cancer area
Average Annual Number of HPV Attributable HPV 16/18 Attributable cases (Estimated) (Estimated)
Cervix Vulva Vagina Penis Anus (women) Anus (men) Oropharynx (women) Oropharynx (men) Total (women) Total (men)
11,967 3,136 729 1,046 3,089 1,678 2,370
11,500 1,600 500 400 2,900 1,600 1,500
9,100 1,400 400 300 2,700 1,500 1,400
9,356
5,900
5,600
21,291 12,080
18,000 7,900
15,000 7,600
3
Cytologic Presenta6on
HPV
HPV
10-11-2016
HPV
Negative
4
HPV Life Cycle
The HPV viron infect epithelial 6ssues through micro-abrasions
10-11-2016
5
E1 and E2 build messenger RNA
10-11-2016
6
As the host epithelial cells divide and differen6ate, a transcrip6on cascade takes place
10-11-2016
7
E6 and E7 interfere with genes that regulate normal cell division
10-11-2016
8
HPV Virus
10-11-2016
9
P53 is responsible for repairing DNA damaged cells
10-11-2016
10
E6 binds with p53
10-11-2016
11
L1 and L2 genes help create viral capsid proteins
10-11-2016
12
The en6re HPV life cycle depends strictly on the process of epithelial cells differen6a6on
10-11-2016
13
Molecular Techniques Molecular Techniques Not Amplified 1. 2. 3. 4. 5. 6.
Nucleic Acid probe tests Southern blot hybridization Dot Blot hybridization In Situ Hybridization IHC Micro-arrays (“DNA chips”)
Amplified 1. Target Amplification (PCR) 2. Signal Amplification (branched-probe technology) a) Hybrid Capture (Digene’s HC II) b) Branched DNA Amplificatin c) Cleavage – Based Amplificaton d) Cycling Probe 3. Probe Amplification a) Ligase Chain Reaction b) Stand Displacement Amplification c) Beta Replicase
10-11-2016
14
Qiagen/Digene “HybridCapture”
Method
This nucleic acid hybridiza6on method u6lizes a DNA probe cocktail specific for intermediate/high risk serotypes (types 16,18,31,33,35,39,45,51,52,56,58,59,68). Results are reported as “not detected” or as “detected” for high-risk HPV serotype. The specific serotype(s) cannot be reported. Concordance with polymerase chain reac6on (PCR) results is es6mated to be 83%.
10-11-2016
15
Cervista The CervistaTM HPV HR test is an in vitro diagnos6c test for the qualita6ve detec6on of DNA from 14 high-risk Human Papillomavirus (HPV) types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) in cervical specimens. The CervistaTM HPV HR test cannot determine the specific HPV type present. 10-11-2016
16
Roche Cobas 4800 • The cobas HPV Test allows HPV 16 and 18 genotyping concurrently with high-risk HPV tes6ng. It
individually iden6fies genotypes 16 and 18, the two highest-risk HPV genotypes responsible for more than 70 percent of cervical cancer cases, while simultaneously detec6ng 12 other high risk HPV genotoypes. The approval was based on data from the ATHENA study involving more than 47,000 women in the US. Results demonstrated that 1 in 10 women, age 30 and older, who tested posi6ve for HPV 16 and/or 18 by the cobas HPV Test actually had cervical precancer even though they showed normal results with the Pap test.
10-11-2016
17
Panther • The Ap6ma HPV assay (Hologic Gen-Probe, San Diego, CA, USA) is an in vitro nucleic acid amplifica6on test designed to detect human papillomavirus (HPV) E6/E7 mRNA from 14 high-risk HPV types (i.e., types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) as a pool in cervical samples collected in preserva6ve medium. The Ap6ma HPV assay is based on target capture following cell lysis, with subsequent transcrip6on-mediated amplifica6on and probe hybridiza6on protec6on for the detec6on of E6/E7 mRNA expression in one measurement. The assay can be run on the fully automated Panther and TIGRIS direct tube sampling (DTS) systems (Hologic Gen-Probe, San Diego, CA) or the semiautomated DTS system. The Ap6ma HPV assay has been approved by the FDA for tes6ng of women ≥21 years of age whose Pap tests show atypical squamous cells of undetermined significance (ASC-US) and for screening of women ≥30 years of age as an adjunct to Pap tes6ng
10-11-2016
18
Panther
10-11-2016
19
IHC
10-11-2016
20
ISH
10-11-2016
21
HPV Virus
HPV H&E
10-11-2016
HPV pap stain
HPV H&E
22
HPV Tes6ng Methods • • • • •
Biopsy (H&E) Pap Smear ( Pap stain) Biopsy and Pap Smear (IHC Stain) Biopsy and Pap Smear (ISH Stain) Liquid based cell sample (DNA tes6ng)
10-11-2016
23
Producing a Good H&E • • • • • • • •
Cut at 4 microns Always use a clean water bath Do not dry slides more than 30 minutes Do not exceed 65 degrees C Use fresh xylene or rotate xylene Use fresh alcohol or rotate alcohols Use fresh stains Don’t forget to QC coverslipping
10-11-2016
24
Producing a Good IHC Stain • Follow guidelines for cukng, drying and deparaffiniza6on • If using automa6on follow manufacturers guidelines • Make sure equipment is clean and well maintained • QC all slides • Use a quality control 10-11-2016
25
Producing a good ISH • Keep everything as clean as possible • Remember you are more likely to contaminate than be contaminated • Follow manufactures guidelines • QC all slides • Competency tes6ng is a must
10-11-2016
26
