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HUMAN !PAPILLOMAVIRUS (HPV) SQUAMOUS CELL CARCINOGENESIS INFECTIONS IN OF THE GENITAL AND

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HUMAN !PAPILLOMAVIRUS (HPV) SQUAMOUS CELL CARCINOGENESIS INFECTIONS IN OF THE GENITAL AND AER()DIGESTIVE TRACT. (PROJECT !, Ii and !11) Karl Syrpnen, M.D., Ph.D. & Stina Syrji~nen, D.D.S., Ph.D. Kuopio and Turku Papillomavirus Research Groups Rauno M&ntyj~i, M.D., Ph.D., 8eppo Saadkosld, M.D., Ph.D., Chang Fuju, M.D., Ph.D., Sinikka Parkkin~n, Ph.D., Merja Yliskoski, M.D., Ph.D., Martti V&yrynen, M.D., Ph.D., Vesa Kataja, M.D., Ph.D., Jad Kellokoski, D.D.S., Ph.D., Marita Hippel~inen, M.D., Aria Tervahauta, M.$c., Hongxiu ~Ji, M.D., Ph.D., Mir~a Puran~n, D.D.$., Katdina kappalainen, M.D., Kaisa Kurvinen, M.$c.,iSakari Hietanen, M.D. Reidar Grenman M.D., Ph.D, Seija Grenman, M.D., Ph.D., Riitta Lei~nola-V~rtanen D.D.S., Media Riihel~ D.D.S., Re~tu Agrawal, M.$c., Aino Laatikainen, Ph.0., Tapio Nurmi, Ph.D., Department of Pathology; Department of Obstetrics & Gynecology; Department of Clinical Microbiology; A.I,Virtanen institute; University of Kuopio; Laboratory of Molecular Virology, MediCity, University of Turku, Finland. BATCo document for Mayo Clinic 28 March 2002
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1 PROJECT ! (KUOPIO) BIOLOGICAL SIGNIFICANCE OF GYNECOLOGICAL HUMAN PAPILLOMAVIRUS INFECTIONS. A PROSPECTIVE FOLLOW-UP AND PARTNER STUDY. 1.1NTRODUCnON Work~. ~ HPV is thought to be widespread in 1be popula~o~ L~e olt~ v~al ~ HPV can sensie~ tes~ (e.g. ¢iose based on recombinar~ D~) app~cable to a large-scale screan~g f='U~e risk groups, es wel as fo~." serological HPV tests capat~ of disclosing the early ~ of cervical cancer. ~ by adequate tmat~." of the precancer (CIN) ~ as we~l as by ama~ging a carefully conducte~ follow-up of The views of ~e genital wazt (Condylomata acumlnaIa) as an Innocuous squamous cell lesion caused by human pal~'llomavirus ~HPV) have been subjected to complete raspprisal since the morphological description in 1976 of two new HPV leslor~ in the uterine cewtx, cun'ently known as flat and andophytic (inverted) condylomas (Meisels et al 1976; Purcia an~ Savia 197~. It was soon demonstr~ed that especially the flat condylomas are freguently associated with all gravies of cervical tntraepithellal neoplasia (C1N), carcinoma in situ (CIS} and not infrequently, with invasive squamous cell cardnomas of the uterine cervix (Syrj~nan 1979; Kirkup st al 1982; Grunebaum et al 1983). Epidemlologlcal studi~s from many countries have consistently shown thet the incfdence and prevalence of CIN lesions are continuously increasing espoc~ly in the younger age groups of sexually active women. Uke HPV infections, there is arr~ple epidemiological documentation available to suggest that cervical cancer is a sexually transmitted disease (STD) (Fenoglio and Ferenczy 1982; Bdnton 1986; Doll 1986; Vessey 1986). In early 1980's, HPV structural proteins (viral antigens) were demonstrated in a substantial percentage of CIN lesions (Jenson et al 1980; Kurman et al 1983). In 1983, DNA-hybridization disclosed HPV 16 and 18 DNA sequences in invasive cervical carcinomas for the first time (D0mt et al 1983; Boshart et al 1984). Following these first reports, vast amount of evidence has accumulated during the past few years which implicates an inttrr~te association of certain HP~ types (HPV 6, 11, 16, 18, 31, 33, 35, 39, 42-45, 50-56) with genital precancer lesions and genital cancer. The concept on the 'low rkk' (HPV 6 and 11) and 'high risk' (HPV 16,18,35,39) HPV types has evolved I'rom the association of these HI~V types w;lh benign (condylomas) and precancer/cancer BATCo document for Mayo Clinic 28 March 2002
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2 lesions, respectively ~(Gissmann et el. 1984). Molecular biology has aided in sorting om the functions of papillomavirus DNA machinery to assess the mechanisms regulating DNA transcription, mRNA transis~on, viral repllca~on and maligni~nt transformation (Howley 1983; Pflster 198~.; Schwarz 1987; McDougall, 1990; Howley 1991). The genome organiz~ion of HPVs is well elucidated, and a number of HPV genomas h~ve been sequenced by now (de Vitlisrs 1989). Ouriundemtanding of the regulatory mechan~'ns involved in transcription and cell transformaIio~ has substantially increased during the past couple of yearn (]~cDougail 1990; Howley 1991; Lambert 1991). Un- doubtedly, the evidence accumulated during the past 15 yearn have made HPV as o~e of the most seriously considered agents impliceled in pathogenasb of human squamous cell carcinoma, that of the lower female genital t~act in pa~cular (Sy~en, Giesmann & Kose 1987; zur Hausen 1977; 1989; 1991). Co~, this virus has y~ (WHO S~c~)~ ~SPECIRC NMS The general Iong-tarmlobjectlve of the current prospective follow-up study conducted in KuopIo since October 1981 is to obtain detailed i~formatlon on the natural histon] of HPV Infections In the female genital tract and of their aseocislions with Intrabpitheliai neoplasia and genital squamous cell cancer in both sexes. The specific aims are as 1) To fully alu@:late the complex natural histoq/of female genital tract HPV infections by colposcopy, PAP smears, end punch bippsias dudng a k:mg.term prospective follow-up without therapy. The rates of spontaneous regression, persisten~ and pmgrassion as well as disease fluctuation and recurrence ~tar treatment are determined by ck~e mon~odng of the lesions at 6-month intsvab. The significance of the follow.up data Is analysed using the life-table technlque an~ survival analysis with covadates (Cox model). 2) To identify (i.~ the biopsies) the biological risk factors predisposing the HPV lesions to progression towards cewical cancer;, the favors analysed so far include the HPV type (detected by different hybridization techniques and PCR), lesion morphology, histological grade, amount and ploidy of DNA (cytomel]y), histoquantltative paramaims (automatic Image anal~tsis). 3} To alucldat~ the intracellular events of HPV Infections by assessing the presence of viral DNA, mRNAs, expression of HPV O~F-coded proteins, growth factom, estrogen receptor, TGF, ectlvation of cellular oncogenes (c-onc), and 0nactivati.~n) of antt-oncogenes (Fib and p53). Expression of p53 as related to different HPV types, and the role of its mutations in the disease outcome are analysed. 4) To assess tl~e host immune reactions affecting the natural h~ory of HPV infections by analysing a) the immunoragulato~y gen~es by complete HI.A-typing, b) the In situ. interactions of different immunocompetent cell subpopulations in HF~. lesions by confocai laser scanning microscope (CLSM), and c) the immunocompetent cell subpopulations in perij~heral blood by flow cytomeby. 5) To analyse the humoral HPV type-specific antibody response and its significance in disease outcome, by using EUSA test and synthetic pept~des to different HPV ORF-encoded proteins in serial serum se.mples collected over the 10-year period (1981-1993). This part of the study is made In collaboration with German Cancer Research Centre (Heidelberg, Germany: Prof. Lutz Giasmann), as well as with Prof. V. Vonka (??? Prague, Czeck Republic). 6) To assess the potential factors acting synergistically witl~ HPV in squamous cell cacinogenesls, e.g. coexistence of other infectious agents (Chlamy(Jia, EBV, HSV, CMV), as well as the smoking. 7) To analyse by questionnaire, epidemiologicai dsk factors pertinent to transmission of genital HPV infections, among cthsrs the role.of sexual I;ehav~our and smoking, both being blaJmed for cervical cancer. The prevalence BATCo document for Mayo Clinic 28 March 2002 0 0 O~
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incidence figureslof genital HPV Infections are being monitored by analysing the data derived from the annual mass-screening prog ....mmrne in Kuopio province. 8) To dstermi~e the role of male sexual partners in transmission of genital HPV infections by examining the .sexual partners of th~ follow-up women by peniscopy, punch biopsy and HPV DNA tTpfng. Another modes of transmission, i.e., HP~' infections in children born to mothers with genital HPV Infections are surveyed as well (see PROJECT 9) To test the I~j-term ef~ of the current b'eatrnent practice available for HPV infections, by randomizing the Treatment Group~ women into four groups t~ea~ed by either a) conization, b) cryosurgePj, c) CO~_-Iaeer vaporization, or d) ~stemic interferon. Also the potential novel therapeutic approaches (see below) are easily testable In these wom.'.'en after extensive testlng in vitro. 10) To test the." applicability and efficacy of oligodeexynucleotidee (antisanse oligos for HPV 16/18 E6 and F_/7 mRNA) as inhibit~rs of viral translation. This could be a novel therapeutic approach to eradicate HPV. Before in vivo testing, extensive experiments are mt~de in vitro using Ilposome technology with HeLa and CaSId cell lines. 11) To contlnde the developmental wod¢ towards rapid molecular te~s for HPV infectiohs, by improving the current DNA-techniqu~ ~, and launching novel Innovations (e.g. microtiter plate-based hybridization), as well as exploring the i~otentials for serologic diagnosis by testing the antibody response against the fusion proteins and synthetic psptide~ coded by different HPV ORFs. This project has now moved to Tu~ku Univedsty, MediCity. 12) Based oK the experience gathered up during the long-term prospective folk>w-up, to establish a commonly accaptabl~ diagnos~c criteria for a) clinical, b) subcllnica~ and c) latent HPV Infections as well as an optimal treatment pra~ice for these increasingly common ~ectlons. Of major Importance will be the availability of diagnostic (serologlcaJ or hybridization) tests applicable In large-scale screening of the risk groups, i.e., women and men with an increased risk of their HPV infections to develop Into inveslve carcinomas. 3.BACt(GROUND Despite a Iongihistory as a disease, it was not until 1954 that the venereal transmission of the genital wars was firmly establlshec~..~ in a study of servicemen returning from the Korean War. Vulvar and other venereal wa~ls appeared in the wiv~ of these servicemen after the incubation period of 4 to 6 weeks (Barrett et aI 1954; Odel Viral particles I~a~d been found In the skin waxts In 1949, but it took some two decades to disclose them In 1981). the ganitaJ warts, which was done in 1968 (Dunn and Ogllvle 1968). The structure of the viral particles in these two lesions proved to be id~entical, the agent being currently known as Human papiltomavtrus (HPV) (Rowson and Mahy 1967). This led the wo~rkem to suppose that all types of human warts were caused by one and the same type of HPV, their va.dable m~ology being ascribed to their diff=ent anatomical location (zur Hausen 1977). This concept has been entirely revi~ed during the past 15 years, however, following the dlscoveP/of many different types and subtypes of HPV, eac.~ shown to possess a pred'deot~n site of infection in man (see the Textbook by Syrj~en, Gissmann & Koss 1987)... At the same t~e, the previous views of the genital wart as an Innocuous squamous cell lesion have been subjected to complete reapprisal since the morphological descriputlon in 1976 of two new HPV lesions in the uterine cervix, currently known as flat and endophytlo (inverted) condylomas (Meiseis et al 1976; Purola and Savia 1977). It was soon demonstrated on light micrcecpy that all three distinct types of condylomes in the genital tract a~e freguer~.~ associaled with intmepithelisl neoplasta (C.JN), carcinoma in s'rtu (CIS) and less frequently with invasive squamous cell carcinomas (SyrJEnen 1979; K]rl~p et al 19~2; Gruneb~um et al 1983). Epidemiological studies from mart/coun~.'es have consistently shown that the incidence and prevalence of CIN lesions are increasing especially in ¢,e ycu~.cer age @'cups of sexuP.Jly active women. Like HPV infections, there is ample epidemiologica] ! BATCo document for Mayo Clinic 28 March 2002
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documenta~on available to suggest that cewical cancer is a sexually txansmlttsd disease (STD) (Fenogllo and Ferenczy 1982; Brinton 1986; Doll 1986; Vessey 1986). Since early 1980's, HPV structural proteins {viral antigens) have been demonstra~e~ d by immunoh~ochemlcel means in CIN lesions (Jenson et at 1980; Kurman et at 1983). Finally, in 1983, DNA-~ybridiza~on disclosed HPV 16 and 18 DNA sequences in invasive cervical squamous cell cetcinomes for the first time (DOmt at a] 1983; Boshart ~t ai 1984). Following thes~ reports, vast amount of evidence has accumulated indicating the intimate association of certatn HPV types (HF~/6, 11, 16, 18, 31, 33, 35, 39, 42--45, 50-56) with CIN and genital cancer. The concept on the 'low dsk' (HPV 6:anti 11) and 'high risk' (HPV 16,18,35,39) HPV types has evolved from the preferential association of these HPV types with benign (condyiomas) and precancer/cancer lesions, respectively (Gissmenn at ai 1984). Further sup .~l~t is provided by the increasing number of reports on malignant conversion of HPV.kcluced laryngeal papillomas, i glant condyiomas, Bowenoid papuiosis, end cutaneous lesions of epidermodysplasia verruciformis (EV). Upi to 80% of all CIN and 90% of the analyzed squamous cell carcinomas of the cervix have shown to contain HPVIDNA sequences, mainly HPV 16 and 18, and less frequently HPV 31, 33 and 35 (zur Hausen 198~; 1991). In edcr~tic~ to the pdmaty tumors, HPV DNA has also been found in the~" lymph node metastesses~Ceil lines derived from Ce~icai cancers such as Hal.a, CaSki and SiHa cells, have been shown to cor'~aln HPV DNA actively trar~crlbed (z~.:r Hausen 1989; 1991; Howley 1991). • Experimental ~udias have demonstrated tha~ some HPVs are capable of transforming rodent cells and Immortatlzing human f~reskin and cewical ker'afinocytes (McDougall 1990; Howlsy 1991). E6-E70RFe seems to be the main transforming .~enes in HPVs (Smotkin & Wsttsteln 1986; Baker at al 1987; Shirasawa et al 19~8; zur Hausen 1989; 1991; McDoug~ 1990; Howley 1991). These regions are regularly transcribed and expressed in cervic~ carcinomas and in canCer-derived cell lines. These same regions am also demonstrated to be required for In vitro. transformation of mde~ cells and for immortalization of human keratlnocytes and cewlcel eptthelhal ceils (Smotkin & Wettstein 1986; Baker st ai 1987; Shiresmva at al 1988; zur Hausen 1989; 1991; McDougall 1990; Howisy 1991; Howley 1983; Pfiater 1.984; Schw'arz 1987; Lamber 1991)o New Insights into the mechanisms of HPV-essociated tmnsform~on have d~dved from the recent findings mat these transforming viral proteins can interact with the known tumor suppresS=' r genes, e.g., Rb and p53, presumably causing an inactivation of these tumor suppressor genes and consequent~, leading to an uncontrol~d proliferation of the infected cells (MOnger et al 1989; $cheffner at ai 1990; Levine 199~, Vousdan st ai 1991). At present, a substantial amount of evidence is available to suggest that specifc HPV lypesiare necessary but as single agents insufficient in oncogenesis, synergistic actions with other initiating events seem t~ be required (zur Hausen 1989;, 1991; How~ey 1991). The genome organization of paplllomaviruses is well elucidated, and a number of HPV geflomas have been sequenced by now (de Villlers 1989). Our understanding of the regulato,-y mechanisms invoked in transcription and cell transformation has;;substantislly increased dudng the past couple of years 0VIcDougail 1990; Howley 1991; Lambe~t 1991). Data o::n HPV-coded proteins and their possible role in cell transfon~ation and as potential immunogens are just e~erging (Androphy st al 1985; Baker st al 1987; Jenison st ai 1988; U et al 1988). One of the most unexplored areas in HPV research is the serological diagnosis of these Infections (GaJlovray 1990). Serological tests for d~tection of HPV type-specific ant=bodies have been explored only recently, because there exists no in vitro culture system for the replication of paplliomavirusas (Galloway 1990). For the production of viral antigens, the proteins have to be expressed in hetaroiogous vector systems (e.g.E. cob3. In fact, HPV proteins fused to procaryotic peptidas have been produced in bacteria and used as antigens in Western blot experiments (U at al 1987; Jeniscn st ~1 1988; Strike et at 1989; Jc~.hmus-Kudielka et ai 1989; Mann et a] 1990). Altsmatively, the sercreactive regions of the ~J-,dividua] proteins are chemically' synthesized and employed in peptide EUSAs (Dillner et ~,' 1989; K~-chna~ at ai 1990). P[elin~nary data from a German study suggest that HPV 16 E6-specific antibodies 0 O~ BATCo document for Mayo Clinic 28 March 2002
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5 ere more prevalem in :women suffering from GIN lesions, and hence may present a rns~er for the risk to develop cervical cancer (M.Mu~er and L.Giesmann, tmpublished data). 80 far, no inform~tion is available on the development of antibodies In individ~ual p~e~s. Despite the abpv. e progress made in the field of basic and applied research, the final establishment of HPV as an etiological agent of cervical cancer still awaits, however. Many of the kay issues still remain to be settled; a) molecula~ mechanbrr~ of malignant lza.~orn'~tion, b} prevalence and biological s]gniflcence of subclinical and latent HPV infections, c) risk ~ctors predisposing the women to clinical HPV infections, d) the significance of host Immune response in e~. the viral Infection, e) the rote of serological HPV dbgnosis in predicting the disease outcome, f) lack of e .'.a~ markers of an increased risk for cervical cancer, g) the role of male partner as a reservoire of HPV, h) the eventual non-sexual (e.g. vertical) tmr~miesion of HPV, ~ the feasibility of examination and treatment of both sexual parmers; only a few to mention. S|rr~ady, many of the above experimental results await confirmation in human beings by Id=ng-term, well-controlled follow-up studies. Epidemlologicai studies ere still too few to permit define conclusions on the factors regulating the nmural histo~ of genital HPV infections, most Importantly, their progression to an Inv~sive cancer (Bd~on 1986; Vessey 1986; Munoz et ai 1988). On the other hand, the natur~i histonj, of CIN has beer~ well characterized by a number of carefully conducted follow-up studies, which u~equlvocally show that CIN is a truly precancerous lesion capable of progressing to an invesive cervical cancer, if left uneradlcat~d (Koss et!ai lS63; Naelell et al 1986; de BnJx et al 1983; 0st0r 1993). Because of so~na inevitable limitattor~ aesoofated with any re~ surveys, the only reliable approach elucidate the nstur~ hlsto~ of HPV infections is to conduct a ~ prcspec~m cohod study. Such was started in Kuop|o iaimoest 12 yesm ago, in October 1981, to establish the rate of regression, persistence and progression of these l~ections, unaffected by therapeutic intervention (SyrJ~nen et ai 1985; Syrj~en et ai 1987; see also the Appendix Ilstt~ the published reports). 4.SlGNIRCANCE OF ,'~-iE KUOP~O STUDY The Kuopio Fo~:~HJp Project is based on the largest cohort of women ever followed-up prospectively for HPV lesions. The follov~.up is based on regular mon~toring of the women at 6-mor~h ir=e~als. The project has been on progress for over !1 years by now, and more than 1.000 women and 350 men are currently included in the dudng these 11 yeas. ,~'This project has already signlt~.,antly contributed (and will continue to do so), to the following generel..ob[ect'.wes.ln Re field of os~cal carcinoqanesis: 4.1.Cancer Prever~on ; As indicated a .l~ve, one of the major long-term objectives of the project is the Identification of patients a~ Increased risk for ce~.pai cancer. This is accomplished by mu~ple ways. A clinically manifest HPV irCectJon itseff has been shown to I~ a risk factor for cendcal cancer. Thus, the mere Identification of these women and their prospective follow-up by close monitoring ensures the early detection of progression towards cancer. By identifying (and eliminating) the epidemiological factors Increasing the risk to acquire a clinical HPV infection (a prerequisite for cancer development), a clear contdbc~on towards cancer prevention can be made. An attempt towards the same goal has been made ustng the Mass-Screenlng Programme of the Finnish Cancer Society to monitor the prevalence of clinical HPV infections in Kuopio province over the 12-year period 1981-1992 (>85.000 samples analysed). As in other tumors, a stro0g research focus rests upon identification of the prognostic factors predicting the disease outcome (i.e., identify the p~ents with adverse prognosis). By conducting the prospective follow-up, the factors can be identified which predispose the HPV~nfected women to clin~_.ai progression. HPV type and lesion grade have O~ / BATCo document for M yo Clini
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6 been identified as su~'h risk factors by now. Quantitative p~ttolog (automatic image analysis), and confocal l~ssr scanning microscop~ provide new means to explore other such factors by examining the samples (e.g. frozen biopsies). Olher dsk factors must exist, however, and it is anticipated tha~ serological studies might identify some of ff~ase. Needles to ~ thaz during this 11-year duration of the Kuopio follow-up project, a unique tissue and serum bank has been ccolis~ed; which is easily accessible to analysis by any of the molecular biological techn~quas. This enables us to immediately test a) any new technique introduced in HPV research, and b) awida va~ety of hypothesis presented to explain ~e pathoganetic mechanisms of HPV in genital carcinogenesis. 4~_F_a~ ~agno~ = The detestloni of cervical precancer lesions by PAP smear is the best example that medical science has employed so far in ~early detection of cancer, made p0es~le to prevent the disease In countfiss where covering nation-wide mass-,scr~ning programmas are effective. Thus, the above listed items CLe., HPV detection by PAP smears) are equally .~eninent to this objective as well However, our study has established that over 60% of clinical HPV infections will eventually regress spontar~ously when followed-up for long enough (>7 years). Thus, a real challoncje for asdy d~ection of the risk groups will be to identify (at earliest possible stage) the women whose HPV lesions will eventually!progress towards cancer. Accordingly, one of the central alms of the Kuopio project has been to develop the diagr~c te~s capable of identifying such patients. As a result of this work, the first tests labelled in situ hybridization kits) developed in our isbomtoP/became on the market in early 1990 (Biohit HPV screening and typlnglkit). These tests can be applied to a large scale screening of women for the high-risk HPV types, known to possess.~' a significant risk for progression (i.e., 5-fold for HPV 16 as compared with HPV 6 or HPV 11). An urgand need ~, however, to identify additional rna~ers for early detection of the women at increased risk for oswicai canc~-. Serological anlalysee ere the next on focus to Idant~ry such markers. Recently, a higher prevalence of HPV 16 and HPV 18 E7-sp .~c antibodies was observed in sere of een~cal cancer patients as compared to age-matched controls. Prslimina~y tiara suggest that HPV 16 E6-speoific ar~lx~dias are more prevalent in women with CIN lesions, aand thus might present a marker for the dsk to develop cewlcal cancer. If this is true, an applicable test could be developed to distinct ~he groups of women with different risk. By a recently started concerted action, a possibility is now available to ~ the unique serum bank collected in Kuopio by uslng the sophislP.,ated technology and reagents evalulabis ini D~=Z, Heidelberg and in Institute of Hematology, Prague (synthetic peptides) to explore the HPV type-specific anl~o, dy profile (e.g. the tluctuation of the tltres over a long pedod) in individual patients. These analyses should not cinly provide basic insights into the natural histonj of genital HPV Infectlons but it is expected that serological ma~kers can be identified for the cancer-risk as well as for prognosis of an already existing disease. No specific cl~emstherapy exists for HPV Infectkm~ Albeit a viral infection, the treatment is still based on eb~ive and destructi~e measures. The seisct~on of o~mal treatment as well as the efficacy of different treatment modalitles is the matter of c~ispute. The present study design permits us to test the long-term efftcacy of the current treatment prac'dce available for HPV infections, by randomizing the Treatment Group women into four groups treated by either a) conizailon, b) c,~osurgery, c) CO2-1aser vaporization, or cl) systemic interferon. At least the intermediate- term cure rates after coniz=~.ion, cryotherapy and laser seem promising, over 90% of the treated women being disease-free at 20-months ~ follow-up. Si~'lady, the potential novel therapeutic approaches a~e essily testable in these womer~ it is es..~, ntiat, however, to compare the ~ cure rates w~h the spontaneous regression rate, which seems to be su~st~J~iai indeed (>65.% in 7 yearn of follow-up). BATCo document for Mayo Clinic 28 March 2002
