HPV és cervix carcinoma kapcsolata

New Developments in Human Papillomavirus Testing Debrecen, October 15 2005 Dr Attila Lorincz Digene Hello. Good afternoon, everyone. In today’s presentation I am going to focus on the clinical value of HPV DNA testing for general population screening. My principal emphasis will be on the Hybrid Capture 2 test but I will also talk in more general terms about HPV testing by PCR and in situ hybridization and also on key aspects of HPV test validation. In the latter part of the presentation I will discuss the clinical data that support the use of HC2 as an adjunct to the Pap test and the endorsements of Hybrid Capture HPV testing from professional medical organizations in the US. Photos: Lagoon nebula; HPV virions (courtesy A.Bennett Jenson, MD)

STEROID HORMON FOKOZOTT HPV ÉS ONCOGEN TRANSCRIPTIO p53 GÉN PRODUKTUM DEGRADÁCIÓJA + A G1/S SEJTCIKLUS BLOKKOLÁSÁNAK HIBÁJA CERVIX CARCINOMA Moodly és mtsai. Int.J.Gyn.Ca. 2003,13, 103.

Kiterjedése és felszíni strukturáltsága változatos, és eltér a klasszikus, a preblastomatózisokat jelző koloszkópos .elváltozásoktól,amint azt a következő 2 kolposzkópos kép mutatja.Lehet a teljes transzformációs zónát körkörösen érintő, mint ezen a képen

A CARCINOGENESIS ÚTJA A HPV E 6 ONCOPROTEINJE KÖTŐDVE A p53 TUMOR SZUPRESSZOR GÉNHEZ, EGY PROTEAZE RENDSZER KÖZREMŰKÖDÉSÉVEL STIMULÁLJA ANNAK DEGRADÁCIÓJÁT A STEROID HORMONOK FOKOZZÁK AZ E6 ÉS E7 HPV ONCOGEN EXPRESSZIÓJÁT, ÍGY GÁTOLJÁK AZ APOPTOSIST. EREDMÉNY: CARCINOGENESIS

SMITH TANULMÁNY EREDMÉNYEI A cervix ca a fejllődő országokban a második leggyakoribb malignoma (a világon a harmadik) a HPV „high-risk” típusa a kialakulás fő elősegítője az oralis contraceptivumok hosszas adagolás esetén (5 év ) fokozzák a HPV pozitív nők cervix ca kockázatát - befolyásoló tényezők: partnerek száma, paritas, dohányzás, barrier módszer HPV + OC = kiemelt szűrő program (négyszeres kockázat) (Smith és munkatársai, Lancet 2003. 12531 nő adatai) (Miller és munkatársai cáfolják a tanulmány néhány megállapítását)

A HPV és a sejt A HPV-fertőzés kialakulása receptorral kötődik (hámfajlagos) a bazális sejtekbe jut, mert csak az osztódni képes sejtekben tud szaporodni szabad DNS (episzoma), 50-100 másolat, kifejeződés csekély Parabazális-intermedier sejtek – a korai gének kifejeződése Érett hámsejtek – késői gének kifejeződése, virionok építése

MORENO TANULMÁNY Cervix ca: 1853 kontroll: 1916 HPV poz.: 1676 HPV poz.: 255 HPV + OC ( > 5 év) 3-5-szörös kockázat (Lancet 2003. 359.1085)

HPV Fertőzéshez Társult Daganatok1 Lokalizáció Bizonyos HPV típusokkal társulás (%) Cervix* ≥95% Vagina* 50% Vulva* >50% Penis Anus >70% Oropharyngealis 20% Bőr squamosus sejtes 90%† Key Point High-risk HPV has been identified in several different types of malignancies. Background HPV has been studied most extensively in the context of cervical cancer, but it is associated with several other types of malignancies as well. High-risk HPV is associated with 50% of vaginal carcinomas, vaginal intraepithelial neoplasias, and penile carcinomas, >50% of vulvar carcinomas, and >70% of anal carcinomas.1 Viral DNA from high-risk HPV types has been found in 20% of tumors localized to the tongue and tonsils.1 Ninety percent of cutaneous squamous cell carcinomas from immunocompromised renal transplant recipients display high-risk HPV.1 Mortality due to these HPV-associated cancers is high, estimated at up to 15,800 in the United States alone in 2003.2 References 1. Gonzalez Intxaurraga MA, Stankovic R, Sorli R, Trevisan G. HPV and carcinogenesis. Acta Dermatovenerol. 2002;11:1–8. 2. American Cancer Society. Cancer Facts & Figures. 2003. Atlanta, Ga: American Cancer Society; 2003:1–48. *Includes cancer and intraepithelial neoplasia †Immunocompromised patients 1.Gonzalez Intxaurraga MA, Stankovic R, Sorli R, Trevisan G. Acta Dermatovenerol. 2002;11:1–8.