Molecular tes6ng • • • • • •
Keep molecular lab CLEAN No cardboard in molecular room Change gloves onen, onen, onen Use hepa filtra6on system to clean air Use good pipekng techniques Follow manufacturer guidelines
10-11-2016
27
CPT Codes • 88305 Level IV Surgical Pathology, gross and microscopic exam • 88342 Immunohistochemistry, each an6body • 88365 In situ hybridiza6on (eg. FISH) each probe • 87621 Infec6ous agent detec6on, direct probe technique, HPV tes6ng • *Add 87621 X2 (Genotyping: 16/18 High Risk)
10-11-2016
28
HPV types • • • • • • •
Common warts, 2, 7 Plantar warts, 1,2,4,63 Anogenital warts, 6,11,42.44 Anal lesions, 6,16,18,31,53,58 Genital Cancers, Highest risk, 16,18,31,45 Genital Cancers, high risk, 33,35,39,51,52,56,58,59 Genital Cancers, probably high risk, 26,53,66,68,73,82
10-11-2016
29
Cervical Biopsy
10-11-2016
30
NSH Your request to have the workshop, HPV (Human Papillomavirus) for the year 2016 has been approved. NSH staff has added the scheduled session to the NSH Contact Hour Portal so that your attendees can self-report following the meeting. At the Conclusion of the Meeting The following items are to be shared with attendees and returned to NSH following the meeting. Missed deadlines may result in NSH revoking the approved contact hours for the session.
1. Session Evaluations: Evaluation Forms: Each attendee should complete an evaluation form. Forms should be collected at the conclusion of the session and attached to the corresponding Evaluation Summary. • Evaluation Summary Form: An evaluation summary form has been provided. It is the responsibility of the Meeting Coordinator to complete the summary calculations for each session and return it with the evaluation forms. 2. Email Text for Post Meeting (August Email Template): As attendees will be responsible for adding contact hours to their records attached is a sample email you can send explaining how it works. Please feel free to edit it as you deem appropriate. Sending this email is not required but recommended. All paperwork is due to NSH within 30 days of completion of the meeting. Contact hour certificates are immediately available once attendees enter their hours. If NSH does not receive the check in roster or session evaluations access to contact hour certificates will be denied. •
10-11-2016
31
NSH CEUs • Thank you for aHending HPV (Human Papillomavirus) . This email is a reminder that to receive a contact hour cer6ficate for the mee6ng you must report your aHendance to NSH through the NSH Contact Hour Portal - ce.nsh.org. •
Instruc6ons for adding your hours to your account are located below. If you have any ques6ons, please contact the NSH office, 443-535-4060.
•
Kind regards,
•
INSERT NAME
• 1.
Direc&ons for Submi0ng Your Hours to the NSH Contact Hour Portal Login to NSH Contact Hour Portal – ce.nsh.org (do not use www prior to the web address) a. First Time to the Site? – complete the “Not Yet Registered” form to create your contact hour portal user account. Once you have created a user account you will be asked to complete the user profile form (your name, address etc). To complete the profile and access hours from previous events you will need your NSH Customer ID number in the first step. b. Finding or Gekng an NSH Customer ID# - You can log into “My NSH” from nsh.org and find your Customer ID # on the profile page. You can access a short video that explains where to find the number. http://nsh.adobeconnect.com/p3i97kyn5ev/. If you are not a member of NSH or have not aHended an event or purchased a book from NSH in the past you may need to create an account through the “My NSH” portal on nsh.org to get your Customer ID#. Here is a short video to explain the process: http://nsh.adobeconnect.com/p1zo0yivh8k/ Once you are logged in select “Session Tracker” from the top naviga&on Add your Contact Hours for an event a. Step 1: Year: Select the year in which the event was held. b. Step 2: Event Title: All events approved in a specific year for contact hours by NSH are available to you to in this drop down list. Select the name of the event you aHended: Aurora Diagnos.cs Webinar Series. c. Step 3: Session Title: Select the workshop or webinar you aHended: HPV (Human Papillomavirus). The number of contact hours awarded for this session is preset and cannot be edited. d. Step 4: Add: Click on Add and your workshop or webinar will appear in your session log listed below. Print your contact hour cer&ficate a. Click on the box next to each session you would like to appear on your cer6ficate. b. Make sure you click all of the sessions from one event if you want them to appear on one cer6ficate. c. If you want all of your hours for a specific year you will need to click on all of the sessions from that year.
2. 3.
4.
10-11-2016
32
AAPC Let me know you are a coder and I will send you the AAPC cer6ficate which has the Index number you will use to claim your credit through AAPC.
10-11-2016
33
The End
10-11-2016
34