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7 A novel apprqa, ch to potent~ eradic~Jon ~ HPV is to develop therapeutic measures capable of interrupting the vi~ functions pei se. The most promt.~ng means to accomplish this is ~e use of oligonucisotides as inhibitom Of Iransl~on, i.e., tha!complemanta~ or anJJ.sanse base sequences targeted to specific sense sequences in the viral mRNA. Thus, exp~..ion of epec~c protein can be regu~ed or inhibited. In the presem study, the inhibition of HPV 16 and HPV 18 onc~prctein expression will be essessed by anlj-sense oligonucleo~les in vitro before in v~vo applications. This approach is detailed in PROJECT III. (F~ fuil~ ~ the made" is ~ to ~ APPENDIX 5.i~(~=ERIMENTAL DESIC-=N AND METHODS The present ~oject was started in Oct. 198t, as a long-term collaborative study, currently contributed by six different institutions iniKuopio. These institutions Include; (I) Laboratory of P~thoiogy, Finnish Cancer Society, Kuopio, (II) Department of Gynecology and Ol:~letdcs, Kuopio University Hospital, (111) Department of Clinical Microbiology, Univemity of Kuopio, !IV) Department of Clinical P~'~gy, Kuopio University Hospital, (V) Department of Pathology, Ur~ of Kuopio,I and (Vt) Department of Biotecl'mology & Biochernlstnj', University of Kuopio. (RGUI:~ 1: Since eady 1 .9~O's, a regular and fluilful coEabor=Jon in special subprojects has been created with a number of prominent Papillo~avirus research laboratories In Europe. The first to start was the Department of Pathoiogy, Univers~y of Siena, ~ (Prof. Piero Tosi and his group). Regular contributions have been made by the Department of Detm~toiogy, Kar~linska Institute, Stockholm, Sweden (Dr. Geo yon Krogh), as well as the II Department of Gynecology & Obstet~ cs, University of Bologna, Italy (Dr. $ilvano Costa). During the past few months, a Concerted Action (submitted forlBIOMED I PROGRAMME of EC) was set up between the Kuopio Group and scientists In German Cancer Rese.~ch Center, (DKFZ) Heidelberg, Germany (Prof. Lutz Gissmann), and Institute of Hematology, Prague, Czech Repuplic (Prof. Vladimir Vonka), to enable the extensive analysis of HPV an~bodies and the development of serol ...qgica! tests for HPV, utilizing the unique serum bank collected in Kuopio since 1981. The tasks of these collaboratingi centers are listed in CHART 1 ~ Role of Each Padieipent in the I~). The woman ~e selected for the study on the basis of their routine cervical Papanicolaou (PAP) smeaJ~, These are taken by th.~ attending physicians in communal health centers, in private offices or in regional hospitals within the respo .nslbil~ area of Kuopio Unive~lty Hospital. PAP smears from this area are referred to screening In the Labo~ory o~ Patl~ology, Finnish Cancer Society (1), in Kuopio. The principal investigator as the director of Is responsible for exa~nination of the PAP smears horn the area of referral of the pa~ents enrolled in the follow-up study. In (I), all the smears are routinely screened by quaJIfied cytotechnicians, and whenever changes consistent with HPV infection are found, the smear w~ll be referred to examination by the principal investigator, who made the decisi~ whether or not the woman was invited in the study. This decision is |nvadably based on demonstration of unequivocally chamcter'~ic cytopathlo changes of HPV infection, accompanied or not by changes due to CIN. Each patient will receive (through the attending physician) an invitation along with her PAP smear report. The call for study comprises the letter of ~witatJon, g~ng a short background of HPV infections and their assoc'k-~.ion w~h ClN lesions, a shc~t outl|ne e/the follow-up stu~, as well as the summary of the examinations which the patient BATCo document for Mayo Clinic 28 March 2002
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will be subjected Io. A.: phone number is enclosed, to which the patient should contact to mal(e an appointment for her first examin~on = the Outpatient Department of Gynecology and Obstetrics, Kuopio University Hospital All the patients attend the clinic at 6Lmonth intervals. This is accomplished by fixing the dale of the next attendance at each ~isit. In cases of failure to attend, the patient will be contacted twice by renewed invit~on directly from the clint~. This procedure has yielded a hlgh raze of continuous pa.rtlcip~on In the study, as Indicated by the fact that of tl~e 530 patients (included in the follow-up sedes A and B), less ~an 5% of women have terminated the follow~p dudng the >11 years when this project has continued. As determined from the pdmap/PAP smear (Le. the one on which the invitation was based), the women are divided into 1) those l~.senting with cytological changes consistent with HPV end CIN (HPV-C1N), and Into 2) those with cytopathic cheng.~ of HPV only (HPV-NCIN). Accordingly, during the first four years of this project (1981-1985)0 a total of 530 wome~ ~Were enrolled in the study, end allocated Into two groups to be followed.up without treatment: those with HPV-C;IN I.~sions (~ A), followed-up by COllX~copy, PAP smear and punch biopsy; and those with HPV-NCIN lesions ( .G~oup B), followed-up by colposcopy and PAP smears (RGURE2: RECRLIITMEN~). Starting from .the late 1985, the third series of women has been enrolled in the folicw-up study. This sedes Is deslgnsted as the T~reatme~ Group, currently comprising 530 women with HPV-C~N lesicns. The seisctio~ of these women are kl. entlcai with those of the Group A women, Le., PAP smear changes consistent with HPV and CIN (HPV-CIN). Thes.,e patients are randomized according to three different treatment modalitias; conizatlon, CO laser vaporization andi cryotherepy. Additional series of 120 women received interferon trs~ment (systemic), and yet another (highly select,~d) sedes of 20 women was trusted with topical interferon. The follow-up of the Treatment Group women is con~lucted identically with the patients in Group A, using colposcopy, PAP smear and punch biopsy, repeeted at 6~onth intervals in (11) (RGUFIE El: ORGANIZATION OF THE TREATMENT SERIE~]. As the fourth .,series of patients in the project, the male sexual partners (Partner Gn:~p) of the women In the Treatment Group hav.~ been recruited for examination, treatment and follow-up since 1986. So far, genital HPV lesions of over 350 m."en have been examined by peniscopy (after acetic acid staining) and biopsy, Insludlng HPV DNA typing by In situ I~bridization and PCFL The lesions am treated by CO2-iaser vaporization whenever appropriate. Control the treatm .e~t Is conducted by paniscopy (with eventual biopsy) at 6-month Intervals by gynecologists working at (11). As th.~ most recent approach, the prevalence of male genital HPV infections was clarified by examining (by penisc~pj and biopsy) a coh(~ of cons~pts in two garrisons In eastern Finland ~uoplo and KontJorenta) during th.~ latter half of 1992. It is to be emp~hasized, however, that the follow-up schedule of Group A and Group B women has been flexible, and permits t~ansfer of the patients from one group to another. This is done 1) whenever considered necessan~ to quarantee that the lesion will not escape detection, when likely to make progression, or 2) to avoid unnecessen/biops|es ~mm the epithelium shown to be normal in two subsequent examinations. Noteworthy is also the policy that the l~ions of Group A and Group B women are invariably eradicated by coniz~ion, whenever progression into carcir~oma in situ (CIS) Is diagnosed. In addition tO the above listed follow-up and treatment groups, there is a small series of women diagnosed and treated for an invasive cervical or vulvar carcinomas at (iI), of which both frozen samples and paraffin blocks are available for study. This material is specially used for the assessment of p53, its mutations end associations with HPV, as explained elsewhere (PROJECT 111). HPV 16 DNA isolated from these lesions has been analysed for point mutations end deletions by Dr. H4J. Bernhard (Singapore University, Biotechnology Institute), whose group is conducting ~z global genetic analysis of HPV 16 to assess the evolutionar~j p~Zzways of this virus in different races. BATCo document for Mayo Clinic 28 March 2002
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9 Further analyses of the DNA from these lesions can be focused e.g., on the DNA methylation stetus etc. 5.1.4.P~ of Paiant Samples Both the PAP s~nears and punch biopsies of the lesions are ro~nety processed into HE-stained esotions among the daily routirte of the Department of Pathology, Kuopio University Hosplta~ (IV} to ensure the prompt diagnosis necessa~ for an adequate and safe follow-up. All the PAP smeam and biopsies are examined by a single pathoiogist (the pdncl ~l~al investigator), to guarantee that the diagnostic criteria remain unchanged throughout the study. The intmobserver vadatlon is controlled by kappa s~tistlcs. The frozen specimens and those for electron micrescopy are pr~ ed in the Department of Pathology, Univemity of Kuopio (V}. ~ HPV typing by Southern blot and in sit___g_u hybddiz~tion, PCR amplification techniques as well as immunoh~ochemical techniques are routinely done in 0,q. At each visit, s~rum samples are taken and submitted to the Department of Clinical Microbiology 010 for analyses of the ant~ titres for HSV, CMV, and V'ZV. These samples will be subjected to ark~yals for type- specie HPV antibodi~ as well. Similarly, samples of cenfcal and urethral swabs a~e taken at each time for isolation of Chlamydia trachoma~tis and HSV in (111). The entire proje~ has been computerized since 1985, a full-tlme operator being msponsibla for maintaining the data files at the ma~n computer (VAX) of the Unlvemtty Computer Center. Thus, the data of the tests completed in institutions 01)(III)(IV)~V) are addedd into the computer files for immediate access on daily besl,s. 5ZMETHODS The gross morphology of the classical genltaJ wart makes it easy to recognize. Problems are faced, however, when the diagnosis of ~he fiat and endophytic condylomas Is attempted by gross appearance. This is because In the majority of osses, t~e fiat and inverted condylornas are indiscemlbis by the naked eye, making necessary the applicatlon of other measures to settle the proper diagnose. Colposcopy is !the basic clinical technique used in, diagnosis of genital HPV Infections In both sexes. Practiced according to~ routine procedures by experienced gynecologists, all patients of the present study are subjectecl to colposcoW at each visit in the clinic. The colposcopic patterns are classified according to the nomenclature acceptediin 1990 by the IFCPC (Intematloni]! Federation for ColposcoW and Cewlcal Pathology), into one cf the following c~egorles; normal, warty, punctatlon, mosalclsm, leukoplakla or combinallon. Normal pattern denotes the cervix, wheie the lransformatlon zone crz) Is entirely visible, with no signs of CIN or other atypfa. Warty pattern includes florid ahd spiked condylomas, as well as the condylomatous ce~govaginitis (V~y~/nen 1986). In combination category a~e classified the cases with two or more different patterns. Leukoplakla patttem also comprises the aceto-white lesions. sz3.c'yto~xjy ~ In making the cytodiagnosis of HPV, the criteda exhaustively described in a large number of reports since C:> 1976 ~eisels et ai 1976; Purola et al 1977; SyrjCqen 1979) are used. As the most reliable sign of HPV infec~on, C~ ko~ecyte is currently considered to represent the cytopathio effect of HPV (Syrj~en, Gissmann & Koss 1987). In O'~ the present study, assessment of all the PAP smears is made by the same cytopathoiogist Codncipal investigator), ~ usin.c :he class~c.~_J Papanicol~u~J grading for the premalignancy (scale from normal to invasive carcinoma). Consist- P,O BATCo document for Mayo Clinic 28 March 2002
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10 eflcy of the grading ~ controlled by calcula~ng ~ irt,-=obse~er variation by the kappa statistics. 5.2.4.Hismiogy ~ A directed pun~ch biopsy is a key method in HPV diagnosis. In the present study, a colposcopically directed punch biopsy is tarsi1 from all p~ttents, except for those allocated into Group B on the basis of their fi~t PAP s~eam. Biopsy is tal~en at the first clinical axam~ and repeated at all follow-up visits at 6-month intervals, because shown to b~ the basic means in mordtodng the ~nical coume of these infections. In the present study, assessment of the punch biopsies is always made by the same pathologist Codncipa] investigator). When changes consistent with CIN ~e encountered admixed with or adjacent to HPV lesions, the lesions are called HPV-CIN in contrast to HPV-NCIN for those HPV lesion without concomitant CIN, The grade of CIN is assessed using the commonly accepted ~riteda for CIN !, II and II1. On special occasions, CIS (carcinoma in situ) is separated as an own entity. Consisten .dy of the grading is controlled by calculating the Intraobserver variation by the kappa statl~os. Standard 5-/~, HE-stained tissue sections am used for histoquantitatlva mesurements. A semlautomated interactive image a~a~." ser 0bes Kontron i} and more rec~, the automatic Image analyesr (Quantlmet 570) are used. Both instrument~s~ are available at the Depa~ment of Pathology. The methodology described by Tosl et el. is used. Representative ~elds (the most cellular areas) are chosen, and 50 nuclei are measured. Stareoiogy is ..L~ctized by measuring and counting points and profiles in esct~ns; the pa~em of grid to be used is related to ~ism or anisotropism of the tissue. Potnt counting is performed on a projection microscopy. Using a 42-point Wei~l grid (line lenght 1.5 cm) at a final magnifica~on of 200.250X, or on a video oveday system (Prodit), using a 42-p0~.nt Wsibei grid (line Isnght 2,5 cm) at a final magn~catlon of 300X (objective 10X); in ibis way it is possible to stu~ ~ereclogical features such as volume percentages, surface densltios, shape factor, and others. Mitoses are counted in consecutive high power fields using a 40X objective with a numeric aperture of 0.75 (magnif'cation 400X, fi~d diameter 450 ~m). Countlng should be started at the point within the marked area with ~e highest mitotic dense. Only distinct mitotic figures are ~ counted, Sections, 5 ~ithick, are cut from e~ch block and stained for DNA with the Feulgen method. The integrated optical dens~ of th~ nuclei of at least 100 cells in the ep'~hellal popul~d~on is measured in each case by cytophotomeW. The o~lcel portion of the analysis system consists of a Zelss microscope with a Dage video camera (Michigan City, Indian~), A ,560-nm illumination source and a 1,000X magnification are used (Tosi et el.). Histograms of ~he nuclear DNA content of the epithe~el ceils are prepared. The DNA index (ratio of the DNA content of the first ma~or peak to the diploid standard) Is calculat~ for the epithelial cells. Dlpioldy is defined as a DNA index of 0.9 to 1.~. Hypodipioidy is defined as a DNA index less than 0.9, and hyperdiploldy as a DNA index gre=er than 1.1. The I~eginnlng of the S phase Is defined to be two standard deviations above the modal diploid peak. The percentage ~ epithelial cells with nuclear DNA content above this value, representing ceils In the $ phase plus G2.M phases (S+G2M) of the cell cycle, is calculated In each case. 5Z6.1mmunohistochemis~y lmmunohistochemical staining is performed using the APAAP method (Alkaline phosphatase anti-alkaline phosphatase} or ABC ~echn|que to detect the expression of cytokaratlns. 1353. TGF, c-myc, c-HA-ras. PCNA cErbB- 2 in cewical lesions. 52_7.Eva~ua~on of HPV type-spec~c proteins BATCo document for Mayo Clinic 28 March 2002
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11 The early OREs are involved in viral DNA mpl'~:a~on, plasmid maintenance, and transforma~on (Schwarz 1987). The ~ ORFsi L1 and 1_2 encode the ma;,or and minor capeld proteins, respectively,. Evidence is mraiisbis on HPV type 6b to suggest that L1 and 1_2 proteins are si~ong~y antigenic, and readily recogn'~zable by appropriate antisar~ in the genital "~'azts (Jenison et al 198~). Analysis of E6-E7 gane expression of HPV 16 and 18 in associ~Jon with anogenitai carcin<~genesls is important because these two ORFs are regularly expressed in HPV-positive cervical cancem and the deriv~l cell lines (Smotkin & Wattstsin 1986; Baker et al 1987; Shimsawa et al 1988; zur Hausen 1989;, 19~1; McDouga]l 1990; Howley 1991). E6 and E7 genes a~e both necessary and sufficient for the effiolent transformmton of rodent cells and immortalization of human squamous epithelial ceils (zur Hausen 1989; 1991; McDougail I~0; Hov~]ey ~91). Our group ~ recently been involved in developing polyolonai and mon~clonal antibodies against synthstio peptides corresponding to an HPV 16 E6 protein. This piece of work has been completed in collaboration with the workers in Sclevo R~earch Center, Siena, Italy (Drs. Bugnoll, Covacci, Leoncini and Petmcca), and those working at the Depa~ment of Evotutive Biology (Dr. Palling, and Department of Patbol~:jy (Drs. Tosi and Clntodno), University' of Siena, Italy, Our i most recent work is focused on a synthetic peptlde con~esponding to 147-1,58 AA (NH2.F~RTRRETQLP~'C)OH) of the HPV E60RF, obtained by solid-phase synthesis and proved homogeneous by ar~cal HPLC. T~..~ e synthetic peptlde was used for Immunization of rabbits. The specificity of the polyclonal antibedles against thelsynthe~c pe~de was tested by Western blot, using as antigen the HPV 16 E6 fusion protein. In addition, two rnor~Iona] antibodies generated in mouse hybrtdomas (DA ?_8.3 and DA ~8.4) were selected for Expression of ~PV 16 E6 protein was studied Immunohistochemtca]ly In a series of HPV 16-associaled cervical lesions, as well as HPV 6- or HPV 11 -induced genital warts. The protsin expression wlil be compared to the concomitant mRNA expression. The observed differences between HPV 16 and HPV' 6/11 lesions in the expression of E6 protein and ~idetec~on by ff=e three a.~bodles might help explaining some c~ the differences In their biological behavior, s~esting that Aerations in transcriptional pottem might play a key role in the development of malignancy (Shimsa~.: et al. 1988). If this concept proves to be valid more generally, the use of our E6 protein antibodies would have..: important appti~ as Immunoreagents with prognostic value. Work Is on progress to seamh for additional ,antibodies that s~ould be specifl~ for E6 protein of a single HPV type only,. TEM was used, between 1981-1985 to confirm the presence of viral particles in HPV lesions as one of the means to assess the b~loglca] bai~aviour of HPV Infections. Because providing little additional informetJon about the biology of HPV In the ~genital tract, TEM as a time-consuming technique was abandoned In our routine follow-up protocol : TEM will have ~ew applications, however, when immunoelectron mlcroscoplc technlclues are applied to anal- ysis of the subcellular iocalLzatlon of HPV-encoded proteins and oncogene products (either nuclear, cytoplasmic or membrane) associated with HPV lesions. The potential of Immunoelectron microscopy to precisely locate v~ra] components, even though they are not assembled as e recognizable structures inside the infected cell, opened the possibility to study the basic and still unresolVed mechanisms involved in vlra~ Infection and morphogenesis (cerrascosa 1988). Furthermore, the opportunity to use electron microscopy for in situ DNA and RNA hybridization studies using genes or gene fragments, as well as artifidal DNA and RNA sequences as probes, is also currently possible (Binder st ai 1986~. This technique rosyhave significant implications in the studies on HPV life cycle within the cell. BATCo document for Mayo Clinic 28 March 2002
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12 Adequate ass~.ssment of HPV infections is not possible without DNA technology. HPV typing is based on the detection of diffeiences in the viral DNA, and the cloned vim] DNA probes of the specific HPV types (68 recognized by now) de routinely used in hybddtzation t~sts to study a vadst~ of human squamous cell lesions. While describing HPV 6 and HPV 11 predominantly in benign lesions, and HPV 16 and HPV 18 in high-grade CIN and IrNasive cancer leslo~s, DNA hybddiza~on techniques have created the concept on the 'low risk', and 'high rlsk', HPV types (DOrat at El 1983; Basha,'t et al 1984; Giesmann et al 1984). Furthermore, DNA techniques allow the detection of the phys!cal state of HPVs In clinical specimens. A variety of D~A-hybddization techniques were adopted In the project in 1984. As one of the specific alms of the project, i.e. to ~evelop thsse techniques app~ical~e In routine diagnostic use, the subsequent modificalions and refinements of thb DNA-hybddlzation techniques have been developed since 1985, and detailed In a sedas of publications. Accordin.':gly, the following DNA-hybridiz~an techniques in vadous modifications are currently Included to analyse the biopsi~s and PAP smears derived from the four petiant groups: Southern blot, in situ hybridization, dot blot hybridlzatlo.h, and filter in situ hybddiz~on. Different techniques show different advantages and in H~ detection. The det~s how to perform as well as the advantaces and l]mltatlons of these disadvantages methods have been .~xtenslvely discussed in the recent Iltera~re (Syrj~nsn S. 1990). In our project, ithe biopsies are routinely typed using In sltu hybridization with HPV 6,11,16,18,31,33 and 42 DNA probes. HPV D~A probes of all the known HPV types have been recently acquired from DKFZ and other sources, and they er~ currently available for testing of the samples. In doubtful cases, the HPV type Is confirmed by Southern blot hyb~dlzstion using a frozen biopsy, routinely taken from all patients. As an Intimate!part of the Kuoplo project, diagnostic tests have been developed in collaboration with Finnish blotechno~ Indust~ (BIOHIT Co. and LOCUS Co.). AS a result of this collaboration, a series of diagnostic kits (e~g, Blohit In sltu HPV.' soreening and typing kits), based o~ in situ hybridization or dot-blot hybridizal~O~l with biotin- labelled HPV probes, ihave been launched in the market since 1990. At the moment, six different commercial kits are available for rapid,and reliable, typs-specific diagnosb of HPV and EBV. This project have now moved to Turku Univeristy, Medic~. O~urrently, in situ hybridiza~on tests for CMV and HSV are under the development. They will be launched In the markpt next year. A development of novel diagnostic tests utilizing e.g. mlcrotitre plate-based hybridization technoI',~jy will he started in the year 1994. The polymera~el chain reaction (PCR) introduced in 1985 ls an in vitro method for primer-directed enzymat~ amplification of specifi~ DNA or RNA fragments with remarkable frequency. Over the past few years, PCR has been used extensively for HPV identification from ex/o~ated cendcal cells and biopsies, either on fresh or fixed samples. Because PCR can an~.plify the HPV DNA of low copy number HPV infections, it is the only means to make the diagnosis of subclinic~l.~ and latent HPV infection~ Developmental work has also been conducted to refine the PCR methods into diagnostic use, including the quarttitation of the technique. All samples remaining HPV-negative with the hybridization techrilques are anabjsed with PCFL Furthermore, all cytological samples from the male urethra a~e subjected to PCR. &2.10,Dev~op~ant of ~okxj~,l tests for HPV ¢FBcjnosis Serological test for the detection of HPV type-specific antibodies have been avai]abla only for a few years because them exists no in vitro culture system for replica~on of papillomav/ruses. For the production of viral antigens, the proteins have to be expressed in I~aterologous vector systems (e.g. ~ (Liet al 1987; Jenison et al 1968; Stdke et al 1989; Jochmus-Kudlalka at al 198.9;, Mann st al 1990). Alternatively, the seroreactive regions of the indh, iduaJ proteins are chemicalty synthesized and employed in pept~de EUSAS (Diliner et a] 1989; Krchnak et BATCo document for Mayo Clinic 28 March 2002
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a] 1990}. Using these, two approaches, a high prevalence of HPV 16 and HPV 18 E7.spscific antibodies was observed in sere fTOn'~ cervical cancer patients as compared to ege-mmched controls (Jochmus-Kudislka st a11989; Krchnak et al 1990; Mann st al 1990; Muller et al 1990; 199'2). Preiimina.,y data suggest that HPV 16 E6-specifio antibodies are more p~revetent in women with CIN lesions, and hence may present a marker for the risk to develop centicat cancer (M.Mu..'ller and LGissmann, unpublished data). AntJbodlss directed to the HPV 16 E4 protein prevail in adults attending ~ STD clinic or in women presenting with an abnornal PAP-smear due to a productive HPV infection. Antibodies t~ additional viral proteins (e.g. E5, L1} were investigated but so far the results have been less striking. The analysis Of the Kuoplo serum bank should not o~ly provide basic inslghts into the natural history of genital HPV Infect~ons~ but it is expected that serological rna~ers can be Identified for the cancer-risk as well as for prognosis of an alre.~.~ly existing disease. The following peptldes shall be used for EUSA using the procedure described by Muller ~al (1990): HPV 16 E4 (KPSPWAPKKHRRLS), HPV 16 E6,/1 (MHQKRTAMFQDPQERPRKLPQLC) and E6/2 (MFQDPQE~PRKLPQLCTELQTTIHDIILEC), HPV 16 E7jl (PTLHEYMLDLQPETTDLYCYEQLNDSSEEE), and E-'7/2 (NDSSEEEDEI.:DGPAGQAEPDRAHYN), HPV 18 E6/1 (MARFEDPTRRPYKLPDI.), E6/2 (FYSRIRELRH- YSDSVYGD), E6/3 .;~HLNEK-RRFHNIAGHYR), E6/4 (NRARQERLQRRRETQV) and HPV 18 E7 (SDSEEENDE- IDGVNHQHLPARRAE'.PQRP0. Seroreactive reglons of HPV 11 are presently being identified and the respective peptidas shall be Incf~uded in the study. These stud,s are conducted b~ coilabocafion w~h Prof. Lulz Gissmann'$ group in ~ ~ and that of Dr. V. Vonka in Prague, Czech, who ere internationally recognized experts In this field. : Moisculs~ medhanisrns responsible for cell transformation by PVs and the subsequent ma~gnant conversion are emerging (Howi~ 1983; Gissmann 1984; Pfistar 1984; Schwarz 1987, Campo 1988). Better understanding of the interact~on bstwes~ HPV and cellular oncoganes.umloubl~ has important implications in the cell transformation process in general (P~o." u et al 1984; 1987; 1988). The cellular oncogens can be activated by several mechanisms, Including chromoso~..al translocstlons, po~ mutatlons, reanangements in DNA, and oncogene amplification, In several cervical c, arci~oma cell lines, HPV DNA has been shown capable of integrating close to cellular oncogenas such as c-src, c-myc, iand ~raf. thus indlcatlng the possibility that interference with the expression ofcellular proto- oncogenes may contours to the malignant phenotype (Campo 1988). Cewical carcinomas containing HPV 16 and 18 DNA have shown ~ correlation between amplification of both c-mvc and c-Ha-ras-1 and advanced tumor stage. In early invasive cervt~,a~ carcinomas c-rnvc expression is closely correlated with prognosis; the 18-month relapse- free suwival of womeh with normal c-mvc expression was 90%, as compared with 49% for those with high c-nwc expression (Rlou et al:1987; 1988). Moreover, it has been shown that HPV 16 can coopsrate with the res oncogene product in lcansformir~g prima~y rat kidney cells (Campo 1988). These data st~ngly suggest that cellular oncoge~es, activated through a variety of mechanisms might be involved in cell trar~formation by paplllomavfruses. No data me available on the dynamics of c-oncogene activation in HPV lesions, Le. we do not know yet, whether such an activation of c-oncogene invariably results in an aggressive clinical behaviour of HPV lesions. However, such questions can be solved by analysing the c_-oncogane activalion In our prospectively collected biopsy materiaL A sedes of studies will be completed in which the selected series of biopsies CHPV 16,18,31,33) are tested for amplification of the major ceiluler oncoganes. For that purpose, DNA-probes of the rna~or oncogens have been acquired, suitable for testing by Southern blot, Northern bk~t and/or in situ hybridization techniques. Hybridization tests are correlated with immunohistochemically detectable expression • of these oncogens Llsing the appropriate antisera to these o~coproteins. The literature and our projects on BATCo document for Mayo Clinic 28 March 2002