HUMAN PAPILLOMAVÍRUS >130 ismert típus2 30–40 anogenitalis2,3 DNS vírus1 >130 ismert típus2 30–40 anogenitalis2,3 15–20 oncogen*,2,3 tipus, pl. 16, 18, 31, 33, 35, 39, 45, 51, 52, 584 A HPV 16 (54%) és a HPV 18 (13%) felelős a méhnyakrákos esetek többségéért.5 Nem oncogen† típusok: 6, 11, 40, 42, 43, 44, 544 A HPV 6 és 11 a condyloma leggyakoribb kórokozója.3 Key Point There are many different types of HPV; of the 15–20 oncogenic types, HPV 16 and HPV 18 account for the majority of cervical cancers. Background Papillomaviruses such as HPV are nonenveloped, double-stranded DNA viruses.1 More than 100 HPV types have been detected,2 with >80 types sequenced and classified.3 Approximately 30–40 types of HPV are anogenital, of which 15–20 types are oncogenic.2,3 HPV Types 16 and 18 are oncogenic and account for about two thirds of all cervical cancers—the next 5 most prevalent types (31, 33, 45, 52, 58) account for only an additional 18% of cases.4 Other oncogenic HPV types include 35, 39, 51, and 56.5 HPV Types 6 and 11 are nononcogenic and are associated with external genital warts.3 References 1. Howley PM. Papillomavirinae: The viruses and their replication. In: Fields BN, Knipe DM, Howley PM, eds. Fields Virology. 3rd ed. Philadelphia, Pa: Lippincott-Raven; 1996:2045–2076. 2. Schiffman M, Castle PE. Human papillomavirus: Epidemiology and public health. Arch Pathol Lab Med. 2003;127:930–934. 3. Wiley DJ, Douglas J, Beutner K, et al. External genital warts: Diagnosis, treatment, and prevention. Clin Infect Dis. 2002;35(suppl 2):S210–S224. 4. Clifford GM, Smith JS, Aguado T, Franceschi S. Comparison of HPV type distribution in high-grade cervical lesions and cervical cancer: A meta-analysis. Br J Cancer. 2003:89;101–105. 5. Muñoz N, Bosch FX, de Sanjosé S, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med. 2003;348:518–527. *High risk; †Low risk 1. Howley PM. In: Fields BN, Knipe DM, Howley PM, eds. Philadelphia, Pa: Lippincott-Raven; 1996:2045–2076. 2. Schiffman M, Castle PE. Arch Pathol Lab Med. 2003;127:930–934. 3. Wiley DJ, Douglas J, Beutner K, et al. Clin Infect Dis. 2002;35(suppl 2):S210–S224. 4. Muñoz N, Bosch FX, de Sanjosé S, et al. N Engl J Med. 2003;348:518–527. 5. Clifford GM, Smith JS, Aguado T, Franceschi S. Br J Cancer. 2003:89;101–105.

A HPV Filogenezise Human genitális mucosa papillomavirusok 39 18 45 11 6 Human genitális mucosa papillomavirusok 33 16 35 31 Állati papillomavirusok Humán bőrfelszíni papillomavirusok Chan et al., J Virology, 69:3074-83 (1995)