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14 antioncogenas p53 an~ RB are .summarized in project IlL I~ has b~en euggested that coex/stant infec~ns by HSV, GMV, Chlamydia (or other infectious agents) in HF~V-infe~ed i:~lents ~nay be involved by pop,ibis syne'cj~o mecl~nlsms in cervical oaroinogen~is. To explore this issue, the petlent~'~ are subjected to oewioai swabs for isolation of HSV and Chismydia, taken at their each attendanc~ in the clin~. All women areitested for HSV and CMV antibodies with EUSA at each clinical attendance, to assess the validity of the hypoth~is presented some years ago that HSV, CMV and HPV could act synerglstlcaily in cendcal ceminogenesis, the te~ts being completed at the Department of Clinical Microbiology. A varying degree of impairment of cell-medla~ed Immunity (CMl) has been detected in patients with skin warts and genital warts, prong that susceptibility to infection by a particular HPV type is related to the degree of CIVIl defect. Prior to the r~..~.ults reported from the present project in the mid 1980's, no approaches were available to assess the nature of ~ch a defect. Since the bagi~ntng of the present project, the analysis of the punch biopsies using monoclonai antibodies for immunocompetent !cells and their subsets was included in the protocol Cnjostat ssotions were stained for the Langerhans ceils, Naibral Killer cells, B cells and T cell subsets. A panel of following monoclonal ar~ibodies am used: Leu-lO, OKT-3, ..OKT-4, OKT-6, OKT-8, and HNK-1 (Leu-7). The percentages of the positive cells in the lesions am established, and ~ OKT-4+ / OKT-8+ (1" helper/t" suppressor cell) ratio is calculated in each specimen. As a more rec~.~ nt approach, these monoclonaJ artdbodles, h~ve been used to assess the above immuno- competent cell subsets~ in the periphe~ bic~¢l, using the FACS (Fluorescence Activated Cell Sorter) available at the Dspadment of ClirdcaiiMicrobiology. 5.2.15.HLA types as ~ moclif~,m of bnmunc~nc~ms A variety of human diseases are currently known to be linked with certain HLA antigens, (i.e. Class !, II, and III), the products of theiMajor Histocompetiblllty Complex (MHC). MHC products are known to ragulete the important immunological functlo~s, previously suspected to be Involved In HPV Infections: Class I! molecules, HLA.DR, -DP, and -DQ, are the Immune response, Ir determinants involved in the presentalion of antigen in the early phase of the thymus.dependent Imr~une response, and control of the Immune suppression, Is determinants; Class I, HLA-A,B,C molecules are also Ir~olved in thymus-dependent immune responses at a later stage, e.g., in the lysis of virus infected target cells by! cytotoxic killer T cells; ar¢l Class III, complement molecules are involved in the handling of immune complexes. Since 1986, HLA antigens have been typed in women included in Group A and Group B. HI.A-A, B, C and -DR antigens are determined by standard two stage micmlymphocytotoxicity tests, and HLA-D typing is done in mixed lymphocyle cult(Jres (MLC) using a panel of local homozygous typing cells. The frequencies of 49 different HLA antigens are determined in HPV patients in a large series of appropriately selected controls. The differences between the patients and controls are compare~ using the Mann-Whitney U-test. This study has been completed for 100 raJ'Mornly selected women so far, and some significant HLA/HPV associetions were disciosed, e.g., the signif'cant link of HLA~DR5 with a) the high grade CIN, b) with the high risk HPV type 16, and c) with clin'cal BATCo document for Mayo Clinic 28 March 2002
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15 progression (report s~bmitted to J. Infect. Dis.). Future studies will be focused on evaJuating the regulatory role of HLA antigens in cert~n specific p~Jent groups with different disease outcome. To analyse th.~ role of sexual beh~viour In the tmnsmisslon of ce~cal HPV infections, the pa~ents are requested for these ~its by a detailed qusstk~nnairs. The pretested form consists of 37 questions covering the entire field of sexual .~ablls as detailed in the previous application in 1985. Similar data are being collected from age-matched he~hy ~-.ontrol women by the same questionnaire. This is accomplished by mailing the form to the women who on cytol~glcal screening in the Laboratory of Pathology, Finnish Cancer Society (I) present with an HPV-negative PAP sn~ear, as previously described. By this approach, a case.control design is made, providing valuable information ~ the risk factors predisposing the women to clinical HPV infections. The bulk of these results are Included In.. the M.D.(Ph.D.) Thes~s of Dr. Vee~ Kataja, which was subjected to public examination a few months ago (See App~.~.: ndix for Publications). The main focus from here on will be the more detailed analysis of these vast amount of data, .e.g., the role of smoking and hormonal factors. 5~_lZi/lass~mmi~ Programme for ger.~l HI~ inf~c~m Epidamlologi~ reports from most countr~ss unequivocally show that the incidence of genital warts has been steadlty increasing thrd,.ughout the 1970's and 1980's, especiaily among young people (Chueng et aL 1984). Reliable incidence and prevaie~c~ e figures for the cervical HPV Infections In unselected femaie populations were not ava|'labis until the present proje~t.~, however. As a part of the'~present project, a special mass-screening programme to define the Incidence and prevalence of cervical HPV Infect.tons in unselected Finnish female population was completed dudng a two-year pedod 1985-1986, by inviting ~he entire age group of 22-year-old women resident in Kuopio province for screening by PAP smear& By this meanS., it was possible to establish the incidence end prevalence of genital HPVJnfactlons in an unsalected female popul~on. The statistical l~mant of the data permitted estima, ons of other important parametres, e.g., the lif~-time risk of acquiring an HPV infection. It will be of interest to analyse the screening results of this cohort (womenlbom in 1963) after 10 years, Le., when attending the normal mase-screardng starting from The routine m~screening programme for cervical preeencer resions is conducted by the Finnish Cancer Society. The ennually ~ccumulating data from this programme in Kuopio province can be utilized to monitor the prevaisnce and trends ~f clinical HPV infections in an unsalected, random population, based o~ some 8.000 annual PAP smeam. Using thi~ unique opportunity, some significant trends and fluctuations in the disease prevalence have been noticed, as rece~ly reported. The data of the. project have been computerized since 1985, permitting the use of an unlimited variety of statistical tests to enaly~e the vast amount of data obtained by the above described techniques. Some of these tests are conventional, including the Student's t-test, Chi-square test, end Mann-Whitney U-test. Some am less frequently used, advocating • short description here. Estimation of reproducibility ~n diagnostic histopathology is p(~ssible by applying the kappa statJstic. This statistic takes into account the contribution of chance agreements. In general, kappa (K) can be thought of as an observed agreement not accounted for by chence, cl'rvided by possible agreement not accounted for by chance (Cohen 1960; Landis et al 1977). This test is used to evaluate the intraobserver variation in the grading of PAP 0 0 i BATCo document for Mayo Clinic 28 March 2002
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16 smeam and punch bldpsies by the one and the same pathologist (the principai inves~gstor}. The reproducibility of his grading proved to be substantial, e.g. for PAP smeas:. K = 0.756 (0.560-0.952), for biopsies: K = 0.779 (0.656-0.902) (the ~5 ~, confidence limits in the brackets). It is customa~ in the follow-up studies to apply the life-table or survhral table analysis in estimating the outcome probabil~ ~ an event of interest. This method is applicable in many types of studies with a time-to- response outcome. Tl~.e ability to use data from cases for which the response has not yet occurred distinquishes the techniques from other statistical methodology. Such dsta are called incomplete or censored, and may arise from loss of fotl~w-up, death from causes other than the one under study, or no response before the end of the study. To describe the foliow:..-up events, i.e. spontaneous regression, (persistence), and progression of the HPV lesions, the me-table analysis ;. has been adopted. This technique permits the stratifica~on of the cases according to parameters of interest,:e.g. HPV type, grades of CIN, PAP smear, colposcopy etc. To compare the survival curves created by the Iife-tal01e analysis, the generaJized Wilcoxon's test applied by the SPSS-X programme is a valid • . method. Another, mor~ f~equently used method for this purpose is the log-rank test (Mantel 1966). The advantage of the latter is that the icompadson of the groups is equally valid throughout the follow-up. On the other hand, the generalized Wilcoxon's~ test puts more emphasis on the early events leaving the most recent follow-up (with possibly only few events) undeiemphasized (Peto et ai 1977). In the present project, the number of events starts declln]ng after 70 months, beyo ..r~d which relatively few patients have been followed-up so far. Thus, with the use of the Isttar test, the survival curveS of the present study can be regarded most reliable dudng the 70-month obsarv~ion period at the moment. Survival analys~ with covariates (Cox~s model) analyzes survival data for which the time-to-response Is influanoed by other exp.lanatory variables, These explanatory variables, often called prognostic factors or covadatee, usually represent eithe~ inherent differences among the study subjects (age, se~ grade of GIN, PAP smear, HPV type etc.) or constitute a se~ of one or more indicator variables representing different trea~nent groups (e.g., conization, laser, cryotherapy, interferon). The covadates may also desodbe changes in a patients prognoslIc status as a function of time. The ~ox's proportional hazards regression model presumes dasth rates (events of interest) may be modelled as log-linear funct~ns of the covadates. As one of the iong-ten'n aims of our research programme, we are Wing to produce a set of prognc~Jc factors which have an !influence on the progression or regression of the HPV Infections. This is accomplished by entering the different v~r~ables into the Cox's model by the stepwise procedure, as made possible In the BMDP computer software. The statistical modelling of the data was recently discussed by Dr, Kataja~ in his Thesis. Aspec=) As detailed abo~/e for the subjects in Group A and B, the HPV-positive PAP smear Is the single and the only criteria of the patient inclusion in the follow-up study. The same Is true with the recruitment of the women into the Treatment Group. On~ the women presented with HPV-positive smeam containing changes ~ent with GIN (HPV-CIN} are Invited in the Treatment Group. This is in agreement with the standards in Finland, according to which all women with cytological abnormalities severe enough to suggest a GIN should be referred to colposcopio (and additiona~j examinations, independent of whether HPV~ in'ion is present or not. All the patients included in the follow-up and treatment cohorts are representetJves of the female population living in the area covered by the Kuop|o University Hospital. The recruitment of the subjects also into ihe Treatrnant Group is accomplished by an invitmicn letter, malisd along with the pathologist's report of the PAP smear, in the same way as into Groups A aJ'¢l B. Again, it is completely up to the patient to decide if she waots to terminate the follow-up at any stage of the study. BATCo document for Mayo Clinic 28 March 2002
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17 The collec~onlaf the research and d'mgnosl~ matedaJ In the presem study is invariably accomplished by utilizing well established techniqes in wondwide di=~jnostJc use to monitor cervical HPV infections and precancer lesions. All the met~ds used to examine the p~tients and to obtain the rasearch and diagnostic rnaterial are those in common diagnostic use. The tresuttent modalitiss Instituted to the women in the TreaUnent Group include established treaiment !echnJqes; conization, laser therapy, c~yotherapy, as well as interferon, the efficacy of which is currently being explored in many multicenter studies. As in any diagnostic and therapeutio clinical work, certain minor risks exist, which are unpredictable. It is completely clear, however, that the patients in the present three sedes are not subjected to any risk major than due to the diagnostic procedures used (coiposcopy, PAP smear, punch biopsy, cervical swab, lsarum sampling, interview anonymously by a questionnaire), as well as due to the therapeutic procedures (coniz=lo~, laser, c~jo, ~ Interferon). No alternative procedures are available to conduct an adequate prospective follow-up ~ cenfcal HPV lesions (either non-treated or treated) other than those used in the present study, and repeated ~ intewals regarded safe to ensure that none of the HPV lesions wilt escape early detection and proper treatment, !whenever rna~ing clinical progress. The above dlag~nostlc and therapeutic protocols also apply to the male sexual padnsm ~ncluded in the Partner Group. This is also tn~,e with their continuous monRorlng by the gynecologists responsible for their examination in the Outpatient Departr~ent of Gynecology and Obstebtcs, Kuopio University Hospital As previously e. mphasized, the risks to which the present patients are subjected are limited to those due to the application of the icommonly accepted dlagnostio and therapeutic procedures. The anticipated benefit of the pa/ients while includec~., in the most carefully controlled prospective follow-up of the cewtcal HPV Infections ever run, highly exeeds the nag!hie risks to be expected from the use of colposcopy, PAP smears, punch biopsies, cervical swabs and serum sa~pling ~s well as ¢onizatlon, laser or cnJo (and Interferon) trea~ent. This benefit will be fully accentuated, when the!follow-up results are compared with the cllnlcal outcome of the HPV lesions In non-controlled or even in rnass-scme~ed female population, subjected .to PAP smears Irregularly or ~t 5-year Intervals, respectively. This is evident also foi the males, now being examined and treated, but usually presenting with subclinical lesions Indiscernible without ~niscopy, and as such forming a potential reservoir of HPV Infection to be transmitted to their sexual partners. 7.LITERATURE GR~D ~ " A~drophy, EJ., SchOlar0 J~'~., Lowy, DR, Identittca~on of the protein encoded by the E6 transforming gone of bovine papillomavirus. Science 1985; 2~: 442-445. Baker, CC., Phelps, WC,! Undgre~, V., Braun, MJ., Gonde, MA., Howley, PM...~zuctur~d and t~u~crlpth:~aJ analy=L~ of human papiilomavirus tT~e 16 sequence~ in cewi~d cercinom= cell lines. J. Virol, 1987; 61: 962-971. Baker, SJ., Feeron, ER., .Nigro, JM., Hamilton, BR`, Preisinger, AG., Jessup, JM., van Tulnen, P., Ledbetter, DH., Badcer, DF., Nakamura, Y., White, R`0 ~.ogelstein, B. Chromosome 17 deletion~ and p53 gene rnutat~ons in ¢olorect~l camlnome. Science 1989; 244, 217-221. B~ett, THJ., Silbat, JDo, ~¢G|nley, JP. Genit~ w~'t~-a venereal disease. JAMA 1954; 154: 333-334. Blnd~, M.0 Tourmente, $.~ Roth, J., Ranaud, M., Gehdng, WJ. In situ hybridization ~t the elation micro=cope of transcripts on-ultrathin seddons of Lowic~l K4M-emb~Ided ti~ue using bio~nylded probes and protein ,~gold complexe¢ J Cell Biol 1986; 102: 1646-1653. Beshart, M.. Gissmann, L~ Ikenberg, H., K]einheinz, A., Scheuden, W. end zur H~usen, H. A new t~pa of papillomm/irus DNA, its pre~ence in genita] cancer biopsies and in cell line= derived from ¢ewical cancer. EMBO J. 1984; 3: 1151-1157. Brabant, M.. McConlogue, L, van D~Jen Wetter,J, T., Coffino, P. Mouse ornlthlne deca~boxylase gena: cloning, st~cture, and exprasion. Prec. NStL AceS. SoL USA lg88; 85: :2200-2204. Brirdon, LA. Current ¢pldemiologlcai studies-Emerging hypothe~,e=. Banbu~y Rap. 19~6; 21: 17-28. Campo, MS. Viral and cellular oncogenes in papillomavirus-aa.'Kci=ted concern. Br. Jo Cancer 1988; 58(SuppI.DO: ~a. JL Immunoebctmn microscopical studies on vtruse~ E]ect~on Microsc. Ray. 1988; 1: 1-16. Chuang, T-Y.. Party, HO.. Kurlar, d, LT. and llstmp, DM. Condyloma acuminatum in P, oche=ter. ~ 1950-1978. L Epldemiology BATCo document for Mayo Clinic 28 March 2002
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18 and clinlc~l festUre~..~Jch. DermatoL 1964; 120: 469-475. Cohen, J. A coelficient ~ agreement for nom~a/scades. Educ. PsychoL Mess 1960; 20: 37-46. Crook, T., Wreds, D.. Vo.iJ.sdan, K}-L p53 point muta~on in HPV negative human cendcal carcinoma cell lines. Oncogane 1991; 6: 873~75. de Bmx. J., OI1]1. G., C~oi.s.~ant, O., Cochard, B., Ionesco, M. Lesions condyicm~tausas du ¢o| uterin: evolution chez 2466 p~entes. Bull ~ncer 1983; 70: 410-422. de V~ers, E-M. Het~og '.~neity of the human pepillomaviru= group. J. Vim|. 1989; 65: Dillner, J., Dillner, L, Rol~b, J., Willerr~, J., Jones, L, Lanca~er, W., Smith~ R., Lamer, FL A synthello pep~le defines a serologic IgA rasponse to ~ human pepil|omavirus-encoded nuclear antigen expressed in virus-cenying c~vicel neopl~sia. Prop.. NatL Aced. Sci. USA. 1989; 86: 3838-3841. Doll, R. Implice~ons of e~iclemicicgk~d evidence for future progress. Banbu~ ROp. 1986; 21: 321-332. Dunn, AEG., Ogi~e, M~M. Intzanucleer virua pad/¢lce In human genital wart t~ue: Obsewaffons on the ultrastructure of the epiderrnd layer..'J. Ultrast~Jct. Res. 1968; 22: 282-295. D~mt, M., Gls=mann L, I .~nberg, H., zur Heusen, H. A paplllomavlms DNA from a cervical carcinoma and ite prevalence in cancer biopsy samples from d~erent geographio regions. ProP.. Nat]. Acad. Sci. USA 1983; 80: 3812-3814. Dyson, N., Howl~y, P.M~, Munger, K., and Hadow, E. "The human pepillom~viru=-16 E7 oncoprotain is able to bind to the retinoblastoma, gpne produuL Science 1969; 243: 934,-936, Fenogllo, CM. and Feran.~=cy, A. L=ffo|ogi~ factors in cendcal neop/asia. Semln. Oncof. 1982; 9: 34,9-372. Rnlay, CA., Winds, PW.,i Levine, AJ. The p53 proto-on¢ogene can act as a suppressor of bansform=d~on. Cell 1989; 57(7): 1083-1093. Galloway, OA. HPV serok~ly: an update. Papillomavirus Pep. 19~0; 1{2): 1-3. Gis~mann, L Papillomav~t.~.~ee and their associaffon with ~ in animals and in man, Cancer SUP,'. 1984; 3: 161-181. Gmnsbaum, AN., Sedlis, IA., Sillman, F., Fruchter, R., Stenek, S., Boyce, J. Asan~a~ion of Human papillomevtru= Infection with cewical In'oaep~etlal neoplesl¢ Obstet. Qyne~oL 1983; 82: 448-455. Holl,~tein, M., Sidrm~ky, ,D., VogeL~tein, B., Harrb, CC. p35 muta~ona In human cencem. Science 1991; 253: 49.53. Howlay, PM. The molecu~" biology of papIllomavlrus lnu~lonna~on. Am, J. PsthoL 1983; 113: 414-421. Howl=j, PM. Role of the human pap=3lomeNiruaee in hurmm c, ar~ar. C, anoer Re~ lS91; 51: 5019-8022. Jenson, AB., Roseothal, JD., Olson, C., Pass, F., Lancaster, WD., Shah, K. Immunologic relatedness of pepil|ommr]ruces from different species.iJNCI 1980; 64: 4,95-500. Janl~on, SA., Rrzlalf, JM.,I Langanberg, A., Galloway, DA. Ident~ce~on of immunoreactive antigens of human i:~plllomsvirus type 6b by using Esc~'ierichic co~e~ed fusion proteins. J. Viro|. 1968; 62: 2115.2123. Jochmua-Kudialka, I., S¢ .h~eider, A., Braun, FL, K]mmig, R., Koldovsky, U., Schneweis, KF_, Seedorf, K., Gbem~nn, L Antibodies ag=inst the human...' pepillomevims type 16 esdy prcd~ns In human sere: conel~n of ~ reactivity with cervlcal cancer. J. Na~. Cancer lnst, 1989;, 81: 1698-1704. K]rkup, W., Evan~ AS., B~ugh, AK., Davb, JA. O'Loughlln T, Wilkln.~on G0 Monegh~n JM. Cervical inl~aepithellal neoplasie~ and "w~ atyida: a ..dudy of colposcepi~, hl~;olog|cel and ¢'ytologl~d eharaoted~ce. Brit. J. Obstet. Gyne¢of. 1982; 89:571- Kondoh, G., Mutate, Y., A~zasa. K., Yutsudo, M., Hakura, A. Very Idgh incidence of germ cell tumorigenesis (seminomegan~s|a) In human p=pillo ..ht~vlrua type 16 ~'~nsgan|¢ mice. J, V]roL 1991; 65: 3335-3339. Koe=, LG., Stew=l, FW., ~oote, FW., Jordan, MJ., Bader, GM., Day, E. Some hi.stoicgice] aspects of behavior of eptdermoid carcinoma in altu:and related ioslons of the utedne cewix. A long.tram prospe~Jve ~tudy. Cancer 1963; 9: 1160-1211. Kmhnak, V., Vegner, J., $~hankova, A., I~cm~r, M., Ritterova, L, Vonke V. Syntheff= peptidas dedvod from E'7 region of human papIIImavinJ= type, 16 used as anffgens in ELISA. J. Gen. V~rol. 1990; 71: 2719-2724, Kumlan, RJ., Janlon, AB,~ Lancaster, WD. Papillomaviru= infeotlon of the cervtx.lL Relationship to int~aepithelial neoplasie based on ~e presence ~ apeciffc viral sbuctural proteins. Am. J. Surg. PathoL 1983; 7: 39-52. Lambed. PF. P=pil|om=vi .d~ DNA replic=tion. J. Virol. 1991; 6~: 3417-3420. I.~'~di~ JR., Koch GG. Th~ measurement of ob=ewer agreement for ¢otegor~J data. B|ometdce. 1977; 33: 159-174. Levine, AJ. The p53 protein and ~ |nterect~ons with the oncegane products of the sm~d| DNA tumor viruses. Virology 1~0; 177: 419-426. Levlne, AJ., Momand. J. Tumor suppressor genes: the p53 and ret]noblastoma sensit~ genes and gana products. Biochim. Bicphy~ Act= 1990; 1032: 119-136. Lay|ha, AJ., Momand, J., Finley, CA. The p53 turnout suppre..~sor gane. Nature 1~91; 351: 453A56. U, C,-CH., S~aho KV, Seth, A., Giidan, RV. |dentificetlon of the human papiilom=virus type 6b L1 open reading frame protein In condylomas a~.d qorrespondLng ardibodias in human set,, J. V]roL 1967; 61: 26~4-2690. Ma~q, VM.. de Leo, SL. I~renes. M.. Bdnton, LA., Rawls, JA., Green, M., Reeves, WC., Raw~ WE. Occurrence of IgA a~l IgG a.'~bodies to select pept~des representing human papiilomav~ma type 16 among cewical c~J~.cer cases and controls. BATCo document for Mayo Clinic 28 March 2002
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19 Cancer Re~. 19S0; 50: 7815-7819. Manos, MNL, Ting, Y., W~ight, DK., Lewis, AJ., Broker, "l'R., WolinsbJ, SM. The use of polymerase chain reaclJon amplifica~on for the d~t~-tion of ~:enit~ human papillom~viruses. Cancer ceils 1989; 7: 209-214. Men~l, N. Evaiu~JJon of =uwivaJ data end two Rank order ~a~-ti~s arising in its consider~on. Cancer Chemother. Rep. 1966; 50; 163.170. McDougaa, JM. HPV imr~odallz~dion and L, ansforma~an. Papillovirus Rep. 1990; 1(3): 1-4. Melt|s, A., Fertin, FL, Roy, M. Con~/Iomatou~ I~slons of cenrbt and vagina. L Cytologic patterns. Acta C~ol. 1976; 20: 505. 509. Meichsr~, W., van den ~mle, A., We|boomers, J., de Bruin, M., Burger, M., Herbdnk, P., Meljer, C., Undeman, J., Quint, W. Increased dete~.:on rote of human pepi~mavims in cervical eerapes by the polymorase chain reac~on ~ compered to modified FISH ~d Southern blot analyeb. J. Meal. ViroL 1989; 27: 329-,335. Munro,, D~L, Rovinskl, B., Bemstein, A., Benchlmol, ~. Loss of • Idghly conserved domain on [053 as • result of gane deist|on during Frlend.vir~s-induced erythroisukemi,, Oncog~n~ 1988; 2, 621-624. Mulls, M., Gau~epohl, H.i de Mar~noff, G., F,'ank, R., Br~eur, R., G~mann, L IderflJfice~Jon of seroreactive reglone of the human pap~lomavirus t~pe 16 prtoeins E4, ES, E7 and L1. J. Gan. V~rol. 1980; 71: 2709-2717. Muller, M., V~dl, RP., gun, Y., Guerrem, E., Hill, PM., Shah, F., Boach, IX., Munoz, N., Glssmann, L, Shah, KV. Antibodisa to HPV-E~ and L=7 ~roteine as merke~s for HPV-16-a~zcis~ed inves~ve cen~ce] cancer. V'~rology 1992; 187: 508-514. MOnger, K., Wernsss, BA., Dysork N., Phelps, WC., Howlw, PM. Complex formaSon of human pepillom~w~rus E7 proteins with the retlnoblsstom.:a tumor suppressor gane product. EMBO J. 1~; 8: 4099-4109. Munez, N., Besch, X., Y~or, JM. Doe~ Human papllk)n~vlru~a~ cause cervtc~d cencedt The stme of ~e epidemlologi¢~d evidence. Br. J. Cancer 1 ..9~; 57: 1-5. Na~el~ K., Roger, V., Ne~: ell, M. Behavior of mild ceP~.aJ dysplasia during long,term follow-up. Obstet. GynecoL 1986; 67: 669. Ntgro, JM., Baker, SJ,, PieisInger, AC., Jessup, JM., Ho~t~tter, R., Cl~my, K., Blgner, SH., D~v[dson, H., BaStiOn, S., Devilee, P., Giover. T,, Collie, FS., Weston, A., Meda~ R., Harris, CC., Vogalstein, i~. Muta~ons in the p53 gane occur in diverse human tumor ty ..1:~. Nature 1989; 342, 705.708. Nuovo, GJ., MacConnall, iP., Forde, A., Dalvenn~ P. {:)e~e~on of human papillomavirus DNA in formeJin-fixed lis~Jee by in sllu hybridS.at|on a~te~ arnpliflca~on by pofym~a~ chain r~'tiorh Am. J. Pa~oL 1991; 139: 847-854. (Oriel, JD. Genital wa.,ts..Bex. Transm. Dis. 1981; 8: 326-329. PakC-'no, JJ., E~senberg, ~Ud., J~nne, OA. Protetn-DNA lnterac~lon~ in the cAMP responsive promoter region of the routine ore|thine decacboxylase g~ne. NuPJal~ Acids Ree. 1991; 19:. 3821-~27. Peto, R., Rk~, MC., An-n~ge, P., Bres[ow, NE., Cox, DR., Howaxd, ~V., Mental, N., McPhenJon, ~, Peto, J., Bmith, PG.: Dealng end analysis of .r~ndomized clinical ~a~ requL, ing prolonged observation of each p,~iant. I!. Analysis and e0mmpis¢ Br, J. ~ 1977; 35: I~ter, I-L Biology and bI~homis~y of papillomavimses. Rev. Phyd, oL Blo~hem. Pharrn,,col. 1964; 99: 112-181. Parole, E, ,~avia, F_ Cyto!ogy of gynscolog~¢ condyloma ac~umir~um. Act~ CyCof. 1~77; 21: 26~1. R]ou, G., i~nols, M., To~jman, I., Duttonqu~, V., Od~, G. Preeance da genorne~ de papillomav]nJs e~ amplifl~dton does oncogen~ c-mvd et c-He-ms dane do~ cencem envahls~nts du col de luterus. C.R. Aeod. So. Paris ~er. I]], 1984; 29<J: Riou, G., Banols, M., Le, IMQ., George, M., Le Dou~ V., H~e,C. ~rnvc proto-oncogene expression and prognosis In eady cacinorn~ of the ~erine cewix. Lancet 1967; [: 761-763. Rlou, G,, Ba~ote, M., She~g, Z-M, Duvillmd, P,, Lhomme, C, Soma~o deletions and muta~ons of the ¢-Ha-ras gene In human cenn~al cancers. Dncogene 1~; 3: 329-333. Rowson, !0~., Ma~, BW~. Human Papeva (Wa~ vires. Bac~'loL Rev. 1967; 31: 110-131. • ~'..halfner, M., Munger, K.i Byme, JC., Howe|y, PM. ~ ~tate of th~ p63 and retinoblastoma genes in human cervical cell Pro~ Na~. APed. Sc~. USA 1991; 88: 5.523-5527. Scheffnsr, M., Wemess, BA., Huibregtse, JM., Levine, AJ., Howe|y, PM. The E6 oncoprotein encoded by human paplllomavirus types 16 end 18 promotes the degenera~on of p~.3.. Cell 1990; 63: 1129-1136. Schwarz, E. Transcription of pepillomavin~s genomes, in. Syrian, K., Gissmann, L & Koss, LG. (eds). Papillomaviruses and Human Disease. Springer-Verlag, Heidelberg, 1987; pp. °~hirasawa, H., Tom,a, T., '.Kubota, }<., Ka.sai, T., Sekiya, $., Ta~am~zawa, H., Simizu, B. Transcriptional differences of the Human papillornavinJS type 16 genome between precencemu,, I~kx~e and invas~ve c~rcinomas. J. ViroL 1988; 62: 1022-1027. Smotidn, D., Wettstein, FO, Transcription of human papBornavirue type 16 early genes in a cen~d cancer and • cancer derived ~ line and ider~-~on of the E7 protein. Proc. Nat|. Acad. SoL USA 1986; 63: ~. Std~e, DG., Bonnez. W.. Rose, RC., Reichmann, RC. Expre~don in Escher~chia coli of seven DNA fragments comprising the complete L1 and !;2 open readlng.frames of human pepiliornavirus type 6b and localization of 'common ~ntigen" region. BATCo document for Mayo Clinic 28 March 2002
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J. Gen. V~rol. 1~); 70: ~ Syrj~en, KJ. Morpholoi]ic survey of ~he condylom~toes le~ions in dyspP~tJc and neopl~.~Jo epithelium of the uterine cervix. Arch. 5yr]&"~en, K., V~.yn/nenl M., Ca_~'en, 0., M~.rdyj~'vi, R., Pyd~nsn, S., 'Ylt=k(>skl, M. Morphologic~ and immun~hf~ochemicd evidence of h .'..umsn paplllom,~ virus [I..IPV) involvement |n the dysp~est~ lesions of the uterine cervix, Int. d. GynasceL Obstet. 19~3; 2;1: 261-269. Syrj/~nen, K., de Villie~, E-M., V~yrynan, M., M~dyj~uvt, R., Parkklnen, S., Samikoski, S., Cast,an, O. CeNica] papillomavlrus infection progressing to inv~ive canner in le== ~ lhree years. Lancet 1985; h 510-511. Syrj~nen, KJ., Gissman~, L, Ko.~, LG., eds. Papillomavimle= and human disee.se. Heidelberg, Spdnger-Verleg, 1987. Syrj~nen, KJ., M~ntyj~u~ i, R., V~ynjnen, M., Syrj~nsn, ~., P~kldnsn, S., Yiieskoski, M., Saarikoaki, S., Sarkklnen, H., Nurmi, T., Castran, O. As.~ess." |ng the biological potent]a] of human papgfomsvirus infections in cervical carcinogenesis. C¢,~er Cells 1987; 5: 2~1-2~. Syrj~nen, SM. Basic concepts a~d pracl~cal epplic~t~ons of recombinant DNA techniques in detection of human papillomavims infeotJons. APHI~ 1990; 98: 95-110. V~yrynen, M. Nature] h!sto~y of cewlcal HW lnfect~or~. Colpescopi¢ assessment ~uld biological considerations. Publications of the Unlvereity o~f Kuop|o, Odg. Rep. 1986; 2: 1.83. Veasey, MP. Ep~demio]~y of cewica] cancer:. Role of hormonal factors, cigarette smoking a~d coupatlon. Banbu~y Rep. 1986; 21: 29-43. ; Vousden, KH., Wrede, I~., Crook, T. HPV ormoprotein furwtlon: releasing 1he brakes on cell growth ¢ord~ol. Papillommdrus Rep. lSSl; 2(1): Wemeas, BA., Levine, .=AJ., Howley, PM. The E6 proteins encoded by human papillomavirus types 16 and 18 c~n complex p.53 in vitro. Sclencb 1990; 248: 76-79. Woodruff, JD., Peterso~, WF, Condyloma arcumin~a of'he ~m~bc Am. J. Ob~tet. Gynecol. lg.58; 75: 1354-1362. Wrede, D., T',~y, JA., C~ok, T., La~e, D., Vou~ KH. ~n of RB and p53 prtoa]ns in HPV-positive and HPV-neg=dive c~eaica] carcinoma cell Ilne=. Mol. Caminog. 1991; 4: 171-175. zur Heu~en, H. Humani papillomaviruses and their po~ible role in squamoue cell cer¢inomes. Curt. Top. Microbiol. Immunol. 1977;78: 1-30. i zur Hau=en, FL Papillor~avimses as caminomsv~ruse¢ Adv. ViroL Oncei. 1989; 8: 1-26. zur Heu=en, H. Paplllomaviruses in ~mogenitei ¢~mcer as a model to understand the role of viruses in huma~ cencers. Cancer Re¢ 1989; 49: 1677-4681. zur Hausen, H. Hum~n xapillomaviruses in the pa~ogenasis of anogenita] cancer. Virology 1991; 184: 9-13. zlJr Hausen, H. VIru~e~ 1 human cancers. Science 1991; 254:1167.1173. BATCo document for Mayo Clinic 28 March 2002
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21 PROJECT I! (KUOPIO) ; HUMAN PAPILLOMAVIRUS (HPV) INFECTIONS IN CARCINOGENESIS OF THE AERODIGESTIVE 1.1.BACI(GROUND ! People's Flapub~ of!Ctt'B, whereas it is a ra~e ~ in Western counbk~ including F'mland. Eviderca has been Our i~sErntna~ data ~ that new es ye~ unmcogn~ HPV types might be involved in a subslzm~ prolxxl~ tmnor~ge~e~ (elff~'mutationsor~ bycomt:dex~3w~HPV16E6~) amin~olved One of the mo~t~ intriguing features of esophag~ cancer (ECa) is Its remarkable geographic variation (1,2"3). In most parts of the v~,orid, Incidence rates per 100,000 are around 2.5 to 5.0 for males and 1.5 to 2.5 for females. In ce~n areas, howe~ar, the Incidence rates are markedly higher, showing up to 500-fold differences from one area to another, from one ,¢~ount~y to another and between ethnic groups in the same countnj (1,2,3). The epidemiological data available indicat~ that a particularly high risk exists In the People's Republic of China, Sigapore, Iran, the USSR, Puerto Rico, Chile, S~erland, France anti South Africa (1,2,3). Similarly, extremely high rotes of EC, a have been reported in the north ~ of China, the Caspian littoral of Iran and the Transkei area of South Africa. In China, the a~nual deaths due to esophageal cancer account for 27% of all cancer deaths among males and 20% in females, ,r'~nking the leading cause of death from cancers, second only to that of stomacll cancer (1). In Unxlan (a county w~ a population of 800,000 in Henan Province of North-China), the age-adjusted incidence rates were 161.33/10s in ma)as and 102.88/10s in females during 1971-19741 The deaths clue to esophageal cancer in this area account for 16% ~f all deaths and 65% of deaths from cancers (1). Similar epidemiologlcal pattern of ECa was noted in Iran and So~th Africa, where up to 180 and 246/10s new cases of ECa a year have been recorded, respectively (2,3). Alth.~ugh the Incidence and mortality rates In the above high-risk countries slightly fluctuate from year to year, they do ~ot appear to decline to any signhlcant extent. The cause of tl~ese major geographic vadvations In the Incidence of ECa are not fully understood. No single etiological factor could'account for the dramaZlcal variation of ECa in all high-incidence areas. The range of difference in rates between high-.and Iow-dsk areas and the dramatic d~versity of incidence even within discrete geographical areas between sexes and among ethnic groups suggest a predominant role for some environmental factors in esophageal carcinogenesis. The esophagus is one of the most frequent body sites in contact with environmental factors, and is a major route of ent~ for foreign, often harmful and noxious agents into human body. Such cleleterious agents may include pathogenic micro-organisms, chemical irritants, environmental pollutions or food addi~es. It seems hihly probable that the pathogenesis of ECa is in some way associated with these factors. Comparisons of the dietary and cu.~ral habits of p.eople from geographically distinct high-incidence areas BATCo document for Mayo Clinic 28 March 2002
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have revealed van] I'~l~e.' In common to suggest a similar Induction mechanism. It remains possible that each of the high-risk areas have t~elr regional peculiarities which are probably of etiological slgn~licance. Cigarette smoking and excess alcohol intake! m~" be the major risk factom in some areas (e.g., Western countries and South ~), especially when theseitwo factors are combined (3). However, these ager~s do not appear to be a ma~or problem in China and Iran (1,2i. In China, attention has been focused en spes~c nutritional deficiencies including those of vitamins A,B, and G, as well as ce=~in mineral and nitresamines formed in moldy fcodstuffs (1). In Iran, the same natritlonal deficiencies! were also noted, and in addition, opium tar was blamed (2). Eviderlce is also accumulating to suggest an etioiog~l role of micro-organisms in esophageal carcinogenesis. Fung~J contamination and infection may be involved in the pathogenesis of ECa by producing nllrosamines and their!precumom in the infected food or producing mycotoxins. Laboratory tests have demonstrated that some common s~ciss of fungus belonging to Fusarium, Geotrlchum, Asperglllus and other genera not only could reduce nitrates io nitrites, but could also decompose proteins and increase the amount of amines in food, consequently promot .l~g the formation of nitrosamines. The mutaganlcity and tumodgenlcity of several fongl Isolated from the grain in the ~igh.rlsk areas have been demonetmted by both" in vitr...__.~o and in vivo studies (1). Like fungi, certain becterla may !also be associated with ECa by producing carcinogenic chemic~s and increeslng cell proliferation by stimul~ng the inflammatory process. It seems Ikety !that certain turnout viruses capable of tra.qsforming cells in vitro do play a role In growth regulation in vtvo, and do contribute to the oncogenIc process. Of these viruses, HPV, HSV, CMV end EBV have been Implicated in th~ pathogenesis of a vadety of human cancers, and they produce tumors in animals and | transform cells In vitro.iThese viruses have been also demonstra~d to infect esophageal epithelium (4), and therefore should be seriously c~nsidered as potential etiological i~gents of EOa, although the mechanisms of association of these viruses with EC.~ is unclear as yet. I~.I-tPV INVOLVEME]~II".: IN ESOPHAGEAL CARCINOGENESIS HPV infections nave been reported to Induce benign squamous cell papillomas in a number of body sites, Including the anogen" ~ tract, urethra, sklr~ lerynx, tracheobronch~ mucosa" nasal cavity-paranasal elnuses, oral cavity, conjunctive and esophagus (5,6). Besides these benign lesions, strong evidence has also accumulated in the ~ few yearn implicating an etiological role for HPV In the development of squamous precancer lesions and carcinomas, including ~ose of the anoganital tract, skin and the upper aerodigestive tract (5,00. According to o, urrent understanding, specific HPV types are necessary but Insufficient in oncogenests, synergistic actions w~ other initiating events being required (7). The evidence in favor of this concept includes the following: (a) the rep~ed appllc~on of methyicholanfhrene or tar to cottontail rabbIt papiliornavirus-lnduced papillomas of domestl~ rabbits caused them to conve~t morn frequently and earlier into carcinomas; Co) bovine paplllomavirus-induced. alimentary papillomas in cattle showed an increased tendency to malignant transformation in animals feeding on:the bracken fern which contains potent r..am..inogens; (c) juvenile la.,yngeal paplllomatosls converted into squamous cell carcinomas aller therapeutic X-ray irradi~on with a latency period of 5 to 40 yearn; (d) about 30~ of EV (epidermodysplasia verruciformis) patients developed skin cancers on an average of 24 years after the onset of the c~isesse, mainly at sun-exposed sites; (e) specific types of HPVs can induce immortalization in cultured human foreskin and ce~vicel epithelial cells, but the immortal cells are not tumorigenic when transplanted to athymic mice, indicating that additional factors are necessa~ for the malignant conversion. The most conviqcing evidence so far for a papiliomavirus etiology of esophageal cancer has been obtained in the cattle studied in Scottish Highlands, which is a remad~ably high-'~cidence area for squamous cell carcinoma of the upper alimentary, tract in c~lJe, that of esophagus in parlicular (8,9). In cattle, squamous cell papillomas of BATCo document for Mayo Clinic 28 March 2002
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the upper alimentanj ~ (including the tongue, palate, phanjn~, esophagus and rumen) am known to be caused by bovine papilloma~i,'rus type ¢ (BPV 4) infeetJon (8,9). Persistent and widespread papillomatosis and even cercinomas could bel experimentally induced with BPV 4 in these animals (9,10). Necropsy examinations heve revealed that carcinomas am usuaJly preceded by squamous cell papillomas In animals feeding on bracken fern; 96% of cancer beadnc~ animals showed benign papillomes in their aJimentary t~act, and 40% of the anirna~s showed more than 15 paplllom., as (9). In some i .nst'.ances, the progression from benign papillomas to cercinomas could be de~ly identified. Inge~-~ of bracken fern has been demon~r~ed es a c,~i~l fa~o~ in 1he malign~'~ convemion of these p~illom~s. 1t h~ al,.~o been demon~rated that the he.ken fem ~nt~ias o~oinogenic ager~ ~ immuno- suppressants. High c~py number BPV 4 DNA sequences could be detected in papillomas, but no viral DNA or viral antigens were found i~ e~er naturally occurring cancers or in experimental ones, indicating that the viral genomes am not required for the maintenance of the malignant stets= These deta suggested that (a) BPV 4 may execute one of the early events in Cell transformation, and Its genetlo inforrnstJon may not be required for malignant progression; (b) immunosuppressi .tin caused by the Inges~on of bracken fern allows the spread ~d persistence of BPV-induced -" papillomas; end (c) th~ bracken fem would supply coceminogans and carcinogens, leading to the full transform~on and tumor progression. (8-10). HPV infeot~on i~ human esophagus was first suggested in 198~ by Syrj~en et al (11,12), who found that 40% (24/60) of esophagea!!squamous cell carcinomas presented with histological changes identical to those of genital condylomas. Our grodp was also the first to demonstrate HPV structural proteins in benign esophageal squamous cell papilloma. These ~esults have been confln'ned by othem demonstrating HPV-suggesl~ve lesions, HPV antigens as well as HPV DNA sequences in esopl'~geal squamous cell lesions (13-25). These findings, in alignment with the current evidence on ~ etiological role of HPV in the pathogenesls of squamous cell caminomas at other mucosal sites, led us to censid~r HPV as a potential etiological agent in human esophageal ~minoganesis 1.3.FlEa.r1" 8TIJDIF.~i OF OUR G~OUP To ~e~ the abo~e concep~ on HPV ~ esophagesJ (~ncer, we II~ve i'~enlly ~Ydrlued the work on these lines by analyzing ~ .~r~es of asophege~J precen~er and ~r lesions celle~ed in the high-~k ~e~ in LJnxian~ Chin~. In this work, lig~ microscopy, ~10rid~.~ion and polym~ erase chain resc~lon {PGR) lechnlque~ h~ve been ~oplied on such s~mples in our lal~or*'~ory [~,~,2b'). Hl~ Infe~l .~ns in the esophagus were demonized by ~11 ~ fechnlques. HPV-suggest~ve lesions of ~ the t1~ lype I22/~i) and le~ frequently of en inverted one [2J~} were found in ~ ~ot~J of ~.g.0% (2,~/~1) of the specimen8 an~/zed hi~op~hciogic~lly. Of the 51 formalin-fixed0 p~-~llin-embedded specimens, 43.1% (22/~1) were shown 1o ¢on~n HPViDNA sequencea by in HPV 18 in 8 [1&7%), HPV .18 in 6 ~1 ~.8%), double ~ ~ HPV "11/18 in 1 ~2.0%), and HPV 18/18 in 1. HPV- like pertk',les in the nuclei of koilocy~ollo ~ell~ were demonsl~'~ed in 2 of the HPV.posi~lve by in si~u :~/bridiz~lon. Using the PGR ~e~niqus, addit~on~ 3 samples were found to ~ HPV 11 DNA sequences. Of the ~ DNA s~'np~es extracted from the fresh esophageal c~'~nomas and their surrounding ~esues0 ~ were ~own ~o contain HPV DNA sequences by So.bern 101ot I~l~ridiz~lon under low etrlngency conditions. Eigi~ of them g~ve posiWe hybridiz~ion with the probe oo~kt~'! of HPV ~ypas 11, 18 ~nd 18 under high st~ngency cond~ions. HPV DNA sequences in thase carcinomas appears ~o be prese~ mainly es en integr~ed form. In ~K~ther ~eries s~udied using fiiter in si~u bybridiz~ion. ~ ~o~1 of 53 ~ [~.3%) were shown ~o be HPV DNA-positJve. HPV DNA w~s detected in ~_.~ I23g) of pa~ents w~hou~ c'~ciogic~ ~'~pi~ in ~)% (3/~ w~ mild dysplasi~, in 80.6% (2S/31) BATCo document for Mayo Clinic 28 March 2002
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2,$ with moderate dyspla~.: "a, in 67.9% (19/28) with severe dysplesia, and in 66.7% (4/6) with an invasive squamous cell carcinoma. Our resu~ clearly indicate that HPV Infectiorts in human esophagus do occur, and, indeed they seem to be remarkably prevalent in the hlgh-risk area for esophageal cancer in Henan Province of China. In fact, the morphological feature~ and HPV type distribL~on in esophageal HPV infections are comparable with those described at other mucosal site~. The above resp~ confirm the HPV Involvement in the esophageal squamous cell les~ons, and suggest that HPV infection might I~ave an important etiological role In the pathogenesis of esophageal cancer, most probably acting synerglstlceJly ~ physical, chemical, and/or nutritional factors, previously related with this mallgnar~-y in the high-risk areas of Chiha. Our data are in alignment with the recent observations mp(xted by Willlamson et al (24) and Miller et al (25) f~om the high-incidence areas of South Africa and Alaska NatNes. The above msuls form a People's Republic of ~3hina. it will be of special Interest to clarify whether s~milar mechanisms.es those established for the bovine esoph ..~al carcinogenesis exist in humans as well. Work has ~ on progress In analysing the p53 tumor suppressor gene in our esophageal cancer series as welL These analys~s.' aim 1) to verify the ==us of ~ tumor suppressor gene in esophageal precancerous and cancerous lesions, 2) to analyze the p53 status in esophageal carcinomas with or without HPV Involvement, and 3) to elucidate the ro/e ~ p53 gene In esophageal carcinogenesis. So far, we hav~ analyzed 21 tumor specimens, in which 9 semples had contained HPV DNA sequences in cancer cells, and 12 ~ples were HPV-negativs. To Investigate the status of p53 gene in HPV-positlve carcinomas, the HPV-poaltlve raglans In the sedal sections were accurately marked and dissected off from the adjacent HPV- negative regions. Celiular DNA extracted from these HPV-posltlve cancer cells was then used for p53 gene The p53 exons.,.' previously shown to have a high incidence of mutations were the target sequences and Included exon 5 (code.: ns 126-187), exon 6 (codons 18-224), exon 7 (codons 225-261), exorl 8 (.codons 262-2~0) and exon 9. MutaIion.~,~ of the p53 gene were Identified in 8 of 21 tumors (38%); 3 mutations were "founc~ in exons ,5/6, 2 In exon 7, and ~ In exon 8/9. Noteworthy ls our finding of p53 mutatlons In 2 (22.2%) of the 9 HPV DNA- positive carcinomas, i These prellmin .~ results are cons~'tent with other reports of an 30-50% prevalence of p53 mutations in esophageal squamou~ cell carcinomas, adenocarcinomas as well as In cell lines derived from esophageal cancers (Holisteln et al., 1991; iBennett et el., 19~ Huang et at., 1992). These data sugges~ that p53 mutations represent an important pathway ~for esophageal .carc~ In addition to frequent detection of p53 mutations In HPV- negative esophageal bancers, genetic alterations In the p53 gene were also commonly found in HPV.posltive carcinomas, suggesting the intriguing posalbll~ that p53 mutation and HPV E6 oncoproteln may cooperate In the pathogenesis of someI esophageal carcinomas. As menfionedlabove, inactivation of p53 by binding to E60Rcoprotein and by mlesse~ mutations are two distinct pathways. The ~nechanism of loss of tumoi suppression activity by binding to HF'V E6 trans~rming proteins, differ considerably from~ p53 gene mutations or ~elic losses. Tumors resulted from HPV Infection and consequently E6 expression may co~ain only wild-type p53 gene (Crook et el., 1991; Scheffner et el., 1991). However, mutations or allelic losses in the p53 gene I~rgely derive.from exposure to exogenous carcinogens (Hotistein et al., 1991; Chang et el., 1993). Th~.se two factors may act independently on the cells, but may sometimes also act on the same cell and cooperate w~ each other. Our findings of the presence of l:~th HFV DNA sequences and p53 mutations in the same tumor pro~Je direct evidence for this cooperation. BATCo document for Mayo Clinic 28 March 2002
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To conclude, the results of our esophageal cancer studies provide evidence to support the hypothesis that certain HPV types ar~ essential but not necessarily sufF.,clent for maJJgna~ transformation, synergistic actions with other carcinogenic ag.."ents being required. Exposure to environmental mutagens or carcinogens induces mutation or loss of one wild all.~le of the p53 or RB gene, leading to a reduced concentration of the wild4ype p5,3. Cells with this genetic damage may acquire a selective growth advantage, but still show benign phenotype. If such cells were slmutaneously infeote~i with HPV, the reduced level of the wild-type p5,3 could enhance the ability of E6/E7 proteins to complex with all ~e p53 or RB proteins. However, in patients without HPV infection developing a tumor, the remaining p53 allele r~ust aJso be inactivated e'dher through point muta~ons or allelic losses. As pointed out before, esophageal mucose iS continuously exposed to a large number of environmental carcinogens (Chang etal., 1992b), many of which are kn~vn to eiicit DNA base substitutions and gena mutations either in vitro or in vivo (Holbein et al,, 1991). These factors may act sy~ergistlcally with HPV, leading to the development of carcinomas. This is in agreement with the InCreasing number of reports on a high percentage of mutations or allelio losses of p53 end RB in esophageal carcinomas (Bennett et al., 1991; Hot/stein et al., 1991; Bennett et al., 1992; Huang et aL, 199"2). Indeed, base substlt~ons were particularly frequent (60%) in pa~ents who were heavy users of both tobacco and alccho~ beverages (..~ctlstein etal., 1991), the two best recognized risk factors for esophageal cancer. The presence of frequent muta~onsI of the p53 gene in both HPV~ and -negative esophageal carcinomas suggests that environmental cardn .¢x~ens are the predominant etiological factors of esophageal canoer, and infeotions with HPV may be one of the prlomoting agents in a multistep esophageal cerclnogenesis. 1.4.DESIGN OF THE PRF_.P~NT STUDY To fully und ..errand the role of HPV in the development of esophageal cancer, it is necessa.,y to cam] out a well-organized and .~enslve epldemloioglcal study to assess the prevalence of HPV infections in the high.risk population. It is also ..e~,ential to assess the eventual differences in prevalence of esophageal HPV infections between the high- and low-risk ~eas. Our previous studies incfuded a sedes of biopsies from esophageal precancer lesions and cance~, and all t~ess samples were collected In the high-Incidence area In China (22,23,26). Thus, an exten.~ive series of esophageal samples derived from both the high- and low-incidence areas was regarded mandatory f.,qr further analysis to corfflm~ our previous observations on HPV. Dudng 1990-1991, Dr. Chang F~ju (having returned ito China ~ter graduation in Kuoplo) collected a series of >700 pa~ems wilh esophageal precancer lesions and! cance~s in the high- aml Iow.bcidence areas in the People's Repul~c of Ct,J~a. All blopslas and cllnical data wereitransferred to Kuopio by Dr. Fuju 1~. 1991, and they ere now available in our laboratory for prompt analyses. Thede samples wil~ be subjected to morphologioa] assessment, .liP/DNA In situ hybridization and PCR for HPV amplificJatior~ This series comprises a unique material which undoubtedly will grea'dy benefit our understanding of the ~ole of HPV in esophageal cardnogenesis. 1.4.2.Molecular Ciordng of Unknown HPV Types in Esophageal ~ In alignment with our previous Southern blot hybridization results, recently Ni et al (27) and U et al (28) identified HPV DNA it~ 57.1% (12/21) and 50% (12/24) of esophageal carcinomas derived from the high-risk population for esophagea~ cancer in China. Of interest was our frequent finding that the restriction patterns of HPV DNA were different from the eplsomal prototypes of HPV 6, 11, 16 and 18. This could indicate the integratJon of viral DNA into the host cell genomes. In alignment ~ the observafions in the genital tract (5,6,7J, the integration of viral DNA into cellular DNA has been regarded as an impoclznt event in HPV-oncogenesis. However, our results cannot rule out the possible presence of as yet unidentified HPV types or subtypes in esophageal lesions. 0 O~ BATCo document for Mayo Clinic 28 March 2002
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There is some ~,~iclence to suggest that an HPV 16-related virus, which diffem from the HPV 16 prototype in its enzyme pattern, ~ more closely related to oral prec~rcer lesions and carcinomas (29,30,31). Therefore, further studies should be foX, used on identifying additional HPV types or subtypes detectable in esophageal lesions. Recently, we have su~ssfully cloned and sequenced an HPV DNA fragment (450 base pairs in length) from an esopl'~ageal caminom~ By comparison with the known HPV types sequenced so far, homology of this new virus is less tl~n 50%, suggesting an entlmly new HPV type in esophageal lesions. Further isolation and characterization of these new HPV ty~es will be important and extremely helpful to fully elucidate the role of HPV Infection in esophageal carcinogehesis. 1.4.3.Routes of HPV ll~on in tile Esophag~ HPV tnfectlonelln the anogenitat tract are known to be sexually transmitted (5,6,7). However, the route by which HPV reaches the esophageal mucosa is not known as yet. Several conceivable w~s have been proposed for the occurrence of ,laryngeal and oral HPV infections. These include a) InUapartum infection dudng the passage through the Infected bi..~th canal, b) transplacefltal infection in utero pdor to ~irth, and c) postnatal infection by contact with infected Individuals (32-36). "r'ne recent discovery of HPV DNA In the f~reskin of normal newborn (33), In a high percentage of oral ~ pharyngeal cavity of neonates vaginaily delivered to HPV-infented mothers as well as in the amnlotl~ fluid (34.,35) ..f'~vors the mechanisms for HPV transrrEssion at bidtL Recently, HPV DNA sequences were also found in some fetal es~. phagus in the high-risk areas for esophageal cancer in China (28,36), suggesting that similar mechanisms may be l~olved In esophageal Infections as well. The dlscovenj of the genital HPV typ~s. HPV 6, 11, 16, and 18 in the esop~hagus further supports the hypothesis that esophageal HPV Infection may be acquired from" infected mothers, eithe..r by intrapartum mechanisms or using the transplacental route. For testing this hypothesis, we are planning to an.k~yze a series of esophageal samples (paraffin-embedded fetal specimens or cellular DNAs extracted from fresh f~ esophagus) by multiple HPV DNA detection techniques to explore the possible mutes of esophageal HPV infectlon. 1.4.4.Ge~e~ ~ of U,~ p~ Gene Loss of normal ~ function can be caused by infections with certain tumor viruses. It has been demonstrated that the SV40 large T .~nttgen, the edenovlrus EIB protein and paplllomavirus E6 protein (Levine st el., 1991; Chang st el., 1993) are able t~ bind to p53. The HPV E6 proteins induce an increased rate of p53 degradation. Tumors resulted from this pathS.', ay usually contain only wild-type p53. Notable example includes cer~cal carcinomas, where p53 mutations appe~ to be rare in HPV-associated tumors, but common in the malignancies which am not associaled with HPV i~ectlon (Crook et al., 1991; ScP, effnm et el., 1991). Esophageal cancer is an Intere~ng model to study the mechanisms of p,53-aascciatad tumorigenesis, Esophageal mucosa ~ continuously exposed to envlronrnental carcinogens and chemical irritants, agents including comsupdon of tobacc~ and alcohol, ingestion of mycotoxins end nitrosamines (Chang et el., 1992b). Some of them are known to elicit DNA base substitutions and cause gene mutations either in vitro or in vivo. Indeed, high percentage of mutations or all~llc losses of p53 and RB genes have been recently reported in esophageal cancer (Holl~ein et eL, 1991; Bennett et aL, 1992; Huang et al., 1993). On the other hand, loss of p,53 normal function may result from binding to E6 transforming proteins of HPV. "Fnerefore, it will be interesting to analyze the p,53 status In esophageal carcinomas infected with or without HPVs. An approach is made 1} to verify the st~us of 1o53 tumor suppressor gene in esophageal precancer and cancer lesions, 2) to analyze the 1053 status in esophageal carcinomas with or without HPV .~nvolvement, and 3) to eva~uate the role of p53 gene in esophageal carcinogenesis. BATCo document for Mayo Clinic 28 March 2002