A HPV Fertőzés Természetes Lefolyása1 Fennálló fertőzés CIN 2/3 Invaziv méhnyak-rák CIN 1 Key Point Infection with HPV will typically clear, but some infections with high-risk HPV types may ultimately lead to cervical cancer via a number of intermediate steps.   Background Following initial HPV infection, the course of progression to cervical cancer depends on the type of HPV. Low-risk HPV types (such as HPV 6 or 11) have a negligible risk of progressing but may persist.1 Overall, the majority of HPV infections spontaneously clear within the first 24 months.2 High-risk types (such as types HPV 16 and 18) are often associated with CIN 2 or higher lesions. The strong association of HPV 16 with CIN 2 or greater suggests that lesions caused by this infection evolve to CIN 2 without a prolonged period as CIN 1.1   Approximately 57% of low-grade lesions (CIN 1) will regress, 32% will persist, and 11% will progress. The risk of developing invasive cancer is estimated to be 1% in patients with CIN 1. Approximately 43% of CIN 2 lesions will regress, 35% will persist, 22% will progress to CIN 3, and 5% will progress to invasive cancer. The likelihood of CIN 3 regressing is about 32%, persistence is <56%, and rate of progression to invasive cancer is >12%.3 In studies of women with HPV infection who developed CIN 2 or 3, the initial abnormal smear was interpreted as CIN 2 or 3 in two thirds of cases,1,4 indicating that most CIN 3 lesions do not evolve from CIN 1.1 In a large, prospective study, mean times of progression from mild, moderate, or severe dysplasia to development of cervical intraepithelial neoplasia (CIS) were 58, 38, and 12 months, respectively.5    In general, CIN occurs at least a decade earlier than invasive cancer, supporting a concept of the temporal evolution of cervical cancer.3 Based on a Markov model that approximated age-specific incidence of cervical cancer and HPV infection–associated events, the peak prevalence of low-grade squamous intraepithelial lesions (LSIL) is at 28 years of age, at 42 years of age for HSIL, and at 48 years of age for cervical cancer.6 References 1. Pinto AP, Crum CP. Natural history of cervical neoplasia: Defining progression and its consequence. Clin Obstet Gynecol. 2000;43:352–362. 2. Ho GYF, Bierman R, Beardsley L, Chang CJ, Burk RD. Natural history of cervicovaginal papillomavirus infection in young women. N Engl J Med. 1998;338:423–428. 3. Ostor AG. Natural history of cervical intraepithelial neoplasia: A critical review. Int J Gynecol Pathol. 1993;12:186–192. 4. Koutsky LA, Holmes KK, Critchlow CW, Stevens CE, Paavonen J, et al. A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papillomavirus infection. N Engl J Med. 1992;327:1272–1278. 5. Richart RM, Barron BA. A follow-up study of patients with cervical dysplasia. Am J Obstet Gynecol. 1969;105:386–393. 6. Myers ER, McCrory DC, Nanda K, Bastian L, Matchar DB. Mathematical model for the natural history of human papillomavirus infection and cervical carcinogenesis. Am J Epidemiol. 2000;151:1158–1171. Gyógyult HPV fertőzés 1. Pinto AP, Crum CP. Clin Obstet Gynecol. 2000;43:352–362.

A HPV-fertőzés A HPV-DNS CIN 1 70% A HPV-fertőzés valószínűleg a leggyakoribb nemi érintkezéssel terjedő betegség. Nem klasszikus fertőző betegség; tünetek: nincsenek vagy helyi elváltozások. A HPV-fertőzés a méhnyakrákban gyakori: A HPV-DNS CIN 1 70% kimutatható: CIN 2-3 70-100% méhnyakrák 90-100% – Molekuláris genetikai bizonyítékok: HPV 16/18 E6-fehérje – p53, E7-fehérje – pRb kapcsolódás

A HPV fertőzések előfordulásának jellegzetességei A HPV-fertőzések „szakaszos”-ak, ami azt jelenti, hogy egy adott HPV-fertőzés nem tart egy egész életen át. Feltételezhetően a vírust a szervezet elpusztítja, és a fertőzés megszűnik, de az is lehet, hogy a vírusok elpusztítása nem teljes, a HPV-fertőzés „alvó” állapotba kerül – klinikailag és laboratóriumi vizsgálatokkal sem mutatható ki –, majd újra fellángol.

HPV osztályozás a méhnyakrák kialakulásában játszott szerep alapján Magas onkológiai kockázatú 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82 Valószínüleg magas onkológiai kockázatú 26, 53, 66 Alacsony onkológiai kockázatú 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81 Nem meghatározott onkológiai kockázatú 34, 57, 83 Munoz, 2003