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In addition to IHPV involvement in the pathogenesis of esophageal cancer in the high-risk areas, tha assessment of HPV irifections 8t other muco¢,al sites, including the anoganitai tract, oral cavity and larynx, in this high-risk population w~t~l be apparently interesting. For this purpose, we have collected a series of tissue samples from the uterine ~erv~ penis, vulva, vagina, oral cavity and larynx in the high-risk areas for esophageal cancer in China, during 19~O-1~91. By assessing the HPV types in these samples and comparing the results with the esophagea~ data wile l~e of greal Interest in understanding the role of HPV in pathogenesis of esophageal cancer and their rel~onships..' with carcinomas at the other sites. The material in the present study consists of more than 2.000 esophageal spedmens def~ved from more than 700 ~ suffering .flora a variety of esophageal squamous cell lesions, with pertinent clinical data available. These specimens are derive(~ from both the high-incidence area for esophageal cancer in Unxian cou~y of Henan province in China, and from the~ iow-lnctdence areas of esophageal cancer in China and in Finland. While collected during 19~:J0-|991, these sam.~les were transferred to the files of the Papillomavirus Laboraton], Department of Pathology, Univarsity of Kuoplo. !Part of them were available at the Department of Precancerous Studies, Hanan Medical University, China. The~e specimens have been fixed in formalin and embedded in paraffin. Another series of 100 cases of fresh esepha~ carcinoma specimens derived from the high.incidence areas of China are also available for study, Hlgh-molec ...~ we|ght DNAs will be extracted from these fresh specimens, to be analyzed for the presence of HPV DNA by Southern blot hybridization. The interesting cases which may harbor new HPV type(s) or subtype(s) will be further used foi viral DNA isolation and molecular cloning. In addition, we ihave also collected more than ~ cases of cervical precancer lesions and catch'minas, 65 cases of oral prec=mc~'i lesions and c~=t~x~nas as well as ~5 lapjngeal precancer lesfons and camtnomas from the hlgh-incddence areas in China. These samples will be analyzed for the presence of HPV infection by in situ DNA Fonnalrn-fixed, .~aralfln-embedded esophageal biopsies are cut into 4-.~m-sections and routinely stained with hernstoxylin and eosini (HE) for light microscopy. Epithelial lesions are graded according to the generally accepted criteda into mild dyspl~ia, moderate dysplasla, severe dysplasla, carcinoma in situ and invasive carcinoma. All cases will be eva~ua~ed with ~0ecial emphasis on morphological changes suggestive for HPV-involvement, and classified as fiat, inverted (endop .~c) and pepillomalous (~exophytic) condyiomas according to the criteria described previously (11,12,22~,26). 1.52.HPV-DNA ~ s~u Hyb~d~a~on In situ DNA hybridization on formaiin-fixed, paraffin-embedded tissues using biotin- or 3s$-Iabelled HPV DNA probes is performed as recently described by Syrjzlnsn et al (37). 1.5.3.E]eclmn Mk:mscopy (TEM) TEM on paratlin-embedded materials is performed as described by Widehn and IKindblom (38) with some r,3"l modif'~cations. The areas shown HPV-positive by in situ hybridization are marked, and the co-responding regions in ~ the par~fln b,~ck are cut out with a thin razor blade. The portions of interest ~e deparaffinized in xylene, rehydra~ed (-~ i I BATCo document for Mayo Clinic 28 March 2002
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through graded eth~.' ~ and postF~xed in 1% OsO4 in phosphate buffer for 1 hr. After dehydration, the specimens are embedded in ~ resin and po~ymertzed at 600C overnight. 1/~n-th~ck sections are cut and sUdned with 0.1% toluid'me blue and c.".hecked under the light microscope to confirm the correct regions. Ultr~hin sections are subsequer~_ly cut on b LKB ultcaIome, double-stained with uranyl acetate and lead citrate and examined under a JEOL J-lO0 electmn;:microscope. 1.5.4.Polymerase Ctla~n Reac~on Formalin-fixed,:~ paraffin-embedded biopsies are analyzed by the PCR as described previously (39,40). The specific HPV DNA seql uencas amplified by the PCR are subsquently confirmed by Southern blot hybdd[zation and dot blot hybridization ~vith 3Zp-labelled specific HPV probes as described eadier (38,~). ~.s.,~scx=t~m Sk~ H~(cr~aUon Fresh esopbe~eal biopsies derived from the carcinoma loci and their surrounding t~ssues will be analysed using the Southern blot hybridization under low- and high.~-'trlngancy conditions. After Southerni blot hybddizalion, the interesting. HPV-positive cases am selected for vbal cloning. The conespond]ng areas .bf the positive band are isolated from the electrophoresis. The DNA samples are treated with T4 potymerase and liglated into the enzyme-cut pissmid. Alter transformation of E. coil and colony hybridization, the correct recombinants ~with the viral DNA insert are selected out. The transformants which cordaln recombinant plesmid are mass cultured, and the plasmid DNA is isolaled. The inserted v~ral DNA is cut off with appropriate restriction endonucle~e. Viral DNA is sequenced and compared with the known HPV types. Subsequently, these eventual new HPV isolates can be used as DNA probes to examine the prevalence of these HPV types in the Paraffln-embedi:led sections are used for the routine immunohistochemistry to detect p53 protein expression as described in proje~ IlL DNA proparation: "Rssues containing suitable invasive tumors are marked and are accurately removed using a scapel to scrape tissues fror~ each serial slides. Similarly, areas sl~own HPV-positlve In the ISH slides are marked, and dissected from sedal .~ctlons. DNA is extraoted from the dissected tissues as described in project II1. P~ Ex~ns 5-9 of the p.~ gene are examined for alterations in DNA sequence using PCR amplification from ganomic DNA foilowed by SSCP analysis as described In project III. DNA samples containing altered tumor suppressor genes will be further cloned into plasrnids and subsequently sequenced using the chain-termination method, and the DNA sequences are determined by the automated DNA sequencer which is available in the Department of Biochemistry & Biotechnology, University of Kuop~o. O~ BATCo document for Mayo Clinic 28 March 2002
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1.Yang CS. Research on:. esophageal cancer in China: a review. Cancer Ass 40:2633-2644, 1980 ?_Joint Iran 1 IARC Study {~roup. Esophageal cancer studies in the Caspian li~ond of Iran: result of population studies: a prodome. J Neff Cancer Inst 59: 11~7-1138, 1977 3.Dz~y NF_ Some aspoct~: of the epidemiology of esophageal cancer. Cancer Res 35: 3304-3307, 1975 4.Goff JS. Infectious caus.es of ecephagi~s. Ann Rew Meal 39: 163-169, 1988 5.zur Hau=en H. Papillom.. =viruse~ ,,- cardnomzvtnme=. Adv Virol Oncel 8: 1-26, 1989 6.Syrj~nen KJ, Gissmonr~ L, Koss LG (ads). Papillomavirulee and human disease. Heidelberg, Spdnger-Vedag, 1987 7.Chang F. Role of papil!omavimees. J Clin P~ol 4,3: 269-276, 1990 8.Jan'e~ WFH, NcNeg PEI Gdrnshaw TR, Selrnan IF., Nclntyre WiM. High incidence area of cettJe cencer with a possible irdaradon between an environmant~ carcinogen and a papiilomaviru=. Nature 274: 215-217, 1978 9.Campo MS. Paplllom~ and cancer in cattle. Cancer Sulv 6: 39-54, 1987 10.Qe~kroger J, Chandra~hud L, Janett WFH, McQawle GE, Yeuda]l WA, McCalfe~t RE, Smith KT, Saved= Campo M. Malignant t~ansformaffon of ,~ papill.~ma induced by bovine papitlom~vlrus type 4 in the nude mouse renal cepsule. J Gen Virof 72: 1165- 1168, 1~91 11.Syrj~n~n KJ. Histologi~d change~ Idm~tlcal to those of condylometous lesions found in esophageal squamous cell cendnomes. Arch Geschwulstforsch 5;~: 263-292, 19~2 l?_Syrj~nen K, Pyrh6nen B, Aukes S, Koskeia F. ,~¢lesmons cell papllfoma of the esopbegue: a tumor probably caused by human papilloma virus (HPV). D'..rag Histopethol 5: 291-296, 1982 13.Winkler B, Capo V, Ra:umann W, Avedil MA, La,Oort= R, Reilly S, Green PMFL FI]chart RM, Crum CP. Human popillomavirus Infection of the esophagr~: ,~ cllnlcepa~ologl~ study with demon~'a'don of papil~omsvlrus ~'d~gen by the immunoporoxJda=e technique. Cancer 55: 149-155, 1965 -. 14.Le~m G, Gogusev J, .Farmeud H, Gorce D, Lemal~e JP, Verdk~- A. Presence d'un enffgene viral de group "Paptiloml~ dan= un ¢ondylorne oesophag~en chez I'homma- Gast~oenterol Ctln Blal 9: 166-168, 1985 15.Hills JJ, Margollu= KA~ Markowitz S, isaacson C. Human pap~om~virus infect~on releted to esophageal carcinoma in blade ~ Afdcens. $ Aft Meal J 69: 441-445, 1986 16.de Borge~ P,J, Ace~ec~o F, MIralles E, Mijares P. $quamou= pap,lorna of the esophagus diagnesed by cytology. Report of cese with concurrent occult epidermold ce~clnoma. Acts Cytol 30:. 487-490, 1986 17.Tome=!no R, Nuata R,: Napou G, Morello V: Condylomatou~ leukoplaklc lesion of the e~ophagu~ - a ease study with dsmans~'effon of papil]o .n~viru~ an~gen by the immunoperoxJdese technique. Its] J Gcdroentarol 20: 265.268, 1988 18.HaJe MJ, Uptz'i'R, Pete:rson AC. Association bebveen human popiltomevirus and carcinoma of the oesophagus in South African black. ~outh Aft Med J 7,6: 329-330, 1~69 19.Mod M, Shlmono R, In~ue T, Kuwano H, Sug]mlcht K, Zhang RG. Papilloma.virus and esophageal cancer In the Jepmleee and Chinese. Ga=t~oenterolog~ 84: 1126-1127, 1989 20.Kuisld J, Demeter T, S!erret GF, Shilldll KB. Human papillomav~rus DNA in esophageal carcinoma. Lancet 1i: 683-284, 1986 21.Hordlng M, Hording ~ D,~ugaard $, Norrild B, Faber V. Human papilloms virus type 11 in a falal case of esophages] and bronchial papillomaIesis. ~cand J Infect Dis 21: 2~9-251, 1989 22.Chang F, Syrj~nen $, iShen Q, Ji H, Syrj~nen K. Human popillomavirus (HPV) DNA in esophageal precencer Is.dons and squamous cell cercino~a~.' from China. Int J Cancer 45: 21-25, 1~0 23.Chang F, Shan Q, Zh~u J, Wang C, Wang D, ~yr~n S, Syrj~nan K. Detection of human p=plilomsvtru= DNA In cytologlc specimens de=~ved from ~soph~geal Wecenc~ |e~'~rl= and cencer. Scand J Ges~'oanterol 25:38,3-288,1990 24,William=on AI0 Ja=kiesl= K, Gunning A. The detesffon of human popil~omevirue in oesoph~geal lesions. Anticancer Res 11: 25.Miller BA, Beckman AM, Myereon D, lanier AP, Tan J, ~ M. The detection of human paplllomsv]rus DNA in archival a=ophageal cancer tL~sue~: of Ale=ks Nerves. The Al:~mct= of Papiilomavirue Workshop 1991, Sa,dHe, Wa~hlngtork pp. 155, 1991 26.Chang F, Syrj~ncn S, S.hen Q, Wang L, Wang D, Syrj~man K. Human papillomaviru~ (HPV) lvolvoment In esophageal precencer I~ions and equamoue ce[.I carcinomas as evidenced by d'df~rent DNA-technlques. Am J Gestzoant~rol 1991 (submitted) 27,NI FE, Luo DY, X~an l-r~,, Wsng CJ, Z~ao LS, Z~ou El., Lln Y, Luo CJo Detection of human papillom=virue DNA eequance~ in esophageal precencer led.one and carcinoma= from Yanting of SIchuan province. The Al:~tracts of the Chinese Congress on Esophageal Cancer. pp. :~4, 1990 (in Chinese) 28.11 y, Huang GQ, X]ao I~t', Huang Y]:, Mao T, Dang WJ, Song J, Ning QZ. The physical states of human paplllommdrus DNA in esophageal precancer I~ions and carcinoma=. The Ab,slracts of the Chinese Congrese on Esophageal Cancer. 10p. 24, 1~F30 (in Chinese) 29.L6ning T, ik¢nberg H, ~eckor J, Gb~mann LG, Hoepfer I, zur Hansen H. AnaJysis of oral papillomes, leukoplakies, and Inves]ve carcinomas for human pal~illomav'~n~s types related DNA. J Inv~t Darmetol 84: 417-420, 1985 30.MaPJand N J, Cox MF, Lyna= C, Pdme SS, Meanwal] CA, Scully C. Detection of human papillomavirus DNA in biops|e= of human oral t~sue. Br J C~mcar 56: 245-250, 1987 31.Chang F, Syd~nen S, k .~llokoski J, Syrj~nen K. Human pepillomm/irus (HPV) infections and their essoc~ations with oral dlsea=e. J Oral Pethol Med 20: 30~-317, 1991 32.Mounts P, Shah KV. R~piraton/popillomatosis: etiological rela~on to genital tract papillomavirus. Prog Med Virol 29: 90-114, 1984 33.Roman A, Fife K. Humzm papillom=virus DNA a=sociated with foreskins of normal newborns. J infect Dis 153: 855-8, 1984 34.Sedlacek TV, LindheimS, Eder C, Hasty L, Woodland M, Ludomirsky A, Rando RF. Mechanbm for human pop~llomavirus transmission at birth. Am J Obstet Gynecel 161: 55-59, 1989 35.Smith EM, Johnson SR, Cdpe TP, Pignatari S, Turek I Perlnatal vertical transmission of human papillom~virus and subae¢luent development of respiratory' tract popillomatosis. Ann Otol Rhinol Lmyngol 100: 479-483, 1991 36.Zhou J. Detection of HPV-DNA in the b~opsies from the pet]ents with esophageaJ cancer {abe. J Henan Meal Univ 2: 60, 37.Sy@nan S. Sy~j~nen K. An improved .in sk'u 0NA hybddlz~Jon protocol for detection of human papillom=virus (HPV) DNA sequences in paraffin-embedded biopsies. J V'~rol Meth lZ~: 293-304, 1986 38.Widehn S, K~ndb~om LG. A ~'ap~ and s.;mp~e method for etsctron microscopy of par,~ffin-ernbedded ~Jssue. U]trast~uct Pathol 12: 131-136. BATCo document for Mayo Clinic 28 March 2002
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2.HPV INFECTIONS IN ORAL GAVt'[Y. A POTENTIAL ROUTE FOR VERTIGAL TRANSMISSION OF HPV FROM AN INFECTED MOTHER TO THE C~LD 2.1.~UND a~ ava~ to ~p~ m ~ ro~ of H~V lS (and ~y oS~r ~ types) In o~ ~ := HPV involvement in ~oral mucesal lesions has been consistangy ~ed in verruca vulgarls, condyloma i acum~a~um, squamo~s cell papilioma and focal epithelial ~perptasla (FEH). Recently, HPV DNA has been found In other clinical entlt;e~ as well, including fibrotio hypeq~asla, Ilchen tuber planus, leukoplakia, and squamous cell carcinomas, but whether HPV is a cause or merely a passenger in these lesions remains to be established. Using the sensitive DNA hybridization and PCR methods, HPV DNA has been disclosed also in normal appearing oral mucosa, suggesting the existence of subclinical and latent HPV infections. About one-fifth of the 68 known HPV types (i.e., HPV 1,2,4,6,7,11,13,16,18,30,32,5"D have been demonstrated in oral lesions. Of these, HPV types 13 and 32 have been found exclusively in oral lesions, the other HPV types being present either In the genital tract or in cutaneous lesions as welL BATCo document for Mayo Clinic 28 March 2002
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The buccal mucesa resembles the ganital tract mucosa in many respects, e.g. being moist, covered by nonke~tlnized or sligT~ paraJ(era~nized squamous epithelium, and with a bewi~dering anay of pathogens colonizing It. Thus, it is reasonable to assume that HPV Infections behave in much a similar manner in both these mucosal sites. The role of ora~ cavity as a reservoir of HPV and its transmission to the upper and lower aerodlgestlve tract is 8n unsolved question. Because of the h~ghly pra~es~Jve character of the respire~ory ep'ff.helium, end the ra$ity of HPV.induced lesions in the ns-~J cev~/p~'enesat sinuses, the mouth is more likely to ac~ as a route of HPV to the I~jnx end the nesrb~, mucosel regions. The s~ of ~ mucos~ as a ~ rou~ (~ a sug~ ; Similarly, de~ite the confinuouls~ incressing emounf of data on orel squernous cell lesions, the resesmh focusing on the ~ ~f HPV in oreJ cerch'logens~s Is still frogman.. The mz~3r ur~solved problems Include, a) a mnswing of the ncm~nclature of orel HPV-tnduced lesions, b) a concensus of their h~ologic~l criteria, c) the of HPV in rele~on to ~her or~ microbes, end d) the significance of HPV in the development of oral premnlignent and malign~,'~ 2.;?..ORAL M/V41FESTAIIONS OF HPV kNFEC'IK)NS Oral scluamo~ cell p~pilloma Is a berdgn tumor, most h'equently encountered In patients in the third to fifth decries. In the eer~ ~980's, HPV e~ology of omJ 8C~s wss suggested by dernonstratidn of HPV p~Jcles end HF'V antigens (1,2,3,4), the ~letection rote vanjing flora 40% to 83%. In an extensive study of 140 oral $CPs, HPV ar~gens were found in 41% ~ the specimens (5). DNA hybrkJizatJon studies have revealed the presence of HPV DNA sequences in 33% to 100% of oral papillomas (6,7,8). The HPV types found in these studies include HPV 6, 11 and 16. in addition to these, Syrj~en et aL reported HPV 13 DNA and Eversole et al. HPV 2 DNA in one oral papilloma by ISH (9,10). Quite Rcently, Young and Ming demonstrated some reactivity with HPV 16/18 and 31/33/35 DNA probes In ten oral pep.'illomas with ISH (11). 22z~ acun~a~'n (cA) The first documentation of an oral condyloma w~s published In 1967 by Knapp and Uohara (12). Sub- sequently, only few edd~onal condylomas of the oral cav~y have been specifically reported, albe'rl these lesions have been included in larger sedes of oral HPV lesions studied. Evidence of HPV etiology has been provided by immunohistochemical studies demonstrating ~PV structu~ antigens (2,3,13), -and DNA hybridization studies disclosing HPV DNA (8,9). By immunohistochemistry, 10% to 75% of the lesions studied heve revealed HPV BATCo document for Mayo Clinic 28 March 2002
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32 antigens. HPV 6 and 11 DNA has been detected in up to 85% of the specimens, but also HPV types 2, 13 and 16 have been occasionally reported in CA. Occasionally,:: common wads are found also in the oral cave. HPV involvement has been conclusively demonstrated by DNA hybridization studies. In these studies, skin-aesociated HPV types 2 and 4 have been disclosed (13,14,15). ~When oral and labial W were correlated, 20% and 100% of the specimens, respectively, expressed capsid antigens (14). Correspondingly, 20% and 100% of the oral and labial Ws harbored HPV 2 DNA. in a d~r~"t patient g~oup, HW-trffected sub~ ,~o ar~ Nghly immun~ul~r~-ed, HPV ty~ 7, 13, 18 and 32 h~e I:~n foun6 in t~.eir oral ~rart~ (t 6,17). ~ty, a n~l l~pe, HPV 57 ~ 16ent~:~ in an o~ w~t CI8). NPV types 6 and 16 have ..been reported once in oral warts, but these lesions most probably were either papillomas or condylomas (19). Bec,~a~ use of the discrepancy in the nomancletura and histological criteria of SCP, CA and W, the correlation between these different stuclk~s is extremely difficult. The term FEH..., also known as Hesk's disease, was first introduced by Archard, Heck, Stanley, and Gallup in 1965 (20). This disease is a benign lesion of the oral mucosa, frequenlly found among Eskimos and Indians from No~g'~ ~ $ot~ A~edca, highest pr~m~ ~ ~ ~ ~6% (21). In Cau~ians, this ~ion has been regarded as an InfreqUent finding. Papillomavims-Rke particles were first demonslra~ed in FEH lesions by Lutzner at ~. in ~ (1~). HRV antiger~ were d~'nonff~ted ~o~1~ ~'~e~ls I~ Sy~f~n ~ ~1. (~). In on ~her immunohistochemical ~udy, HPV antigens were revealed in 80% (221. The characterization of HPV type 13 from FEH les~ons was published by Pfister and coworkers In 1983 (23). Subsequently, HPV 13 has been regularly demonstrated in FEH {24}. In 1987,iBeaudenon at al. d~,ril:~ a n~ HPV type 32 in 6 FEH sp~imens [ff~. Sin~ then, ~e resUlcted to oral cavity an~ caused by HPV types t3 anti 32. Oral leukoplaki~ is a whitish les~ histologica~ showing a variety of epithelial changes ranging from harmless epithelial hyperplasia with hyperparakeratosis and hyperortokeratosis to various degrees of epithelial dysplasla, including the in situ and early carcinoma (26). Because a substantial percentage of oral carcinoma appear to be arisen from the areas of leukoplakia, this'entity has traditionally been considered as a precancerous lesion. The etiology of oral teukoplakia is uncertain, but the exogenous agents such as tobacco and alcohol consumption-I:~ BATCo document for Mayo Clinic 28 March 2002
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have been implicated in the development of this lesion. The etiological role of smokeless tobacco has been appreciated only recently (2-/). The possible viral edoicgy was r=st suggested by light microscopic demonet~ion of HPV-suggestive ch.bnges. Subsequently, HPV antigens (28) and HPV 6, 11 and 16 DNA sequences have been disclosed in oral leuk(~plakia lesions (8,29,30). The role of HPV infection in the etiology of oral leukoplakia is still to Uchen planus=; (LP) is one of the most common epithelial diseases affecting oral mucosa. The possible viral etiology is propose~.i by the recent demonstration of HPV in a high percentage of oral lesions. Using immunoperoxJdase te~Lhnique, HPV structural proteins hsve been shown in LP lesions (29). With DNA hybridization m~'J~c~, HPV 11, 16 ~d 16-related types have been found in LP (8,31). Bythe PCR technique, 65% of erosive oral liellan planus samplesi contained HPV 6, 11 or 16 DNA (32). However, Keshima. et al. (29) could not demonstrate HPV 6, 11, 16, 18 or 3.,=1 DNA in their HPV ant~ poel~ve oral LPs, suggesting that another HPV type was involved in these lesions. In Sl~e of the prellminan1 results showing the presence of HPV in LP, the etiological role of this virus remains to be established. Because of ttie great vadety in the c~nlcal use of terms leukoplakla end hyperkeratosis, It is difficult to Interpret retrospectively, what propodion, of lesions named keraiosas have actually been leukoplakias. However, the presence of HPV 2, 6, 11 or 16 DNA in such leelons has bean shown by ISH and SBH (27,31). In addition, papilloma virus partlcl .~s have been found in a tongue flbroma and fibropapillomas (33,34), HPV structural antigens and HPV DNA In fibrous and papillanj hyperplaalas and a fibroma (35,36). A single report has shown HPV 16 DNA tn a glnglval hyperplas.=la (37). Recently, HPV capetd antlgens have been demonstrated In three ameloblastomas and HPV 16 DNA in an ocii.ontogenic keratocyst (38,39). The term subclinical HPV .Infection was originally applied to lesions in the uterine cervix thai could be visualized only Her th~ application of 3-5% acetlc acid and examination of the muco~a under the colposcope (54). To be strict, subclinical or latent HPV infection should be preserved exclusively to denote the epithelial changes, no matter how minor they !are, that cannot be visualized by any of the clinical diagnostic techniques including cytology and histology (55). The ma~ked fluctuation in the clinical course of HPV infections Is a clear indication of the fact that transition from a clinical infection to either a subclinical or a latent infection is a frequent event (56). So far, systematic evaluation of the oral cavity for the presence of subclinical HPV infections have not been completed. Only recently has HPV DNA been disclosed in clinically and histoiogically normal oral ep~elium. The first such report is from Maitland and coworkers in 1987 (31). They found HPV 16.related sequences in 5 out of 12 clin,~cally normal buccal epithelium biopsies. In other studies, HPV DNA was found in 1/17 and 3/33 normal oral BATCo document for Mayo Clinic 28 March 2002
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biopsies corrl~atera] to precancerous or cancerous lesions (7,29). The HPV type was undefined in these studies. By ~e PCR me~ed, Sni]dem et al. could not find HPV DNA in 4 biopsies from normal epithelium (17). On the contrary', HPV 6b and :16 were detected In 17% and 23% of oral scraplngs In adults, and In 24% and 19%, of those in preschool children, respect~valy (57). Thus, it is obvious that latent HPV infections are found in the oral mucosa similarly as they have been demonstrated In normal genital mucosa. Recently, a systematic study on oral HPV infections was started by our group. The first line goals of the study were to assess t~e frequency of HPV Infections on oral mucosa, to evaluate the effectiveness of the various diagnostic tools, and tO evaluate the transmission of genital HPM infections to oral cavity in 334 women with past or present genital HPV Infections. By clinical inspection, only three condylomas/ papillomas were diagnosed. Scrapings of oral mucosa revealed the presence of HPV DNA in 3.8% of the cases when analysed by dot blot hybrid'~ation. This HPV..-positivity represents only the subclinical and clinical HPV Infections but not the latent ones, because the basal cell~ cannot be collected by scrapings. By Southern blot hybridizalk~ 15% of the biopsies taken from clinically normal l~uccal mucosa contained HPV DNA. PCR method increased the HPV-positivity in clinically normal buccal mucos~ up to 22% (108-112). 2.3.HPV INVOLVEMENt" IN ORAL CARCINOGENESIS Oral squamou! cell carcinoma constitutes a major cause of mortal~ in certain regions of the worJd, including areas of Ind~ and South-East Asia (40). The factors thought to be Involved in the pathoganesis of oral cancer Include ¢hemlc~ carcinogens, viral and physical stimuli, chewing of betel quid, as well as consumption of tobacco and alcohol [41~). More recently, the ~'ole of smokeless tobacco in oral carcinogenesis has been an intensely studied subject as well (27). Immunosuppression In oral carcinoma most obviously plays a major role. The possibility of herpes simplex vlru~ (HSV) infection has been suggested, although the etiological role of HSV in oral cancer In now being re-evaluate~ (42). In addition, HPV DNA has been demonstrated in a substantial percentage of premallgnant lesions and cancer of the oral cavity (43). The role of HP~/infection in the etiology of oral cancer was first appreciated in 198,3 by Syrj~en et al (44). In their study of 40 oral Carcinomas, 16 speclrnens showed HPV-suggestive char~ges on light microscopy. Of these, 50% showed positive r~actlvity for HPV structural proteins. The same series of biopsies was recently analyzed for the presence of HPV DNA by ISH- and PCR techniques; 12/40 (30%] disclosed HPV 11, 16 or 18 DNA sequences (45). In 1985, de Villters at al. found HPV 2 and 16 DNA in 3/7 tongue carcinomas (46). Similarly, HPV DNA was found in 3/6 oral carcinomas, HI:n# type 11 and 16 being revealed (30). In another esdes, HPV 2 and 16 DNA were found in 4/18 oral carcinomas, and all positive cases were situated at the floor of the mouth (47). Again, Milde and L0ning repor~ec~ HPV DNA in 4/7 oral carcinomas (48). HPV 2 DNA was disclosed in 2 verrucous carcinoma and BATCo document for Mayo Clinic 28 March 2002
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in one leukoplakia issi.~n with dysplasia (49). Maitland and colt'~x:EaIors (31) found HPV DNA in 7/15 carcinomas ta~an from the late~l border of the tongue or the floor of the mouth. In the same year, ~ case report was published, immunosuppiessecl male developed an invaslve $CC of his tongue, in which HPV 16 DNA was disclosed where (50). In two other stuc~ HPV 2, 6, 11, 16 or 18 DNA was found in 11.8% and 23.5% of carcinoma specimens In a recent review of I~ead and neck carcinomas,/t was concluded that more than 50% of oral and nasal, as well as laryngeal anil esopl~ageal carcinomas are associated with HPV (52). Unusually high percentage (76.4%) of eplsomai HPV 16 se~. uances were disclosed In a sedas of oral epidermoid carcinomas in Taiwan ('/}. The authors highlighted that ~ Vi.:'ral and chemical factors may be involved in the process of carcinogenesis. W.njabu and coworkers anaiyz~l I~h anoganital and orophan]ngeat carcinomas and concluded that HPV DNA sequences are more frequently assod~ted with anoganital than oropha.,yngeal $CCs (53). Akin to genital SCC, s, HPV 33 DNA was disclosed In one orali SCC (2/). On the other hand, Kashima found HPV DNA only in 6/29 SCC, but their demonstrallon of HPV ~- and o-7-ralated sequences emphasizes the fact that HPV types not commonly found in oral lesions may be found .if properly searched for (29). In Finland, about 40 new oral carcinomas and 140 lip cancers are diagnosed eactt year, whlch makes one fifth of all malignancles:.":." in the head and neck region (100). The squamous cell carcinoma, which conatltutes about 80% of oral cancers, d~evalops through the vadous grades of intraep~thelial neoplastic changes over a long time period. Because these; neoplastic lesions are mostly discovered late in the progress of the cr~sease, the 5-year suwival is usually poor.itt appears that oral carcinomas are caused predominantly by long-term exposure to chemical carcinogens. Moreover', there is evidence implicating physical as well as viral stimuli in the genesis of oral neoplasms. Three goul?s of DNA viruses; papov"~druses, edenoviruses and herpesvlruses, have demonstrated their oncoganic pote~al in ihe humans (41). The multkstep process of carcinogenesis can be divided Into initiation, promotion and progression stages. In viral carcinogenesis, lthe viruses have to ~'¢orporate one or more functional genes into the host DNA. Integration of HPV into host DNA:. is a f~equent event In cervical carcinomas, but encountered ~ frequently in oral SCCs (7,63,101). For yet so'he unknown reasons, HPV seems to be associated more frequently with anoganita] than ompharyngeal squamo~Js carcinomas (63). Quite lately, a new cell line from a buccal carcinoma containing closely related HPV 16 sequences was established (102). The finding of HPV DNA in normal mucosa argues against HPV being the sole factor in. oral malignancies (103). Indeed, there is evidence that cofactors are needed for malignant transform~on, e.g., irracl~ion in la~jngeal carcinomas, and sunlight in skin carcinomas in patients with epidermodysplasis verruc'rrcrmis, in addition, smoking is claimed to be frequently associ~ed with many types of rna]ignant tumors. The deve.'.o.oment of carcinomas in patients with immunosuppresslon illustrates that the immune BATCo document for Mayo Clinic 28 March 2002
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system is l~'kely to play an important protective role (50,86). £.3.1.T~ : Genital HPV i .~ons aze currently cor~defed to represent the most prevalent sexually transmitted disease (STD) (58). Concernln~ ~e common hand warts, the viral transmisston by skin to skin contact Is generaJly accepted : (59). An HPV Infact~or~ may be received by direct contact wi~ other children, contaminated household instruments autoinocul~on i19). Serological studies have suggested that HPV infections occur v~y esr~ in life and by are widespread, but only ~ small portion of people will eventually develop a manifest infection (57,60). The priman~ infection may also be acquired during l~e passage through the infected birth canal (61,62). This mode of tzansmisslon has beenlitnked to the development of juver~e laryngeal papillomas. To further complicate this concept, Wa~ts at el. reported ~ case where a young girl with recurrent laryngeal papillorn=osia was delivered by cesarean seot~on by a mother w~th genital wa~ts (63). In adult onset laryngeal papillomas, the role of oral cavity as a possible reservoir of HPV Infe~on has been broug~ Into consideration. It is worth considering that oral-genital sex may be responsible for transmitting HPV from the genital tract to o~ cavity (19). The finding of genital HPV types 16,18,31 ,~3 and ~5 in the oral ~ substant~tes tl~ concept (64). The transmission of the same HPV type between two subjects has been shc~wn ]n children with sexual a~ese (65). The observation of HPV DNA in penile lesions of men whose l:~lners have g~nital HPV lesions further confim~s the role of sexual activity in HPV transmission. The soume of the virus in latent infections Is more troublesome to trace. Recently, it was suggested that normal Individuals must serve as the reservoirlfor at least some of the known HPV types (66). The possibility also exists tl~ oral HPV infections a~e ubiquitoqs like genital HPV irrfsc~ns (67), and remain ~ent in the oral mucosa until being triggered to replicate by chemic~, p~sical, or other microbial agents. Alert from the dare presented above, familial htsto=y witll genetic pr~spos~lon has been suspected in cases of oral FEH (26). 2.~_Assoolation of co*fastom To under-'tend the significance of HPV infection in ora~ mucosal lesions, elucidation of the natural history of the virus would be Of prime importance. Unfortunately, such studies are only evalleble on genital HPV infections (56,77). The follow-up data from these studies suggest that the natural history of HPV is similar to that of the class/eel CIN lesions~ Because of the fac~ ~at the HPV infections occur in women more than 10 yearn younger than BATCo document for Mayo Clinic 28 March 2002
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those with ctessical C~N, it has been concluded that HPV may accelerate the development of CIN lesions. On the other hand, chemical, !physical and microbiaJ agents have been propes~d to intensify the effects of HPV infections. .; Such fi~ctors include .smoking, betel quid chewing, oral con',mcepttve use, epithelial traumas, irrad'~tion, ultraviolat light, other STD microbes, as well as smokeless tobacco (7,78,79). Alcohol seems to be a predisposing factor because of the increased permeabil~ of ora mucosa on ethanol intake (80). It appears that the dsk factors for HPV " infection and carcinoge, nests p~'allel to each other. In the Kuopio follow-up study, the current smoking increased the risk of manifest genital HPV infection by 2.2-fold (81). Whether this was an independent effect ~f smoking or associated with some! confounding f~ctore remains to be seen, however, in fact, the toxic metaboifies of cigarette smoke IT~ve been di~rnonstrated in cervical fluids (82). Clearly, smoking decreased the number of epithelial Langefflans cells (the major arclgervprasant~lg celts) In the normal cewical epithelium (83). A stmgar effect was seen with HPV 16 infection.! In our recent study, how~ar, we could not find any con~atk~ between the smoid~j hal:~s The detection r~te of cervical HPV infection Increases during pregnancy, Implicating that hormonal factors are Involved in the contr(~l of HPV. Whether this Is due to alterations in ca~vical epithelium caused by es~en, or enhancement of virali expression, is not clear (84). On the other hand, pregn~;cy is known to Impair the immunological defenc~ mechanisms. A ¢Jaesical example of this is the epulis gmvidetum of the ginglve. Changes in the gingival t~ssue ~e also seen along the menstrual cycle, but similar effects in other oral mucosal sites (tongue, buccal mucosa, palat(#, llpe) h~ve not been systema~ invas~ated. ]mmunosupprassicn caused by drugs or HIV Infection ate known to! predispose the oral mucosa to HPV infections (85,86). Furthermore, HPV lesions I~ve been reported significantly .~mors often in petlents with chronic hepatic failure and diabetes (59). The amplification of cellular oncogenas In assoc .la~ion with HPV has been observed in ganital ,squamous cell cardnomas, but this subject has not been studied in oH carcinomas as yet. For the ctinl~lan, the best known viral iNection Jn the oral cavity is caused by HSV. The iinvolvement of HSV In oral camin0mas has been apprecieted but so far, HSV has been suggested to act in the init~icn process only (78,87). The possibility of HSV and HPV acting synergistically in oral mucosa is a potential .~echan~sm, however. ~3~3.Mull~:en~ natum It is well appreciated that multiple sites of the genital tract m~y be simultaneously infected by HPV (99). The muificentdc nature of the disease has also been observed in oral cavity, where HPV DNA has been found from normal mucosa adjacent to carcinomatous lesion. ~ phenomenon complicates the eradication of HPV from infected mucosa, because no ctinical facifities exist to detect virus in a latent form at the moment. "t'he multicentrio character of the infection was brought into conskJeration by a study by Kashima and collaborators, who round HPV BATCo document for Mayo Clinic 28 March 2002
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DNA in two oral ieuk~plakia les~ons obtained from a p~Jent with a previous history of juvenge-onset recurrent mspirato~ paplllomatosis (29). ~..I.S'~o"11~'I~11G STy.." Y OF ORAL HFV INFECTIONS IN KIJOPIO 198~1992 The et~ological !ro~e of HPV in benign oral squamous cell lesions is generally accepted. Since early 1980% these viruses have been implicated in etiology of a high proportion of oral squamous cell carcinomas as welL Prompted by this accbmulaIed evidence on the role of HPV In oral mucosal lesions, the first systematic approach to assess the biologlCial significance of HPV infections in the oral cavity was conducted in Kuopio between 198~- 1992 by. Dr. Kellokoski. The specific aims and results of the study were as foltows: 1) To classify the morphological c~ in the blO. ps|es from bucoal mucos~ of women with genital HPV infection, and to eva~e the usefulnes~ of oral scrap~ as well as morphological and histological cdteda in ; assessing HPV infectl~ms in oral mucosa. Flesuls: B~' cFmical ~ the HPV aasodatio~ of tiny le~dor~ is ~ To analyse ..whether acetic acid staining can be applied on the specific diagnosis of oral HPV infections, to further elucldat~ the factors raleted to the acetowhlte staining, Icluding the smoking and dfl~ habils and and cytokeralIn paJtems inlthe epithelium. Resull~ Ace~ acid slairfiog proved to be of no va/ue in ~ of HPV 3) To detsrmlne~the sensitivity of dlffemet hybddlza~on methods in detection of HPV Infection and to oral HPV Infactlons in bucc~l mu....~:~sa in women with genital HPV Infections. Results: D~t blot hybridization de,acted HPV DNA subdirdc~ HPV INec~ons. Wittt Southern b~ ~ 15.1% of biopsies t~ken fn~rn ~ normal bucca] mucosa c~ained HI~ DNA. 4) To assess HPV transmission by correlating the HPV types In the orai mucos~ of the patients and those on the anogenital muc~..,, of their partners. ~ Only a single pal;ent had the same HPV type both in the ge~tal ~.5.ORAL C, AVI'IY AS ~ POTENTIAL ROUTE FOR VERIICAL HPV TRANSMISSION 2.5.1.Aims i BATCo document for Mayo Clinic 28 March 2002
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39 The studies completed as D.D.S.(PhD) Thesis of Dr. Kellokoski heve raised several questions which are the subject Of the present ~tudy as follows: 1] To explore the no:~-sexual routes of HPV transmission by examining HPV DNA in the oropha~yngeal swabs of newborn babies born to mothers with clinical HPV Inf~ of the utedna cervix. :2) To asses th".e clinical significance of such an eventual transmission of HPV (whether a tnJe infection or a passenger) by sequential swabs of infants born to mothem with clinical HPV infecffons of the cervix. 3) To analyse ~e clinical course of o~ HPV infection in the infants, Le., whether the infection disappears or does it remain latent?. 4) To establish! the prevalence of HPV infecllons (e.g. skin wads, oral HPV infections, whether latent, subcllnioal or clinical) In chlldrsn (at the age of 1-10 yrs) born to these mothers with known genital HPV hletory. 5) To corm~ate ~he HPV types in children to those of their parents. 6) How end when HPVdmsltiva serology develops in these children? 2.6.STUDY DESIGN The subjects of ithe present study are the volunfeer children born to women prospectively followed-up for genital HPV laslor~ since 1981 at the Depa,,lmant of Gynecology and Obstetrics, Kuoplo Unlvers~ Hospital (see PROUECT I). A total of ~ children has born to these modem during the 11-year follow-up period. All children will be invited to attend the ;clln~cat examination for their oral HPV Infections. Examinations carded out to these children Include: 1) History of p.revious wa.,ts and herpetic Infections, medications and medical history. This is done with parents, 2) Clinical ex~ination for detection of wart~ and hefl~Jc lesions in hands and face, 3) Clin~al examination of the entire oral muco~.' and pharynx. Photographs are taken from the lesions and all clinical lesions are biopsied, if necessa,-y for treatme~nt, 4) Cytological scrapings are collected from oral mucosa with small brash designed for cell scrspings, for HPV~DNA analyses. If the child is HPV-posltive, then the virus typing data will be correisted to the HPV status of the i~arents. HPV-positive ohlldren will be fcliowed-up for several years with regard to their oral mucosal lesions. The second ~ group consists of 30 newborn babies born to mothers with clinical HPV infeotlon of the cewix. As control groups, a sedes of babies (about 50) born with spontaneous vaginal delivery to healthy (i.e., non- HPV-infectec0 mothers, and 30 babies deliv~ed by elective cesarean se~on will be included in the study. Examinations carried out to this group include: 1) Cytological samples for HPV DNA analysis collected from the genital tract of the mothers just before delivery, 2) Amrdon fluid sample from the nasopharynx is collected from newborns in connection with sucking the airways, 3) Blood sample is collected in connection with cutting the umbilical cord, 4) First control sample from the childrens oral cavity are collected with the smeJ! brush after one to BATCo document for Mayo Clinic 28 March 2002 O~
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4O five ¢~ys from birth, 5) If HPV-DNA is found, children will be followed-up a~t three, six and twelve months. By this study design, the eventual ve~cal tran~lon of HPV can be assessed, and the question wh~her HPV in the newborn .b=~bles is a passenger or a clinically stgnifica~ infective agent can be reliably addressed. ~6.1.Perlb~ent child h~ory The examiner interviews all children (and/or their parents) and fulfills the questionnaire. Special emphasis will ba focused on medica~don and previous warts In hands. The children of the follow-up patients are examined at the dental chair with good illumination. Hands and facial skin will be carefully i.nspected for possible cutaneous warts. Subsequently, the complete dental status and all mucosal changes are r~cordecL The newborn babies with HPV Infection are followed-up three times during the first year (at 6 weeks, 6 mo~ths and one year). Oral mucosa of the children is clinically examined at the age of 6 months and one year. Cytologic scrap~gs will be taken with three sequential swabs from the entire buccal mucosa on both sides from the infants of th~ follow-up patients. CytologP,_,al samples are collected with small brush designed for that purpose and stored ini 0.05M phosphate buffer containing gantamycln. Buffer with gentamycin is also added to amnion fluid samples. The cells are separated from suspension with centrifugation and treated with protelnase for cell lysis and to purify ~e sample from proteins. DNA dissolve and remain in fluid. The sample is stored in .20~ All clinical lssior)s will be biopsied. Half of the biopsy will be processed and st~dned for hematoxylln-eosin (HE) sections. All biopSies are analysed for the presence of the following morphological changes: I'~yper-keratosis, orthokeraIosis, parake~tosis, dyskeratosis, keratohy~ne granules, acanthosis, vacuolized cells, single cell dyskeratosis, muitinuclsated cells, koilocytes, sickle cell koilocytes, spikes, i~ammation, and dysplasia. On mcrphological grounds, the lesions were class~ed as either squamous cell papilloma, fiat condyloma, suggestive fcr HPV infec',Jcn or no Bvidence of HPV or other specific diagnosis. The other half will be frozen for furtl~er Southern b:c: hybridization and PCR analysis. BATCo document for Mayo Clinic 28 March 2002 0 0 0"~
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41 All biopsies will be analysed with Southern blot anaJysls, completed according to routine procedure~ A~ samples of bxfoliatJve cytology am anelysed by PCR. From samples treaIed with protelnsse, ,e-globin is cloned as a control before HPV-DNA amplification. If ~-glob~ amplification shows the presence of human DNA, HPV DNA PCR-amplificalion~ is carded out with HPV-consensus primers. Those primers find HPV 6, 11, 16 and 18. PCR amplfficatJon is done with 20 cycles (denaturation 94-96~, annealing 40-55=C, primer extension 72~C). Reactions are done in 50 ~ volume. An P.J!quct of 10-20 ~1 of the PCR-cloned product is taken for electrophoresis in 3% agarose gel .containing.: athidium bromide to visualize DNA under UV-light. ff positive HPV DNA signals are found in eisctrophoresis, DN~ is transferred to niVocetlulose filter for hybridization. Spssific biotiny~ed oligonucisotides are used as probes. Hybdds are detected with Amershams ECL-technique. HPV-DNA positive PCR-products are dlgested with Pstl and Rsai resffict~on enzymes that cut HPV 6, 11, 16 and 18 in spesific fragments. Fragments are separated with electrophoresb. W'~ this method we can confirm the prescence of specific ~-IPV types in samples positive in PCR or in hybddiz~on. The statistical .~alyses a~e done at the University of Kuopio by the SPSS (Scientific Package for Social Sciences) program ini a VAX 11/785 computer. Pearson's correlation coefficient matdx and Student's Rest, non-parametric Mann-Whitney U-test and Witcoxon Rank Sum Test are used, where applicable. P values less than 0.05 are interpreted st~:'stlcally significant. 2.7JJTERATURE CrTED. 1.Jsnson AB, Link CC, Le~cester WD. Pepillomavlrus effolngy of oral ¢evity papillomas. In: Hooks J, Jorden G, ed=. ViraJ Infection= in oral medicine. Amstsrd.am: E~sevier, 133-146, 1~2. 2.JerBon AB, Lanoz~er WO, Hadmann DP, Sharer EL Frequency and dLstdbutlon of paplllom~w~u= structural antiger~ in verruc~e, multlpie papilloma=, and ~ondylomat- of the oral cavity. Am J Plthol 107: 212-218, 1982. 3.Syr~en K, Happonen .RP0 Syrj~en S, Ca/onius EL Hurna~ I~omvtru~ (HPV) an~gans a~d loca~ ]mmunolngic re~ctJvlty in oral =quamou= cell tumom, and hyperplssics. Scand J Dent Re= ~: 358370, 1984. 4.Wetch TB, Barker BF, Wi!liams C. Peroxldase-antiperox~dase ~v=dualJon of humatt o~al squamous cell papillomas. Oral Surg Oral Meal Oral Pathol 61: 698-606, 1986. 5.P~d~yachee A, V~ Wyk~ CW. Human papillom~ru= (HPV) i~ ~ ~quamou= cell papillomas. J Oral Patho116: 353-355, 1987, 6.Beaudenon S, Prastorlus F, Kremsdorf D, Lutzner M, Wors~aa N, Peh~u-Am=udet G, Odh G. A new type of human papl]lomsvinJ$ associated with oral focal ep~hel~l hyperpla=l=. J Invest Dermatol 88: 130-135, 1987. 7.Chang k'W, Chang CS, L=i KS, Chou MJ, Choo KS. High prey=dents of human papiilomaviru= infection and possible associ~,ion wP~h betel quid chewing and smoking in orcd epidermoid ce~ctnome.~ in Talwan. J Meal V~rol 28: 57-61, 1989. 8.Syd~men SM, SyrjEnen KJ, Lamberg MA. Oete~on of human papillomavbus DNA in o~d mucosal lesion= using in sltu DNA- hybrid~1~on applied on p~raffin s ect~ons. Oral Surg Oral Meal Oral Pathoi 62: 660-667, 19~6. 9.Syrj~nen SM, ~,~Enan KJ, Happonen RP, Larnberg MA. In $1tu DNA hybddiza~on ana.~is of human papi]iomavirus (HPV) sequences in benign ore/muco,sa] lesions. Azch Dermatoi Re= 279: 5zk3-549, 1987. lO.Ever=ole LR, L,~ipi= PJ. Or=d squernous papgloma~: Detecffon of HPV DNA by in situ bybdd'rzation. Oral Surg Oral Meal Oral Pa~hol 65: 545-550, 1988. 11.Young SK, Min I~V. In situ DNA hybridiza~on ~aiy~= of oral papillomas, leukopiakias, and ca~cinomes for human papillomavirus. Oral Surg Oral Med O~al Pathoi 71: 726-729, 1991. BATCo document for Mayo Clinic 28 March 2002
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42 12.1r~lz~pp MJ, Uohc~a GI.: Oral condylom~ acumlnatum. Oral Surg 23:538-545, 1967. 13.Lut;mer M, Kuffer R, BIiJ~chet.Bsrdon C, Croissant O. Different papiliom~viruses asthe causes of oral wad~. Arch Dermatof 118: 14.Evemole LFL LaJpi= PJ, Green TL Human peplliorrmviru= type 2 DNA in or=d and labial vermca vulgari=. J Cut~n Pmhol 14: 319-325, 1987. : 15.Adler-Storthz K. New~d JR, Tessln BA, Yeudall WA, Shillitoe EJ. Identh'ication of human pepillom=vims types in oral verrac,~ vulgads- J Oral Pathol 15; 230-233, 1986. 16.Greenspen D, da Villie~= EM, Gresnspa~ JS, De Souze YG, zur Hausan H. Unusual HPV types in oral wads in asseclet~on wi~ HN infe~ffon. J Oral Pathbl 17: 482-487, 1988. 17.Snfjders PJF, Schulten~ EAJM, Mullink H, ten Kate RW, Jiwa M, van tier Wazd !, Me~er CJLM, We|boomers JMM. Detecffon of human p=pillomavirus an~ Epstaln-Ba~r virus DNA sequancse in oral mucosa of HIV-infected I:~t~ents by the polymemse chain reaction. Am J Pathol 137: 659-666, 1990. 18.de V]/liors EM, Hirsc~Behnam A, yon K~ebeI-Doeberitz C, Neumann CH, zur Heusen H. Two newly iderttffled human Papillom=wiru~ types (HPV 40 and 57) ~olated from mucosal lesions. V~ralogy 17I: 248-253, 1989. 19.Naghashfar 7_, Sswada~ E, Kutcher MJ, Swan~,~r J, Gupta J, Daniel R, Kashlma H, Woodruff JD, Shah K. Identification of genita~ 1~act pepilloms~rusas HP~/-~ and HPV-16 in weds of the oral o¢~'y. J Mad Virol 17: 313~324, 1985. 20.Arch~rd HO, Hack JWi Stanley HR, Gallup NM. Focal epithelial hyperpiasia: an u nusual oral mu¢o~al le,~ion found in Indian children. Oral Surg Oral ,Mad Oral Pathol 20: 201-212, 1965. 21.PraetorhJ~-Clausen F. Rare oral viral disorders (molluscum ~ontagiosum, Ice.el|zeal Icendoac, anthoma, verrucse, condylom~ asuminatum, and focal el~dlel~ hyperplasia). Oral Surg Oral Mad Oral Pathol 34: 604-618, 1972. 22.Prastorius F, Pr=etoriu~ Ch~usen P, Mcgeltoft M. ImmunohLgcoahemical evidence of papillomavirus antigen in focal epithelial hyparplesia. Ta~dlaegebl~Klet 8~: 589-595, 1985. 23.Pfllter H, Hettioh'l, R~nne U, Glssmann L, Chilf G. Characterization of human paplllomavirus type 13 from foe.~d epithelial hypelplasia Hack lesions.! J Viral 47: 363-366, 1983. 24.Hanke RP, Milde-La~g'.~h K, L6ning T, Str6ma~a Koppang H. Human papillom~virus type 13 and focal eplt~lellal hyperplesla of the oral muco~a: DNA .~hybrldtza~on on p~r~rvarnbedded epecimer~. Virchows Arch 411: 193-198, 1987. 25.Henka RP, Guedn-Rev~rchon I, M;lde-Lango~ch K, Str6mme-Koppang H, L6ning T. In situ detec~on of human pepillom~,virue typee 13 and 32 in focal Fpithelkd hyperplesia of the oral mucose. J Oral Pathol Mad 18: 419-421, 1989. 28.~rj~en SM. Humani pepillomavtrus infecffon= in the or~ cavity. In: $~EN K, GISSMANN L, KOSS LG, eds- Pepiltomsvlruce~ ~nd hur~an dbecse. Heidelberg: Spdogar-Vedag, 104-137, 1987. 27.Greet RO Jr, Evemol~ LR, Cro~by LK. Deted~on of hurmm papillomaviruc,-ganomic DNA in oral epithelial dyeplasia=, oral emokelees tobacco-assoc~te¢l I.eukopiakles, and e~ellal ma~gnancles. J OraJ Maxillofa~ Surg 48: 1201-1205, 1990. 28.Lind PO, ~yrJ~nen SM~ Syrj~men K J, Koppeng HS, Aes F_ Local immunoreactivlty and hum=l papillomavlms {HPV) in oral precancer and cancer lesions. Stand J Dent Re= 94: 419-426, 1986. 29.Kcshima HI(, Kut~her M, Kepis T, Levin IS, de Vii|lain EM, Shah K. Human Papillomm~iru~ in squamou= cell carcinoma, I~ukopiakia, Itchen pianu~ and clinically norm~ epithelium of the oral cavity. Ann Otol Rhin La~yng 99: 55-61, 1990, 30.1.6ning T, Ikenberg H, Backer J, Gissma~ln L, Hcepfer I, zur Hausen H. Analysis of oral papillomas, leukoplaldss, and cercinomes for hurrah pea|lion,virus type rel~te<l DNA. J Invest Dermatol 84: 417-420, 1985. 31.M~d=md NJ, Cox MF, iLynas C, Pdme SS, M~vell CA, Scully C. Detection of human Papillomsviru= DNA in biop~|es of human oral ~ue. Br J Cancer ,56: 245-250, 1987. 32.Jontell M, Watts S, Wajlstr6m M, Let, in L, Sloberg K. Human papilloma virus in erosk, e oral lichen pia~u=. J Oral P~dhol Mad 19: 273-277, 1990. 33.Broich G, S¢~ki T. Electron micros¢op~ dare,on of human paplllomsv|rus particles In orsJ proliferative lesions. Bull Tokyo Dent Coil 30: 213-220, 1989. 34.Gro=~ GE, Pfi=ter H, M~ermayer C. Papilloma v~rus p~ff¢le= in a fibrom~ of the tongue. Aria Derm~tovane¢ 60: 315-~18, 1980. 35J~bbey LM, P~ge DG, .,~awyer DR. The clinical and histopathofoglc features of a series of 464 oral squamou= ~ell papiiloma~ Ors] Surg 49: 419-428, 1960. 36.S¢u~ C, Prime S, M .~.Jand N. Papillom~viru~es: Their por~lble role in ond disease. Oral Surg Oral Mad Oral Pa~ol 60: 166-174, 1985. 37.Gibson PE, Seymour M=S. Human pspillomsvirue type 16 DNA at biopsy of gingivni hyperpl~sia after cerdia~ tmnsplant~on. 38.Kahn MA. Amelobl~sto~na in young persons: A ciinicop~thalogio analyais end etiologic investigation. Oral Surg O1~ Mad Oral Pathol 67: 706.715. 1989.; 39.Cox M, Evason J, Scully C. Human papillom~vlms type 16 DNA In an odontogenic kerslocyst. J Oral Pathol Mad 20: 143-145, 1991. 40.Pindborg JJ. Oral can~er and precencer. Hemy Ling, Dorset Preas, Dorchestor, 1 980. 41.Boyd NM, Reade PC. Mechanisme of ce~cinogenesls with padicular reference to the oral mucesa. J Oral P~thol 17: 193-201, 1988. 42.E=kin~=i DP. Oncogani¢ potential of sexually ~ar~mitted viruse.= with special reference to oral cancer. Oral Surg Oral Mad P~ol 64: 35-40, 1967. 43.Syrj~nan SM, Syrj~nan KJ, Hepponen RP. Human papillomvirue {HPV) DNA sequanc~ in oral precencerou= |e=ion= and =quamous cell carcinom~ demonstrated by in situ hybridization. J Oral Pa~,ho| 17: 273.278, 1988. 44.Syrj~nen K, Syrj&nen S, Lamberg M, Pyrh6nan S, Nuutinen J. Morphological and immunoh|steshemicel evidence suggesting human Papillomavirus (H .IW) involvement in oral =quamous cetl catc|nogenesis. Int J Oral Surg 12: 418-424, 1983. 4,5.Chang F, Syrj&nan S, Nuut~nen J, K~j~. J, SyrjEnan K. Dete~-dlon of human papillomavirus (HPV) DNA |n ors] squ~Jllous cell carcinomas by in situ hybridization and polymereee chain re,~ction. Arch Dermatol Res 282: 493-497, 1990. 48.de Vii|lets EM, We|deuce H, Otto H, zur Heusan H. Pap,lomavlms DNA in human tongue carcinomas, lntJ Cancer 36: 575-578, 1~5. 47.claVilliers EM. We~dauer H, Le JY, Neumann C, zur Hau~en H. Papillomaviren in bcnign~n und maligncn Tumeran de= Mundes und dee oberen Resp~ratJonstrakte~. La~ng Rh|nof Otol 65: 177-179, 1986. 4~.Milde K. L6ni='.g T. Detection of pal~iliorrmvirue DNA |n oral papillomas and carcinomas: application of in situ hybddiza~on wi~ i~iotinylated HP.~ 16 probes. J Oral Pathol 15: 292-296, 1986. BATCo document for Mayo Clinic 28 March 2002