A HPV VLP-k Spontán Kialakulása A papillomavírus VLP szerkezeti modellje* L1 Protein (55–57 kD) L1 Capsomer (~280 kD) VLP (~20,000 kD) 72 Capsomer 5 x L1 Key Point Virus-like particles spontaneously assemble when the HPV L1 protein is overexpressed in several different cell types. Virus-like particles consist of 72 capsomeres, each of which is composed of 5 L1 molecules. Background When overexpressed in mammalian,1 insect,2 yeast,3 or bacterial cells,4 the papillomavirus L1 protein spontaneously assembles to form VLPs that are devoid of the oncogenic viral genome.5 High-resolution cryoelectron microscopic analysis has determined that the HPV structure consists of 72 pentameric capsomeres, each composed of 5 L1 molecules.2,6 References Hagensee ME, Yaegashi N, Galloway DA. Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins. J Virol. 1993;67:315–322. Kirnbauer R, Booy F, Cheng N, Lowy DR, Schiller JT. Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc Natl Acad Sci USA. 1992;89:12180–12184. Jansen KU, Rosolowsky M, Schultz LD, et al. Vaccination with yeast-expressed cottontail rabbit papillomavirus (CRPV) virus-like particles protects rabbits from CRPV-induced papilloma formation. Vaccine. 1995;13:1509–1514. Nardelli-Haefliger D, Roden R, Balmelli C, Potts A, Schiller J, De Grandi P. Mucosal but not parenteral immunization with purified human papillomavirus type 16 virus-like particles induces neutralizing titers of antibodies throughout the estrous cycle of mice. J Virol. 1999;73:9609–9613. 5. Roden R, Wu T-C. Preventative and therapeutic vaccines for cervical cancer. Expert Rev Vaccines. 2003;2:495–516. 6. Baker TS, Newcomb WW, Olson NH, Cowsert LM, Olson C, Brown JC. Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and three-dimensional image reconstruction. Biophys J. 1991;60:1445–56. * VLP = Virus-like particle. 1. Kirnbauer R, Booy F, Cheng N, Lowy DR, Schiller JT. Proc Natl Acad Sci USA. 1992;89:12180–12184. 2. Syrjänen KJ, Syrjänen SM. Chichester, United Kingdom: John Wiley & Sons, Inc; 2000:11–51.

HPV L1 Virus-Like-Particle (VLP) Vakcina Szintézis A HPV belseje Üres vírus capsid (VLP) HPV-DNS L1 gén Immunválasz Transzkripció L1 gén plasmidba ültetve Capsid proteinek mRNS Transzláció Eukaryota Sejt

SILGARD™ A Merck quadrivalens HPV L1 VLP vakcinája1 Quadrivalens HPV (6, 11, 16, 18 típusok) L1 VLP vakcina A VLP-k Saccharomyces cerevisiae-ben képződnek Gombák által termet vakcinát felnőttek és gyermekek milliói kaptak. Aluminum adjuváns (225 ug per dózis) 0.5 mL injekció 3 adag 6 hónapon belül Key Point GARDASIL™ is Merck’s quadrivalent HPV L1 VLP vaccine. It is produced in recombinant yeast, like the hepatitis B vaccine, and adsorbed on a proprietary aluminum adjuvant, like the tetanus vaccine. Background Merck’s quadrivalent vaccine has the advantage that it targets 4 HPV types (6, 11, 16 and 18).1 Together, HPV Types 16 and 18 account for about 70% of cervical cancer and high-grade lesions, whereas HPV Types 6 and 11 are associated with approximately 90% of anogenital warts.1 Together, these 4 types are responsible for over 40% of low-grade cervical lesions,2 and HPV 16 and 18 account for over 50% of high-grade lesions.3 The active quadrivalent vaccine is a mixture of 4 recombinant HPV type-specific VLPs consisting of the L1 major capsid proteins of HPV 6, 11, 16, and 18 synthesized in Saccharomyces cerevisiae.1 Expression of the L1 protein in yeast generates noninfectious VLPs that resemble HPV virions.1 A similar method is used for hepatitis B vaccine.4 The 4 VLP types were purified and absorbed onto amorphous aluminum hydroxyphosphate sulfate adjuvant.1 The adjuvant dose was 225 micrograms per dose, and the vaccine injection volume was 0.5 mL, given by intramuscular injection at Day 1, Month 2, and Month 6.1 GARDASIL is a trademark of Merck & Co., Inc., Whitehouse Station, NJ, USA. References 1. Villa LL, Costa RLR, Petta CA, et al. Prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: A randomised double-blind placebo-controlled multicentre phase II efficacy trial. Lancet Oncol. 2005;6:271–278. 2. Clifford GM, Rana RK, Franceschi S, Smith JS, Gough G, Pimenta JM. Human papillomavirus genotype distribution in low-grade cervical lesions: Comparison by geographic region and with cervical cancer. Cancer Epidemiol Biomarkers Prev. 2005;14:1157–1164. 3. Clifford GM, Smith JS, Plummer M, Muñoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: A meta-analysis. Br J Cancer. 2003;88:63–73. 4. Recombivax HB® Hepatitis B Vaccine (Recombinant) prescribing information. Merck & Co, Inc. Whitehouse Station, NJ. 1998. GARDASIL is a trademark of Merck & Co., Inc., Whitehouse Station, NJ, USA. *VLP = Virus-like particle. 1. Villa LL, Costa RL, Petta CA, et al. Lancet Oncol. 2005;6:271–278.

Quadrivalens HPV (6,11,16,18) vakcina Immunogenitási és hatékonysági vizsgálat Randomizált, kettősvak, placebo kontrollált Fázis II vizsgálat 522 bevont fiatal nő 277 vakcina 275 placebo 3 dózis: 0., 2., 6. hónapban 36 hónapos utánkövetés Nőgyógyászati vizsgálatok Cervicovaginalis mintavétel HPV DNS tesztre Ellenanyagszint mérése PAP test

Quadrivalens HPV (6,11,16,18) vakcina Hatékonyság 100% 100% 88% 89% D. Ferris XXII. IPV Kongresszus, Vancouver 2005

MSD Quadrivalens HPV vakcina Eredmények Biztonságosság A mai napig >13,000 nőnek adtak HPV vakcinát A vakcina általánosságban jól tolerálható Immunogenitás A vakcina magas anti-HPV titer választ eredményez A csúcs anti-HPV 6, 11, 16, 18 válasz geometriai átlag-titere 58.7-szerese, mint akiknek természetes infekciójuk volt Hatékonyság Vizsgálatok bizonyították, hogy a HPV-16 vakcina csökkenti a HPV-16 fertőzés kockázatát a HPV-16 seronegatív nőkben Mivel a vizsgálatok jelenleg is zajlanak, ezért egyelőre csak előzetes eredményeket tudok Önökkel megosztani a vakcina biztonságosságáró, immunogenitásáról és hatékonyságáról. A mai napig.. Az oltottak ellenanyagszintjének geometriai átlag titere 58.7x magasabb, mint azon személyeké, akik természetes úton vészelték át az infekciót To date, the vaccine has been administered to over 13,000 subjects. In general the vaccine has been well-tolerated. The vaccine induces high-titer anti-HPV responses. The responses at peak are over 100 times higher than those observed following natural infection. Finally, we have conducted two sets of studies that have shown that the HPV 16 vaccine reduces HPV 16 infection. We focused on HPV 16 in these early studies because it is by far the most dangerous HPV type, being responsible for 50% of cervical cancers.

Points of Intervention With an HPV Vaccine HPV-native Sexual contact 1 = Merck, GSK 2 HPV infection Condyloma 2 CIN 2 Cancer 2 Recurrence 1=VLP vaccine 2=therapeutic vaccine Cure 2 Adapted from Hines et al. 1998. Cur Opin Ob Gyn 10:15

Venn Diagrams Representing Sets in the Cervical Condition Universe HR HPV Normal women are outside all the set circles 1% Future CIN 2/3 ~5 years Current CIN 2/3 Abnormal Cytology P16 positive

Kombinált Fázis II/III Hatékonysági Vizsgálat; a GARDASIL™ elsődleges hatékonysági elemzése Per-Protocol Populáció Átlagos követési idő = 4, 3 és 2 év, a vizsgálattól függően A HPV 16/18 –függő elváltozások elleni hatékonyság GARDASIL Placebo 95% CI n esetek Hatékonyság (%) P érték CIN 2/3 vagy AIS 8487 8460 53 100 (93–100) < 0.001 CIN 2 36 (89–100) CIN 3 vagy AIS 32 (88–100) Key point Results from the combined Phase II/Phase III studies show that GARDASIL™ was 100% efficacious against high-grade precancerous and noninvasive cancerous cervical lesions related to HPV 16 and 18 in a population of female subjects naïve to these types until completion of vaccination. Background The per-protocol analysis included 8487 subjects in the vaccine group and 8460 in the placebo group out of a total population of 20,541 subjects included in efficacy analyses (10,268 and 10,273 in each group, respectively).1 In this combined analysis, prophylactic quadrivalent HPV vaccination prevented HPV 16/18-related CIN 2/3 and AIS. Vaccination was generally well tolerated.1 This intervention is expected to greatly reduce the risk of cervical cancer.1 GARDASIL is a trademark of Merck & Co., Inc., Whitehouse Station, NJ, USA. Reference 1. Data on file, MSD. PP = received 3 vaccinations within 1 year; no major protocol violations; HPV 16/18 sero(-) at day 1 and HPV 16/18 DNA(-) day 1 to month 7; cases counted starting after month 7. CIN = cervical intraepithelial neoplasia; AIS = adenocarcinoma in situ. GARDASIL is a trademark of Merck & Co., Inc., Whitehouse Station, NJ, USA.