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49.Adler-Stort~z K. New, and JR, Tes~in BA, Ye~l W~ Shill~oe ~. Hum~ p=pglomvlms ~ 2 D~ in o~ ve~co~ ~inom¢ J Or~ P~ 15: 4~ 1~. ~bgl DP. ~i~r ~o~ ~piliom~ ~d ~uem~ ~11 ~ino~ ~ffie ~ngu¢ ~h De~l 1~: ~, I~. 51.~ei~ ~ Hom~in OP. Ft~nce ~ hum~ ~g]om~]~ D~ In b~ign ~d p~cero~ oral leukopl~ ~d ~u~ ceil ~ino~ De~ 17~ ~, 1~. ~rg H, F~ ~ ~ PD, ~ P~ Hum~ ~p~ma vires ~ ~d ~ino~ ~ ~e head ~d n~ Clin ~o!~ng~ 13: ~7~; ~.~u ~, Shi~ D, ~nhe~ ~u~ ~dnom= ~ng ~e ~me~ ~n ~on. ~ J Surg P~ 13: ~-~4, 1~. ~.R~ ~ S~ P. ~n~ w~ ~d ~¢~ ~. ~L ~ Im~ ~1~ ind~ f~ ~m~ benign ~pillom~ird i~o~ f~ high grade ce~ i~p~eli~ ~pl~¢ ~ J O~ ~1 1~: 611~8, 1~. ~ ~ Pa~ms ~n~ pr~ ~d e~oid ~m. ~:M~n~o J, ~. Papil~ in hum~ ~o~. R~ ~ in ep~de~ p~m. ~o ~ia Publi~o~ ~m ~en ~ 78: 1~, 1~. ~.Jen~n ~ ~Ping Y, V~ne JM, ~ ~ ~n ~ ~m~n ~, ~l~ay ~ ~e ~ ~e~ gen~ human ~pi~m~l~ ~ in ~u~ ~d ~. JID 1~: ~, 1~. ~m~ ~ F~e ~. H~m~ ~illom~i~: ~ we ~ to ~pe? CIIn MI¢~iol ~ ~ 1~1~, 1~. ~.Gr~ G. Lesi~ ~e G~NN ~ K~ LG~ eds. P~i;;o~ms~ ~d h~ dise~. HeJdel~rg: Spd~er-Vedag, I~-~, 1~7. ~; ~, 1~. , 61.Mou~ P, Sh~ ~. ~r=o~ ~l~m~a: ~o~I~ r~on ~ gen~ ~a~ ~pi~i~ Prog M~ ~ ~: ~114, I~. ~H~en C, ~mud~ ~ rel~onshJp ~ ~nlle ~nge~ papil~m=~ ~d m~ ~n~ aeumin~ J ~pr~ ~ 31: ~, 1~. ~.W~ ~L ~er ~, ~ ~ H~ ~il~m~ D~ ~ ~ ~u~us ~11 ~no~ ~ head ~ n~ O~ Surg ~ M~ ~ P~ol 71~ ~I-~7, I~1. ~.Gi~on P~ G~dn~ ~, B~ ~ H~ pa~ ~ ~ ~ ~ of ¢hil~n. J ~ ~r ~: 1~-1~, 1~. ~Lod~ AT, Reid ~ ~.zur ~ H. P~illo~avi~ Jn ~en~ ~r ~ a ~al to ~de~d ~e role ~ ~ in hum~ ~ ~cer R~ ~: ~1, 1~~, 13 In o~ f~ ep~eli~hyp~l=~= in ~e ~g~'~. J ~ P~ M~ ~: ~1, 1~1. ~.~kme~ RH, ~i= JG, G~ed H. tn ~ ~b~ ~ ~pi~om~i~ D~ in h~d ~d n~k ~u~o~ ~11 ~cinom~ A~ ~o~ng~ He~ ~k Surg 113: 81~1, ~.CI~ ~, M~hem WJG, v~ der Und~ ~, U~ J, ~ WGV. Hum~ p@il~m~i~= d~ in ~m~d~ ~ ~Ino~ ~d ~ ~ ~e ~I~ ~ ~e ~m~ ¢~n rea~. ~ J P~I 1~; ~7~, 1~. 71.T~ ~, ~il~ ~1 81: 1~-1~, 1~. 73.~er ~, H~ M~ UII NL ~ G~ ~ ~ ~l~k W, ~del ~ In v~o ~~n ~ hum~ =~n w~ hum~ 74.~uier ~ ~1 B~ ~e ~ of ~ ~ e~ J Or~ P~ol 14: ~ 1~. 75.S~h~ ~ H~W ~ P~i~= ~11 ~em~o~ ~e ~r On~l 8: ~, 1~. 76.G~I~W DA, Jen~on 1~. hidop~logical, ~olog~c~ ~d coW--opec d~ Eur J Ep~iol 5: 1-7, 1~. ~.zur H=~n H. Hum~ g~¢ ~n ~rg~m ~n ~ vi~ INe~on= or ~ne~ism ~n a vi~ i~on ~d ~ep~e ~ ~ hu~ pap~o~ In i~=~ ce~ ~ I~ J C~er ~: ~, 1~. ~.~uier ~ ~h C~Pen~e~ ~W= ~ ~ ~un~ ~mugh epide~b ~d o~ @~I¢ J Or~ P~I 17: 512~16, 1~. submit. ~n 1M, H~W ~, Smoke ~n~e~ ~ ~ic~ m~. N Engl J Mad 312: 31~16, 1~. ~.~on S~ Hollin~o~ e~ol~ of cewi¢~ in~e~el~ n~pl~¢ ~ immuno~ol~c ~. J Rew~ M~ ~: 61~16, 1~. ~.Ho~u Ji. ~is. Hum~ pap~:¢m~i~s (H~ IN~ons ~ ~ ~ ferule geni~ ~ Un~em~ ~ Kuo~o, 1~1. ~.~en S, Von ~ogn G, Ke~lo~ki J, ~nen ~ Two d~ent hum~ papillom~ims ~ ~ =~¢i=~ w~ oral mu~ I~o~ in ~ HN-~o~;e man. J Oral Pa~ol M~ 18: ~70, 1~. ~.Dem~dck ~, Inoue M, Le~er ~VM, ~ma !, ~gg~ ~ Paul LC. Hu~n papillomaS= ~pe 16 ~ed w~ oral 87.Eglin RP, S~I~ C, Lehner T, W&~-~ P. M~reg~ ~ ~on ~ R~ complementa~ to he~ sim~ vires in h~ o~ ~u~o~ cell ~minom¢ ~c~ 7~7~, 1~. ~.Cillner L More~Lo~z J, D~J~e~ ;. Sero~E~ ~s~ ~ ~p~l~m~ims gmups~¢ ~ in women w~ neopl~= BATCo document for Mayo Clinic 28 March 2002
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of ~e cervix uteri. J Clin Microb~o128: 624-627. 1990. 89.Podolani M, Mardova~i G. Pie~o=emoli P. Cermelli C, Boselll F. Ant:-oodiss to papiliomz~v~rus genus-ard~gens in women with genital wads. Microblol 1.D: 271-279. 1987. 90.Boa IR, Burkhatdt A. interepP~heli~l ¢e~Is of the or~ mucoce. Light and elecCon microscopic observations in gemffrse, ~peciflc pathogen-free and conve~ionaJized mice. J Oral Pathei 9: 65-81, 198G. 91.Nair PNR, Schroeder HF_ Du~-l-a~sociated lymphoid Idssue (DAL'i) of, minor sailvzuy glands and mucosal immunity, lmmunol 57: 171-180, 1986. 92.Ra=p FL, b-'~,~w=on CC, Hoidal JR, Repine JF_ Revemible impairment of the adherence of alveolar macrophage~ from cigarette smoke~. Am Ray P,~sp DL~ 118: 979-986, 1978. 93.Miller LG, Gold=t~in Q, Murphy M, Ginns LQ. Revem~le a,~dion= in lmmunoreguleto~j T cells in smoking. An=riyals by rnonc~onal antJbedie= ~ flow cTtom#~. Ch~'t 82:. 526.529, 1962. 94.F~on M, Edw~'d= A,~Und A, Milton GW, Hersey P. Low rmtund killer-cell activity and immunog~bulln levels assocLsted with smoking in human subje~. Int J Cance~ 23: 603-609, 1979. 95.~auk JJ, Norris K. Tol~cco cornponerd~ induce aJtera~ons in kera~nocyte lipid membrane fluidity and inhibit cell proliferation in culture. J Oral Pa~hol 47: 30-33, 1988. 96,Des SN, Khanna NN, Khanrm S. In vivo and in vitro ob=erva~on of ~ellul=r immune paxamsters in squamous cell carcinoma office oral cavity and ~ ~orrelatJan with t~mor Io~d ~nd progno~¢ Cancer inve~t 4: 207-216, 1986. 97.L6ning T, Burkhardt A~ Plasn~ cel~s end immunogJobulln-synthesis in oral precancer and c~ncer. Virehows Arch A Path Anat Histol 384: 109-120, 197~. 98.zur Hemsan H, intmcellulat =urveill~nce of poml.~ng virsl inf¢cffon~. Lancet 4~q-491, 1986. 98,Syrj~nen KJ, Huma~ I:~pillomaviru~ (Hi=V) infecl~on= end 1heir as=oci~ons with squamous Gel| neoplasi¢ AJch Qeec~wulLsffomch 57: 417-443, 1987. 101.S¢~hnalder-M~mou~, S, Croiss=nt O, Orth (3. int~on of human papillomsvlrus type 16 DNA sequences: a possible early event in the program|on ~f genital tumor=. J V~rol 61: 6295-3298, 1987. 102.Wong DYK, Chang KW, Chan CF, Chang RC~ Char=ctedza~m of two new cell lines dedved f~om oral c~vity human squ~mou= cell cetci~-mm~3C.' 1 and 0C2. J Oral Maxtl~ofa¢ ~urg 48: 385.,.~0, 1990. 103.soully C, Cox MF, Pd.rne S, Maitland NJ. Papillom~Imse=. The current status in ration to oral disee=e. Oral Surg OraJ Mad 104.Nk~dobitak Q, Pit~roff. S, Herbst H, Shepherd P, ~ T, AnagrK~topoulo~ I, Stein I-L Detection of human papillomevtrus type 16 DNA in carcinoma= of~e pa~affne tonaiL J Clin Pathol 48: 918-~21, 1990. 105.0~mw R~, Manias D~ Fang WJ, Zachow }Q~ Fars= AJ. A =uw~y of human cancers for human papillomsvlrus DNA by filter hybrldlzation. Caner 5e: .~-~4, 1987. 106.Padddnan S. Nucleic iacld hybridization tenhnique= in diagncals of human papil|om,~ viru= (HPV) infections of 1he uterine cervix. The=is, Kuoplo: Ur~tve~ity of Kuopio, I~. 107.Southern EM. Detectlo~n of specific sequencss among DNA fragments separated by g~l aleotophoreeis. J Mol BIo198: 50~517, 1975. 108.Kelloko$1d J, Syrj~nan! S, ~yrj~nen K, YIL~koski M. Oral muco~d changes In women with ganit~ HPV infect~on. J Oral Pathol M~d 19: 142-148, 1990. =. 109.Kallokodd J, ~frj~ne~ S. Kataja V, Yl'=skosk| M, ~d~en K. Acetowhlte =taJn|ng and it= s~jnlficance in dk~gnosla of oral muc~:~sa] le~lons in womed with genitaJ HPV infecffo~s. J Oral Patrol Mad 19: 278-283, 1990. 110.Kalloko~d J, ~j~nan~ S Tosi P, CIntodno M, l.K~Jnl P, ~yrjE=~en K. Cytokeralin p~tem in normal and HPV inf~ed oraJ mu~os~ In women with g~nltal HPV infe~ons. J Oral Pa~hol Mad 20¢. 2"6-31, 1991. 111,Ketlokoski J, Syrj~men ~, Yli~koski M, ~yrj~nan K. Dot blot hybddtza~on In detec~on of human pap|llom~rus (HI=V) Infe~tiona in oral cavity in woman ~ genital HPV infec~one. J Oral M~¢roblol Imm 1982. in press. 112.Kellokoski JK, Syrian ,e~n SM, Chang F, Yliskceid M, ~yrj~n~rl KJ. Southern blot hybddfza~on and PCR in human papillom=wfrus (HF~ infec~ons in women with genital HPV Infecl~ons. J Oral Palhol Mad 21: 19~2. In press. 3.HPV AND ~ QF THE RESPIRATORY TRACT 3.1.BACKGROUND in early lSSO's, evider~:~ has been i~ovided ¥~al proteins and HPV DNA) on HW invoNeme~t in inve~ted ~ mucosa, where HPV 6/11 DNA has been found in ber~gn pap~k)mas and, more |mpo~, HPV 16 DNA in squamous ceg ~ Acco~ to a feasible currant by--is, HPV is suggested to act sy~ with different agents ~n ma~jnant transfomTaIk~ am needed, Ix~m~x, the role of HPV (~n re~on to other potert~al factors) ~ng expired in de~ by ~e ~ .g~oup~ The etiology of resp~'-~ory neoplasms are generally con~ered as cigarette smoking excessive alcohol intake, BATCo document for Mayo Clinic 28 March 2002
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and exopsure to chemical carcinogens in the environment (1,2). This ls t~ue with our previous experimental lung cencer research on li~J:~mtory animals (3-6). Using intra~'acheal instillation technique, we have systematically investigated the camihogenic effects of coal tar pitch (Me natural compound which is widely used in many industries, In paving road end rooting the housa} on respiratory tract of rats. The sedal sacrificed study revealed that pitch had cons~picuous damage on the respirato~y system of rats, espec~ly on the bronchiolo*aiveolar areas. tar The lesions induced I:~ it ranged from hypeqSast~ metaptas~ and dysplast~c changes to extensive cancers. These lesions were usually m. uitlf~cal, and were more severe in the rats recisvlng higher dosages of coal tar pitch. The deposition of pitch pa~t~ icles within or adjacent to these lesions could be readily identified. Lung cancers occured In 12.5% (4/3~ and 25% (10140) of the A~ats treated with 13S.56mg and 200rag of coal tar pitch, whereas no tumors were found In control~: rats and the rats which recisved 6.48mg of coal tar pitch. The overall cancer Incidence signif~m~ related to;the cumulative dose of coal tar pitch. The histological types of lung cancers consisted of squamous cell ca~cin .o~as (10 out of the 14 lung cancem), adenocarcinoma (1114), and combined squamous and adeno- ca~dnomas (~14) (3,6). The development of pitctHnduced rat lung cancers appears to derive from the hyperplaslas of bronc.~lo-alveolar epithelium, proceest~ stages of squamous metaptaslas and/or dysplaslas to In recant yearn, I~creasing evidence has baen accumulated that certain types of human papiIlon'~vlrus 0-1PV) play a significant rolel Irl Induction of spe~ typas of human ca~1~ers, e.~ecialiy the anogenital carclnomas~ SCluamous cell carcinomas associaled wI~ epldermodysplasla verrucifomis (EV) leslons and warls In Immunocompromised ~atler~Is as well as caminomas adsing in upper aerodigestIve tract, verrucous carcinomas, and Bowen's disease (7). ,I~ecently more a~tantion has besrt focused on the i~-esence and role of papiIIomaviruses In head and neck tumor, lung tumors and lumors of the digestive tract. The involvement of human papIIIom~vlrus (HPV) irl upper resp~-a~oly t~act tumors 03anig~ papIIlonla and ,squanlous cell carcinoma) has been well demonstrated by epide.mloIogical. UItrasln~ctural and Irnnlunohistochemlcal studies and by molecular I~ybddlzatlon techniques (8-I0). The ...~fore, the research o~I the po~ synerg'~Iic actions between papIIIomevirus Infection and these chernlcal Initan~.,, In respirato~y c~rcIi1ogerlesls would be an Interesting and slgni~,ant subject, as alreedy specu~ by Prof. zu~.Hausen more than 14 yasm' ago lhat =... If ¢en/IcaI cancers were related to ¢ondyloma virus Irlfectlo~ consideration.' of cancer of the oral ¢avi~], the lung, and the bladder should be included in such a dls~ ..." [il) The transitional c~II of inverled papilloma of lhe nose and pa~anasal sinuses Is a well recognized entity, called I~t a variety of names, iDue to II1e fact that the issions frequently contain extensive area of squarnous metaplasia, these tumors have alsoibean called squamoas cell paI~llornas by previous authors. The frequency of thls leslon has beert calcula~ as 1/25 of that reported for the commo~ irdlarnmato~ polyps. It has been known for decades that the transitional papiIIorrtas of the nose end sinuses have some peculiar features in their cilnical beI'~vlour. "I'hese Inciude the lligh rate (30% to 62%) of recurrerlce evan ~Iter adequate therapy, and the well established tendency (some 12~) to undergo malignant transformallon either succeeding the papilloma or concomitantly with it (12,13)o These features In the behevlour of the nasal papitlomas a~e s|milar to lhose documented for the recurrent respira~on/papillomas in the larynx, and suggest a similar infectious etiology (HPV) for lhose two entities. Indeed, this concept gained recenIIy subslantiation by a ~eport, where morphologlcal similarities (l~oilocytosis, papiIIomatosis, dysI(eratosis} to HPV issions elsewhere were emphasized in a typical nasal transitional papliloma (13). In addition, the ission was shown to contain HPV structural proteins when subjected to immunoperoxidase staining (13). Further, 0 BATCo document for Mayo Clinic 28 March 2002
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dysplas'dc areas were disclosed in the exterr~ve papillomatous lesion. These findings were supported qu~te lately by other workers (H.,)5), who isolated and ¢ham~e~zed HFV DNA sequences 0.e., HPV 11-related virus and HPV 57) from nasal lnvert~d papilloma. Further evldenc~ to support the concept on the malignant potential of the inverled nasal papillomas was recently presented in :,our study, where such lesions were analysed using in situ DNA hybddizatlon. HPV DNA of types 11 and 16 wereldisciosed in both benign papillomas and equamous cell carcinomas derived from them (16). Lately, Brendwein et .al (17) fond HPV 16/18 in two cases of inverted papilloma, one with mild dysplesia and one associated w~ invas~e squamous carcinorr~s by using In situ hybridization with RNA probes. Thus, these results clearly show a venj I~igh incidence of HPV 16 and/or HPV 18 in inverted papilloma associated with malignancy. When analysed for DNA aneuploldy, those with HPV 16 Infec~ons and aneuploidy were shown to be significantly associated with mallg=hant transformation (16). Laryngeal papillomas are benign epithelial neoplasms consisting of a central fibrous connective tissue core coverted by squamou~ epithelium. These can produce sedous cllnlcel problems because of their potential for ale~vay obstruction, frequent ~scurrancas, and tenderc'y to spreed throughout the respiratory tract (19~. Increased risk of malignancy has been;reporied. The papillomas have a blphaslo age distn'bution, occurring in both young children and adults. Accordlnglto commonly accepted concepts, laryngeal papillomas can be divided as juvenile and adult- onset types (19). La~n~. geei papillomatosis may occur at any age, but children under the age of 5 years are at the I~jhest risk of develo~ping the disease, 51% being acquired by that age. The age distribution of the adult-onset papillomas usally shows the peak between the ages 20 and 40 years in most studies. Males are affected more fmquanuy than femm~, ~ of c~es (~9). The HPV et~ology of both the juvenile- and adult-onset laryngeal papillomas has been established beyond doubt. Eady expedmeqts were successful to transform the disease with cell-free extracts from man to dogs (20). HPV pafflcles have been ~epeatedly demonstrated In the biopsies of these lesions. Following the adoption of the immunohistochemloal itechniques in HPV research, many studies have been completed to detect HPV structural proteins in the leryng~a~ I papillomas (8,9,19,21). Subsequently, HPV DNA sequences have been firmly documented in IxXh the juvenile, a~.d adult-onset laryngeal papillomas by DNA hybridization technique (8,9,19,22). Of the more than 60 distinct type.~..' described, HPV types 11 and 6 are most strongly assoc~ed with laryngeal papillomas (8,9,19,22). Human pa..piliom.avirus type 11 was originally cloned from a laryngea~ papilloma (26), and has since been found In other such Ipapillomas, as well as in condyioma and mild dyspi~sia of the uterine cerv~. Human papiliomavirus type 6 was originally isolated from a condyioma of the utedne cervix, but has also been documented in significant numbers!of patients with laJTngeaJ papillomas. Malignant conversion of squarnous papilloma of the Imynx has been reported, although it is uncommon. A review of the case reports show that malignant neoplasms are more likely to occur in cases of severe papillomatosts of long duration with lesions spread throughout the respirator/tract (19). Malignant conversion, therefore, seems to be more common in cases of diseses of juvenile onset, but it has also been reported in cases of adult onset disease. The malignant, transformation has been associated with irradiation, and the dsk of this treatment have been generaJly accepted (8,9,19). Majoros et al (24) reported that 14% of cases receiving irradiation exhibited melignant transformation. Radiotherapeutlo management of these lesions if therefore no longer appropriate. Some authors now report malignat changes, even in the absence-of i~Tediation. There may be an association between smoking and conversion to squamous carcinoma, although there are reports of malignant degeneration tn subjects who do not smoke. In laryngeal papillomatosis, therefore, physical as well as chemic~3 ,~actore influence the transition of BATCo document for Mayo Clinic 28 March 2002 0 O~
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papillomas to a mallgt~qt state (7,8,9,19). Tile rsta~onship t~twesn HPV$ and malignant les/ons has also been suggested by the ~clings of HPV DNA in high percentage of laryngeal squamous cell carcinomas and verrucous carcinomas (25,26,27). Among 60 types of HPVs, HPV 30 was originally isolated from the laryngeal carcinoma (25). In the studies canled out in our laboratory, HPV capeid antigens were demonstrated in 36% of la,,yngeal carcinomas (21), and nine to 12 per cent of laryngeal carolnom~s contained at least one type of HPV 6, 11, 16 DNA sequences (26). ~.3.Tr'~heobto~ Mucosa The most comn'~on malignant tumor In the bronchi Is the squamous cell caminoma, which also is the most frequent malignancy o~ the males in most western countries. It is also generally accepted that bronchial squamous ceil caroinoma devel .o~s from the metaptastic squamous epithelial through the different grades of intraepithellal neoplasla and tn situ c~rcinoma. In the rrteraturs, very little attention has been focused on the benign equamous cell lesions of the bronch .'~, e.g., the solitary squamous cell papillomas. It is well recognized tha~ recurrent juvenile- onset laryngeal papillo~nas not infrequently spread Into the trachea and bronchi without malignant conversion even during an extended tim.' e pedod. Interestingly, the ~ttological changes In the cells exfoliated (Le., bronchial washing or sputum specimens} from ; the papillomas proved to be identical with those found In the cervical PAP smears of HPV-infected women (28,L:x3,30). Indeed, characteristic ~oilocytosis, multlnuclea~on and dyskeratosis were identified, suggesting the same etlology (HPV] for the bronchial pap=llomas as well (28,29,30). These early observations suggested that HPV could be involved in bronchial siquamous cell lesions as wall. Confirmatory avid., ance was subsequently presented to substantiate these morphological findings, when HPV 16 DNA was dleolose~..~ In anaplestlc lung c~rcinoma t31). The role of HPV In the development of bronchial c~rclnoma was furtherI a~,sessed by analysing larger sedes of bronchial carcinornas with the DNA hybridization techniques ('32,33). Ind~l HPV types 6 and 16 DNA was disclosed in a number of bronchial carcinoma, as when analysed by In situ hyb ~.r~dization (32,3,3). In this context, reference should be made to poss~le synergistic mechn~srns between the virus and .~hemical carcinogens as suggested previously in bronchial SCluamous cell cemtnogenesis. 3.3.DESIGN OF THE ~LIDY 3.3.1.Systematic E~ of HPV Infections in Respiratory Tumors As mentioned a~e, a substantial amount of evidence has been provided during the past few years implicating an etiologi .~ role of papillomavirus infection In the pathogenesis of respiratory benign and malignant squamous cell lesions: in human beings when analyzed either histopa~hologically or using DNA hybridization techniques. However, m., oat of the previous studies have only included small numbers of biopies and less sensitive DNA detecting techniqqes, further studies am still urgently needed. In this project, we are planning to retrosectlvely analyze a large sedes o~ biopsy specimens derived from the nasal cavity, paranssal sinuses, larynx, tracheobronchlel tract, oral cavity and e.~ophagus by histopathological assessment, DNA in situ hybridization and polymerase chain reaction (PCR) techniques. The samples will be collected from different ames of China and Finland. 3.3.2.Molecula~ Ckming of Unknown HPV Types Although the increasing number of reports on HPV DNA sequences in HPV lesions of the oral cavity and larynx strongly suggest the etiology of these tumors, the large number of negative benign and malignant lesions of the oral cavity, head and neck and the upper aerodigestive tract suggest that specific papillomaviruses are as yet unidentified. Extensive further investigation should be performed to identify and characterize these v'~ses. Only after the BATCo document for Mayo Clinic 28 March 2002
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characterization of such papillomaviruses and study of their interaction with ceilula~ genes, can other co-factors and edd'fdonal viruses be studied to elucidate the mechanism of malignant transformation in tumors of the upper asredigest~ve tract For this purpose,, recently we h~va sucesofully cloned and sequenced a HPV DNA fragment (about 450 psJ~es in length) from One case of esophageal carcinoma ps~ients. By comparsion with the known HPV types which so far have been sequ.:enced, the homology of the vires from the esophagus is less than 50%, Indicating the possible presence of new HPV:type(s) in esophageal lesions. 3.4.MATERIALS AND METHODS The materials ofl the present study will be collected from from the files of Papiliomavims Laboratonj at the Department of Pathoio ..~ly, Univers~ of Kuoplo, Finland, andthe Department of Precancerous Studies, Henan Mc~eical Univmsity, China. So .far, more than 200 cases of respiratory tumors have been available. These specimens h~ve been fixed in formaJln and embedded in paraffin. They will be analyzed for the presence of HPV infections by histol:mthologlcal ass .s.~sment, in situ DNA hybridization and the PCR ampifiica~orL A systematic col.[ection of fresh samples from respiratory lesions will be started in Kuopio University Hospital In the future. DNA will be extracted from these fresh specimens, and will be analyzed for the presence of HPV DNA by Southern blot hybrid~lzatlon. The Interesting cases which may bad,or new HPV type(s) or subtype(s) will be further used for viral DNA Isolation and molecular cloning. ~.4.2.MBthods 3.4.2.1.Morphological ..Evak.mtion: Formalin.fixed, paraffin-embedded biopsies will be cut into 4-,era-sections arid routinely stained with I~ematoxylln and ecsin (HE) for the light microscopy. Epithelial lesions are graded according to the generally ~ccep~ed criteria into normal, mild dysplasla, moderate dysplasia, severe dysplasia, carcinoma in situ and invasive carci.~,~*~oma. The sections on all cases wig be evaluated with special emphasis on morpholc~:jical changes suggestive foi HPV-involvement, being classified into fiat, inverted (or endophytic) and papillomatous (or exophytJc) condyloma~. according to the crlteda described previously (13,21,28,29,30). 3.4Z2.HPV-DNA in ~ Hybddiz~on: In situ DNA hybridizalion on formalin-fixed, paraffin-embedded tissues using either 3SS-labelled or ~otin-labelled HPV DNA probes will be performed as described by SyrjCqen et al (34). 3.4.2.3.Pobjmerase Chdln Reaction: The formalin-fixed, paraffin-embedded biopsies will be fudher analyzed by the PCR ~ described pr~Iously (~,3~). 3.,$~.4.Southem Blot Hybdd'zzat~n: Fresh biopsies der'Ned from both of the carcinoma loci and their surrounding tissues will be analysecJ for HPV DNA using the conventional Southern blot hybddlz~on. 3.4.2.5.V'~al Isola~on and Mokcular Cloning: T~e DNA samples are first hybridized with Southern blot technique, and the interesting HPV positive cases are selected for viral cloning. The corresponding areas of the positive band are isolated from the electrophoresis. The DNA samples are treated with T4 polymerase and ligated into the enzyme- cut plasmide. After transformation of E. coli and colony hybridization, the correct recombinants with the vim] DNA insert are selected out. The transformants which contain recombinant plasmid are mass cultured, and the piesmid DNA is isolated. The inserted vi~J DNA is cut off with apprepriate restriction endonuciease. Vlral DNA is sequenced 0 0 O~ BATCo document for Mayo Clinic 28 March 2002
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and compared wilh the known HPV types. Furthermore, the isolated new viruses can be as DNA probes to detect the prevalence o~ these HPV types in these lesions. 1. E]wood JM, pearson JCG, Semenchv FL Alcohol, smoking and occupa~onal factors in the astrology of cancer ofb~a oral cave, pharyx er~d [~'ynx. Ird J Cane.at 34: 603-612, 1985 2. Tuyn= AJ. Astrology of head and neck cancer: Tobzmco, aicohof and dial Adv Otorhinola~gol 46:89-106, 1991 3. Fuju Chang. Experime.rCsl studies on rst lung caP, car ~nduced by Inbat~haal ins~llation of coal tar pitch. I. Histopathological sludy. MS Thesis, The P.ublice~on of Hene.n Medical University, pp 1-32, 1987 4. Fuju Chang. Ex'pedrne~ studies on rat lung cancer induced by Intmbacheal inst~tlation of co~l ~r pitch. It. HistopethoganesL~ oftha lung cancer. MS 3~ha~|s, The Publica~on of Henan Medk~d University, pp 33-71, 1987 5. Fuju Chang. Experiments/studies on rat lung cancer induced by irdmlracheal Inst~llatlon of coal t~r pitch. IlL The roles of coal tar pitch granulomas in the pa~ogunesis of lung cancer. MS Thesi=, The Publication of Han~n Medical University, pp 72-90, 1987 6. Fuju Chasg, Qingxia:: Zhao, Qingfan Zhu, ~qming Wu, Chert Chun, St~na Syrian, Kad Syrj~nen. invest~ga~ion on the carcinogenic effe,dcs of c~ tar pitoh in rat respiratory tracts by Irrhabacheai Inst~ll~rl~ons. Carcinogenesis 1991. In pr~. 7. Sydtmen KJ. Papillomavlrus infections and cancer. In: 8yrj~n K, Glssmann L, Ko~ LG (ads). Papillomwiruses and human disease. Heidelberg, Spdnger-Verlag, pp 467-503, 1987 8. K-~,hima H, Mounts P= Tumors of the head and neck, lanjnx, lung and esophagus and their possible rela~on to HPV. In." 8yrj~an K, Gissmann L,'Ko~ LG (ads). Pspillomaviruses and Human Disease. Heidelberg, Springer-Vedag, pp 13~-157, 1987 9. Syrj~man S. Epldemio!ogi¢~ and ~linicel features of respiratory HPV le.~on.~ In'. yon Ka'ogh G, Rylander E (ads). Genitoanal Papillom~ Vitae infec~on.~ A Survey for the Clinician. IQulstad, Conpharm AB, pp 261-287, 1989 10. ~"lebberg BIvL Human papil]on~viruses und upper airway oncoganesis. Am J Otole--yngol 11: 370-374, 1990 11. =Jr ~n H. Human papillom~viruses and their possible role in =:lUamOU~ cell cmelnoma¢ Curt Top Mlc~oblol Immunol 78: 1-30, 1977 12. Vrabe~ D. The irNeded Schnalderian Peidl]oma: A clinic.el and pathologic~ study. La,'yngescope 85: 186-220, 1975 13. Syrj~nen KJ, Pyrh6n~n S, Syrj~sn S. Evidence wJgge~l~ng human papilloma vlru~ (HPV) etiology for the squamous cell paplllom~ of the pa~a~as~d= sinus. Arch C-aschwulsfforsch 53: 77.~2, 1983 14. Resider DS, Jahn =A, P=er A, Pater MM. Isolation and ch~aetertza~on of paptl]omavtrus DNA from nasal inverffng (Schneideri~) paidlloma~. Ann Otol Rhino] La~ngal 2: 170.17~, 1987 15. de Villisds E~, HIr~c..' h-Behnern A, yon Knabel Doeberitz C, Neumann C, zur Hausen H: Two newly identffied human Pepillom~viru~ type~ (HPV 40 and 57} ~sola~ed from mucosal lesior,-. Virology 171: 248-253, 1989 16. ~y=j~nen 8, Happens ..h R-P, Virolalnen E, 8iivonon L, Syrj~nen K. Detection of human papillorrmv~ms (HPV) ~ructursl antlgan~ end DNA type= in nacal!inve~d papillomas and qu~mou~ cell careinoma~ of the nasal cavlffe~ and paranesal sinuses. Act Otol~-tgal 104:334-34t, ;1987 17. Brandwein M Stelnbdrg B, Thung S, et el. Human papillomsvtrus 6/11 and 16/18 in Schneldedan Inve=ted papillomas. In sttu hybrld'~ca~on with human papiIIomavlrus RNA probe=. Canner 63:I708-1713, 1989 16. Kiemi PJ0 Joeesuu ~1, Syrj~nen $. Syrj~nen K, 81ivon~ L, Virolalnen F.L Association of DNA aneupIoldy with huma~ papi~lomwime induced r~alignant transformation of sins-neural t~ansitionai papillomas. Otola~ngal Head Neck Surg 100: 563- 567, 1989 19. Moun~ P, Shah KV..i=~esplrslory papillomatoals: Effologica] raison to genital t~act paldllom~vi~lSeS. Prog Mad Virol 29: ~O- 1140 1984 20. Reeler DR, ~now JB...Cell free filtrate transplardatlon of huma~ I=ryngeal papillom= to dogs. I.anjngoscopa 77:397-4160 1967 21. ~/rj~ulen K, ~yrj~nen $, Pyrh6non S. Human papilloma virus (HPV} antigens In lesions of laryngeal equamous cell carcirlom~=. ORL 44: 323-334° 1982 ! 22. Levi JE, De]cole R, AIl~e~ VN, Todoni H, Villa LL: Human paplllomavirue DNA in raspirato=y papillomatasis dete~ed by in situ hybddiz~on and the pa/ymer~e chain reac~on. Am J Pathol 135: 1179-1184, 1989 23. Gl~nann L, Dlehl V, Schultz.Coulon H J, zur ~ H. Mole~ula~ oloning ~nd cher~cterizaiion of hum~ paidlloma virus DNA derived from a laryngeal ~apilloma. J Virol 44: 393-400, 1982 24. Majoros M, Parkhill E=M, Devine KD. Paidllon'm of the lan/nx in children. Am J Burg 108:470-475,1964 25. Kahn T, 8chwarz E, I~nberg H, zur Hausen H. Molecular cloning and characterization of a new human papillomwirus (HPV 30) from ~ I~yngeel car¢!nom¢lnt J Cancer 37: 61-65, 1986 28. b~yrj~nan 8, Syrians ~ M&dyjErvi R, Collan Y, I(~j~ J. Human papillomavirus DNA in squamous call carcinomas of the I~ynx clemens]rated by in situ DNA hybridPzaiion. ORL 49:175-186,1987 27. Aixamson AL Brandsma J, Steinberg B, Winklor B. Verrucou~ carcinoma of'ha larynx; Possible human papillomwims et~ology. Arch Otolaryngol 111: 70~-715, 1985 28. ~yrj~,~an KJ. Condylomatous changes in neoplasl~ bronohi~ epithelium. Repor~ of a case. Respiration 38: 299-304, 1979 29. SyrjEnen ICI. Epithei'.=al lesions =uggestive of • cendylometous odgin found closely associated with Invasivo bronchial squsmous call carcinoma=. Respiral~on 40: 150-160, 1980 ~0. Syrj~men KJ. Bronchial squanlous cell carcinomas a.~ociated with epithelial changes Identical to condyloma~ous lesions of the utedne cervix. Lung 158: 131o142, 1980 3I. Sbemlau A, Gissmanr~ L. I~enberg H, Stark M. Bannesh P, zur Hausen H. Human papillomavlrus type 16 relaIed DNA in an anal=leslie carcinoma of the lung. Cancer 55: 1737-1740, 1985 32. Syrj~nen K, Syrj.~nen ~. Human paidllomavirus DNA in bronchi~d =quamous cell caminomas. Lancet i: 168-1690 1987 33. SyrjEnen K, Syrj~nen E~. Kellokoski J, I~rj8 J, M~tj~uvi FL Human papilloma, virus (HPV) type 6 and 16 DNA sequences in bronchial squamous cell carci~,emas demonstrated by in sltu DNA hybridization. Lung 167: 33-42, 1989 34. Syrj~nen S, Syrj~nan K. An improved in situ DNA hybridization protocol for detection of human papillomavirua (HPV) DNA sequences in paraffin-embedded b~opsie& J Virol Math 14: 293-304, 1986 35. Chang F, Syrj~nen S, Nu~nen J, K~.rj~ J, SyrjSnen K. Detection of human papillomm~rus (HPV) DNA in end squemous cell ca,'cinomas by in situ bybddP~on and palymerase chain reaction. Arch Dermatol Res 282: 493-497, 36. Chang F, Janatuinan E. F~.~ataJnen P, Syrj~nan S, Sy@nen K. Esophageal squamou= cell papillomas: failure to detect human papillomwirus DNA by in s~u .'.-ybridization and polymerasa chain reaction. Scand J Gasb'oanterol 26: 535-543. 1~)1 0 ~J~ O~ BATCo document for Mayo Clinic 28 March 2002
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PROJECT III. ('rURKU) BASIC BIOLOGY OF HPV INFECTIONS IN SQUAMOUS CELL CANCER 1.CELL UNES 1.1.BAC~ND HPV 16 and HPV 18 DNA have been detected in cell lines derived from carcinoma of the uterine cervix (1) and HPV DNA has b~en shown to imrnorta~Be kerstinocytes in vitro (2,3). However, the full understanding of the inherent mechanismsi of initla~on and progression of squamous cell neoplasla requires the study of cells from premaJlgnant lesions !where the factors influencing the progression can be studisd. These studies have been hampered by the sm~Jl number of established long-term cell cultures derived from precancer lesions. An existing cewioal keratlnocyte Sell line Is derived from a mild dysptasla (4). These cells are dependent on 3T3 feeder cell support a~d they co~aJn high copy number of predominantly eplsomal HPV 16 DNA. Another cell line has been recently established from a vulvar Bowanoid papulosis lesion (5). To fully eluoidat~ the factors involved in the inlti~on and progression of cewioel neoplasia, data are needed not only on the cells r~.epresentative of the ma~cjnant phenotype but, Ideally, on the cells where the biology of HPV can be examined in e~y lesions. Premallgnant cell llnee isolated from in vivo lesions would serve this purpose, but the establishment of s~ch cell lines has been dll~ulL We have recently Isolated two cell lines from cancer precursor lesions (6). The first ce.II line, UT-DEC.-1, was derived from an HPV 33-positive rnild vaglnaJ dyspissJa. The other cell Ene, UT-DEC-2, was derived flora an HPV 16-positive moderate vaginal dysplasla. A number of re .l:~orts exL.~ts linking HPVwith cen~ice~ cancer and cancer-derived cell lines (for review see 7,8). Considerably fewer r@orts have focused on the issue whether HPV plays a significant role in squamous cell c~rcinomas at the oth.~r sites of the anogenitaJ bact (9,10). The detection of HPV In genital warts, and pa~iculady in VIN, VAiN and inv~sive carcinomas, suppo~ls the hypothesis that HPV may be responsible for at least part of the carcinomas outs~e the uterine cervix as well (7,10,11,12). "[he detection rate of HPV varies considerably in different sedes of clinical samples. It is currently agreed that the detection of HPV Is st~'ongly dependent on the sensitivity of the meth~:t used, as well as off the representativeness of the samples. There are still no studies on the cell lines derived f~om extracervical genital squamous carcinomas concerning the presence and physical state of HPV. This is due to!the, slmpis reason th= such cell lines have not been available until recently (6,13,14). Using an panel of appmpriat~ DNA techniques, it will be possible to reliably study the rote of HPV as well as oncogenes and antioncogenas |ni Isolated carcinoma cells growing in cell lines. Furthermore, it is important to study the extracervical genital carcinomas to elucidate their similarities and differences to the cervical caminoma. This may eventually lead to a m~re specific treatment of these diseases. 1.2.AIMS OF THE STUp.. Y The purpose of this study is to examine the factors of growth regulation in both premalignant and malignant cells derived from the iower female genital squamous epithelium. The transform~ion of the cell and maintenance of the malignant state are analysed in two premellgnant cell lines derived from the vagina and in 9 malignant cell lines from the vulva. In order to under, and the mechanisms of ~ ma.infanance and ~ of~ ~n 1).HPV aenome in UT-DEC-I- and UT-DEC-2 cells durlnq different staqes of transformation. This study w~ provide BATCo document for Mayo Clinic 28 March 2002
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51 us with information about the regions of the HPV genome that are important to con'dnuous growth of the host cells. 2).F_xpression of HP~ in UT-DEC-1 and UT-DEC-2 cells dudnq the different staqes of transformation. This analysis is a pre~luis'rte for the future studies, where we are wing to modulate the transformed cells towards the normal phenotype by using anlisense transcripts. The final goal is to design new therapeutic strategies against these squamous call ineoplasia. ~.p53 and retinoblastoma aeries in UT-DEC.I and LIT-DEC-2 cells. The antloncogene status of the cells is, to the current opinion, essential before ar~ conclusions can be drawn from the factors influencing abnormal cell prol'~eration. 1).Presence and physical state of HPVs in ceil lines derived from 9 vulvar and one vaqlnal carcinomas. Relatively few re.ports are available on HPV and vulvar carcinoma and even much les~ from the rare pdmary vaglnat carcinoma (13.,1<.). All ef these studies am based on clinical material. Many, if not all, of these studies can be disputed about th.e sensitivity of the methods applied. Similarly, the prapar~ion of the sample may be critical. Slnce the cell lines C~ntaln only malignant cells, the sample preparation does not affect the results. These 10 cell lines are screened fo~ the presence of all known HPV type~ ~.State of p53 and Rb .qenes in HPV-positk, e and -nepl. stive carcinoma cell lines 'from the vulva and vaqina. The primanJ obj.bctlve is to test the hypothesis preesrded on the basis of the findings In cervical cancer ceils, namely: HPV-negative!cancers express mutated tumor suppressor gene, while HPV-posltive cancers express the wild type protein (15,16). Qur non-cewioal (ganliaO carcinoma cell lines sews well for this purpose, since the expected HPV-positlvity is muc~ lower than that in the ce~cal cancers, where HPV DNA is usually present in >90% of cases. In addition, many of the studies based on clinical rnatedal are handicapped because the tumor sample is always a mixture of both mailgnant and nonmaJlgnant cells. One can never be confident which cells are actually being analysed, whereas th~ cell lines constitute malignant cells only. UT-DEC-1 call li~e was dervLsd from a mild vaginal dysplasia (VAIN) biopsied from the dght lateral fomix of a 41-year.old woman.i Since the pdman] treatment by l~ser vaporization, this therapy has been repeated twice because of flat vaginali;condyiomas. At the latest control in 1991, no abnormalities were found. The restriction pattern of HPV 33 changed during the early passages Co7-p9) indicating an integration of a previously episomal HPV DNA at that stage. The integration was confirmed by two-dimensional gel alectrophoresis. The ceil line UT-DEC-1 has been cytogenetically characterized previously (6). The other ceil line, UT-DEC-2 was derived from an HPV 16-positive moderate vaginal dysplasia and has also been characterized previously. 1.3.2. Vulvar cell lines Cell lines used In this study have been estab[Lshed from squamous cell carcinomas of the vulva. These BATCo document for Mayo Clinic 28 March 2002
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carcinomas i"~ve bee.n kanjotyped in short-term cultz.~'e ~ Jn early l:~=%~9es as e,.=ta.biished celt lines (1~,14). The chromosome abnorm~ itles found indir.~ed ~ 1) vulval c~rcinornas ~e ganetic~ly complex, but ~omogeneous; 2} loss of 18q22-c123!and loss of 10q23-q25 may be associated with a poor prognosis, and 3) development and progression of vulval carcinomas appear to result from cumulative effects of altered gene dosage at multiple, consistent loci. 1.3,3, Cullum of c~is. Mo~olayer cultures are maintained under standard media supplemented with serum and spesific nutrients and growth factors when necassa~. 1.3.4.Presence and state of HPV in ceil Presence of HPV DNA in the cell lines is studied with Southern blotting: The e~tmcted DNA is cleaved with spesiflc restriction endonucisases and run In agarose gels. The fragments are transferred to membrane and hybridized. The restri~ion pattern is compared to the prototype HPV. I.~rge fragments that do not fit the prototype sequence Indicate Int .~:jratlon. Integration sites can be revealed by cleaving the DNA by endonuclesses that do not cleave HPV. ff the r~ rictlon fragments differ In size, it Indicates that HPV has Integrated at multiple sites. The losalisatlon can be verified by chromosome.In eltu hybridization. Possible HPV D~IA deletions can be detected by Southern blot from different p~ssages of the call Ilne, ff any fragment decreases iri size it is indicative of deletion. The cleaved DNA is hybridized with subgenomlc fragments to find out which gene~ are deleted and whP,.h genes remain. This is important particularly in premaiignant cell lines. Interesting fragments ~n be amplified with PCR and sequenced. Physical sta/a ~ HPV can also be studied by two dimensional gel electrophoresis. It Is also possible to estimate the HPV cow~. number by comparing the mobility of different HPV structures in the gel. In situ hybridization is used to find out wh...i~ther different cell populalJons exist or whether the copy number is invariably the same in all cells. The gene expression will be atudled by RNA In' situ hybridization by gene-speslflc probes. Transcription Is also studied by Northe~ blot hybddlzallon. Transcriptt~ may also be studied by cloning cDNAs which are produced from mRNAs by revere transcriptase, cDNA is cloned to a bacteriophage. Recombinants can be isolated by using whole gsnomic probe In hybridization. By sequencing these clones it is possible to study the transcription pattern in premaflgnant calls from different passages of the cell lines. 1.4.CURRENT PROGRESS OF THE STUDY The first step of ~is approach was the isoistk~ of two prema~ignant cell lines from dysplestic squamous call" lesions (6). In this work, we have characterized the cell lines by their growth properties, karyotype, tumorigenicity and presence and state of HPV in monolayer culture. We desdbed the selection of abnormally differentiating cells from the surrounding normal cells. Both cell lines seem to be immortal. During the early stages of differentiation, HPV 33 which was initially episomal, spontaneously integrates in the host genome. Chromosomal Instability was noted shortly after this integration event. HPV 33 E6-E70RF:s were also found transcriptionally active. UT-DEC.2 cell line was shown to be HPV 16.positive by PCR. UT-DEC-2 ce~is are considerably less proliferative than UT-DEC-1 cells, and they are also dependent on continuous epidermal growth factor and dexamethasone substitution. It is apparent that secondz,7 changes have taken place dudng the culture. C~ O~ BATCo document for Mayo Clinic 28 March 2002
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53 1.,5.~ OF THE STUDY So far, the studies on the paIhogenesls of HPV-ralated lesfons have relied on analyses done on c~inical samples, comparative studies with an[rnal models and cell ¢~lture studies. Cell culture studies have been based on examining cervical cancer call lines and trar~fectlon stu~es. In malignant cells, the progression to malignant phenotype has already occured, and we can only indirectly es~mate the events at the Initial stages of carcinogenesis as wall as co-fastors k;fluencing it. Transf~ctfon studies are hampered by the fact that the infec'don is transported into ~e cell in a more or less unnatural way. In the present project, we are using cells de~ed from an in vlvo lesion, and the spontaneous progression of the premal~gnant cells is monltored in cond~ons that are as little manipulative as lx~sfbls by using the modem culture techniques. The cancer cell ~nes to be used in this study represent cells from a unique orlgtn of anogenital squamous epithelia, Le., vulva and vagina. Cell lines originating from these s/tes have never been used for HPV research before. The study described here can provide us with information that may help understanding the mechanisms that influence the growth regulation of neoplest~ squamous cells of the lower femaJe genital tract. 1.6.~'i1JRE Cfi'ED 1.13o~hart M, Glssrnann !., Ikenberg H, I~alnhelnz A, Sch~udan W, zur Heu=en. A new type of papillomsvinn DNA, ~ presence in genltzd cermet biopsies and in cell ,nee derived from caned ¢m~cer. EMBO J 3:1161-11570 1984. 2.Durst M, I>tmtlw~-Pel~seveka RT, Boukamp P, Fusentg NE, (~ssmann. Molecular and cytogenMt= anal,/sl= of [mmo,lzdized human pdmmy kemlinocytes obtained after t;'ansfe~on with human peplllomavln,e type 16 DNA. Oncegane 1:251-256, 1987. 3.Kam" P, Mc[)ouga~l JK, HPV-18 lmmoxtallzatlon of human kar~nocyte~ V;roiog~/173:302-310, 1989. 4.b"tank~y MA, Browne HM, Appleby M, Minson AC. Properl~ of • non-tumorigenic human cervioal keratinocyte cell line. Int J Cancer 43:672-676, 1989. 5.~-.hneldar-Maurmu~y S, Crois.~ant O, Orht G. Intagmffon of human pepillomavirus type 16 DNA sequences: a possible earl,/event in t~e progre..~ion of genital tumor~ J Virol 61:3295-6,1967. e.Hl~tanen, ,.~; Auvfnen, F.;=." Gr~nman, S; Ltdcke~a, T; ~d~la, A; Kiorni, P; M,~enp~, J. k;ola~on of two kar~iinocyte cell l~nee derived from HPV~e.: dy=ph~t~¢ vaginal lemons. Int. J, Cancer 52: 391.398,1992. 7.~rj=tnan, KJ. Paplliom~n~ irffections and cmlcer. In: Papiliorn~dru~ and human disease. (Eds: E,'yrj~nan, K; G|esmann, L; Koes, LG) Springer-Vedagi Berlin-Heidelberg, 467-5030 1987. 8.zur H~u=en, I-L Human ~)apiliomm'iruces in the pathoganasl= of anagenital cancer. Vlroicgy 184: 9-13,1991. 9.Bu~,arna, J; Naghashf~r, Z: S~wada, E; Darnel, R; Wooc/ruff, JD; ~ K. The predominance of human peplllemsviru~ type 16 in vulvar neopla=ia. Obste~ Gynecel. 71: 10.Plancer, RS; Hobby, JB. Intreeplthel;al and Invsslve neop~asie of the vulva in as.~ociatlon with human paplliomsvlrue Infe~ion. J. ReprocL Mad. 33, 11.Andersen, W; Franquemont, D; Wllllern~, J; Twler, P; Oum, C, Vulvar squamou~ cell caminoma and papil~om~vlruses: Two =epara~ err~es? ,&an. J. 0bstet. Gynecol. 165, 329-336,1991. 12.BIO~, J; U~o, S-Y; W'~zy~ki0 S; Macri, C; Walker, J; Peaka0 M; Barman, M. Cllnlca] and Histoiogi¢ features of Vulvar caminomes Analyzed for Human pepiliomavirus Statu~ Hurr~ P~'~oL 22: 711-718,1991. 13.Grenman, SE; Van Dyke, DL~ Womhem, MJ; England, B: M~'Cl~t~hey', KD; Hopkins, M; I~bu, VR; Grenman, R; Caray, TF. Pkenotypic char=ctariza~on, k=uyotype an,,lysis and in vil~'o tamoxifen =ens~vity of new Er-nagat;ve vulvar carcinoma cell lines, UM-SCV-1A and UM-SCV-1B. int. J. Cancer 4,5: 920-927,1990. 14.Womh~m, M J; Van Dyke, DL; Granman, SE; Grenman, R; Hopkins, M: Roberts JA, Gasser KM, Schwmtz DR, Cmey, TE. Consi=tant chromosome abnormalities in equamous cell carcinoma olive vulva. Genes, chromosomes a~d Cancer 3:420-432, 1991. 15.Scheffner, M; Munger, K; Byme, JC; Howley,, PM. The state of the p53 and ret~noblestoma genes in human cervical carcinoma cell lines. Proc. Nz=fl. Aced. Sci. U. S. A. 88. 5523-5527,1991. 16.Crook. T; Wrede, D; Tidy', J: M~on, WP; Evans, D; Vousden, KI-L Cicnal F53 mutation in pr~ma~y cervical cancer:, association w~th hurnan-papF~/om~ms-negagve tumours. Lancet 339, 1070-1073,1992. 0 BATCo document for Mayo Clinic 28 March 2002
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~ORGANOT~IC TISSUE CULTURE SYSTEM (RAFT) 2.1.BACKGROUND The mucosot~odhic HPVs produce few vifions in vivo. Nonetheless, HPV11 has been propagated successfully in neonatal tissues implanted in a~hymlc or nude mice (1). Specifically, human foreskin chips experimentally infected with HPV 11 and impl.anted under the renal capsule of athymic mice developed into cysts that are Indistinguishable from genital condylomaya in morphology and in patterns of viral activity (2). The vidons recovered can be sedally passaged in fores~in xenografts in nude mice. HPV 16 vidons have also been visualized by electron microscopy in a cell line derived from a clinical biopsy after being grafted onto the flank of a nude mouse (3). Pdma.nj epithelial cells can be propagated in vitro In the presence of fibroblasts. Although the resulting cell sheets ha~e been used successfully for autologous skin grafts for burned patients, their morphology Is markedly d~fferent from the native epithelium, Indicating that proper cellular differentatlon did not take place, however. During the past ifew yearn, e system dubbed the ~ system h~ been increasingly utilized by epithelial cell biologists and HPV re,ambers because it allows primary human foresld~ ker',~tlnocytes to achieve stmtlf-m~ion and differentatlon morphol?gieeJly similar to the fore~kin in vivo (4.5,6). In this system, dispersed pdmary keratlnocytes are seeded on a dem~. al equivalent consisting of a porous collagen gel containing flbroblests. At confluence, the assembly is raised to ;the medium-air interface. The kera~lnocytes stratify and exhibit a differentiated morphology, generating a living skin equivalent. The system has been used in parl~cular to study the influence of E6 and E7 genes of the high risk; HPV types on the morphological differentt~on of keratinocytes (7',8,9). Recently, a cervical cell line containing episomal HPV-31b has been grown as a raft culture, some transcripts from the E and L regions being detected by Nothem blot hybridization and DNA replication was detected by in situ hybridlz~on (10). Dollar et aL were the first to product HPV vidons in vitro using a modified ralt culture system (11). OF THE STUDY The purpose of Sis study is to use the rat culture system to explore the dynamics of HPV-assoolated carcino- genesis in the vaginal ;and vulval cell lines described above (111.1.3). First, these cell IIn~ are analyzed for growth and differentation pote~ial on the raft culture system at a variety of passage levels to gone'ate squamous ep~halium The second hypothesis implicates that HPV integration pe~ se might be 1ha tdgge~" of the dysplastic alteralJons in squamoue ep~heliu~. This will be studied in the cell lines at passage levels before and efter the integration of HPV DNA. The ~xpredsion of E6 and E7 genes has bean shown to cortege with the cell proliferation In vitro end tumorigenicity in vivo (~2). In this study, the cell lines me used to characterize the changes in HPV gone expression that occur in concert with epithelial differertt~on and/or dysplasia. co-factors are still incomplefely characterized, however. The fourth aim is to study the effects of different factors of growth regulation and their association with HPV-induced carcinogenesis in the cell lines grown on a raft system. Several studies have suggesled ~'~ ~ smoking is a signiffc~ co-factor in HPV-ascoci~ed can:inogenesis ~t ~ fan~e genital tract, ~ of the ursine cerv~ in paJlJcu~r. The established raft culture system can be used to esses~ the effects of nicotine and co~Inine on e~hellal morholo~ with and without the presence cf HPV infection. 2.3.MATEPJALS AND METHODS 2.3.1. Culture of spithelial cell lines and normal can~k;al/txccaJ (or ~omskin) fibroolaats i BATCo document for Mayo Clinic 28 March 2002
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Ep'~helial cells ,'~om different passage levels are grown in DMEM supplemented with 50~l/ml streptomycin, 100Ulml penic~lin and 10% fetal calf serum. Biopsies from a normal cervical and buccal muc~a as well as from the foreskin are transported to laboretory in DMEM ~ntng 100#g/ml streptomycin and 200U/rnl penicillin and 0.5/=g/ml Fungizone. The connective tissue is cut Into small fragments of lmm~ and explent cuRures are started according to standard techniques. For the r'a/t culture, flbroblasts dedved from the explants are f~om their passage. Different fibroblasts (i.e. mucose] or skin) are tested for their effect on epP, J~elial differentatlon as well. Ra~ cultures are generated using the procedure published recently by Shahabecldin (13). In this method, flbroblasts are Incorporated into a dermal substrate (DS) composed of collagen-glycesaminoglycan cross-linked by c~osan on which ep~elial cells are seeded. Shortly, a/ter washing, the pieces of D$ (3cm2) are equitil:~aIed in DMEM a~ 37"C for 2h..With the aid of stainless steel rings, 4x10s f~roblasts are seeded onto the DS and DMEM supplemented with a~blotius and 10% fatal calf serum is added outside the ring Lq each dish. Medium is changed 3 times/week for 2 weeks. Then the DS is turned upside down, and ep~eilal cells are seeded on the DS similarly as done with the fibroblasts. The medium is DMEM supplemented with insulin, epidermal growth factor, hydro- ¢orl~one and ascor~i~= acid. The medium Is changed every second day. After 5 days, when the ceils are reached the confluence, the cu~u. res are lifted into the air-liquid interface using a eta/nles~, steel gdd. Epithelial cells are then allowed to stralify at least for 9 days. The effects of c~.~ ctors on epithelial morhology are studied by treating the raft cultures e.g. ~ retinoio acid, 12-O-tebadecanoyl pl~.rbol-13-acatate, TGF, TNF, cotlnlne or nicotine (and other known carcinogens) for 16 hours every fourth day for 16, days at physiologic concentrations. • 2.3~3.FlxaUon and pmpamtk~ of Ussues Half of the cultur.b is fixed in 10% neutral formalin and embedded in parafF-m.. The other half of ~e culture is frozen until used. Five-F~icmn sections of the ~ft specimens are cut for muntine histology, Immunohlstochemlstry, in situ hybridization an~ PCR. The differenti~o~, of the epithelium grown on the raft is studied by immunohb'tochemisW using a panel of cytokar'~ns. For the I.¢calizatlon of PCNA, a marker of cell proliferation, a polyclonal antibody (P10, Dako-PC, Dakopalts, Denmark) diluted in 1:50 Is used. Protein p53 is detected by using a polyclonai antgx~'y (CM-1, Novocast~ra Laboratori~ Ltd., Newcastle upon Tyne, UK) diluted in 1:1200, Immunohistochemistry is performed with the avk:lin biotin immunoperoxldase (ABC) method according to routine procedures described elsewhere. 2.3.5.1n situ hybrid~ Whole genomio Or exon-specific subgenomio clones in pGEM vectors are used. Riboprobes are synthesized by in vitro transcripticn in the presence of 3SS-labeled UTP. In s~tu hybridtzallon is peformed as described before (~4,~5). 2.3.6.Polymerase chain reaction (PCR) PCR is used tc confirm the presence of HPV DNA and HPV transcripts. In add~on, it is used to amplify exons 5 through 9 and exc~'.s 5 to 22 of the p53 and Rb genes, respectively. The ampl~ed genes are used for the SSCP analysis ~-s descrit:ed in 111.3.3.3. BATCo document for Mayo Clinic 28 March 2002
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To screen the presence of p53 and Rb gone mutations In cells from different passage levels as well as in cells grown on the raft cult .t~e system, PCR-SSCP anaJysis is performed, as will be dascn'bed later in 111.3.3.3. 2.4.CURRENT PRoC__.~tEss' OF THE STUDY The e~psdments, were staded with the cell line UT-DEC-1 derived flora a vaginal mild dysplasia at a late passage 63 (j~63), no~. grown on the raft culture. The first results were recently presented In 8th World Congress of Cervical Pathology and Colposcopy, Chicago, May 12-16, 1993 (16). Morphology of the UT-DEC-1 cell line (p63) In organotyptc culture was abnormal in a number of ways. The 15.20" layer-thick .squar~ ous epithelium was compc~ed of undifferentiated (besalold bjpe) cells (Fcjum 4), and the eplthellal sheets were icomplstely disorganized. On the surface of the epithelium, keratohyaline granules were sometimes present. The entire thickness of the epithelium was dysplestic, occupied by cells w~th a high nuclear- to-cytoplssmic ratio ~ with a high number of abnormal mitotic figures. The rafted cells showed a high proliferative activity in all layers, asdetermined by PC, NA Immunohistochemistry. The raft was HPV 33 DNA-positlve by ~n situ hybrldizal~n. We also .~analyzed the p53 expresslon and found an irregular staining pattern with some Intensely staining cells, while others were only weakly p~sitive, the PCR-SSCP and sequence analysis of the p53 gone revealed no mutations, ~:however. The obsewatlon that these cells have undergone a progression towards a ma~jnant phenotype prompted us to look back the progr :easive steps of transform=ion in this particular cell line. We have now raft cultured the cells of this lineage derived f~om 4 different pessaqe levels In o~der to establish a model whereby the premallgnant cells can be monitored in vit)-o as they gradually lose their regulato~ mechanisms controlling celt pmlifer~on. 2.5.SlGNIRCANC~ OF Til-IE STUDY Our prellminanj results have shown that the raft culture system offers a particularly useful method for the study of HPV-assoclate~l carcinogenesis in vitro. The possibility to modulate the viral life-cycle program offers an opportunity to study ~ iregu~ory mechanisms not possible in vivo. The premelignant and cancer cell lines to be used in this assay repreSent cells from unique origin of the genital epithelium. Cell lines originating from these sites have never been examined before. These cell lines grown on the mJt culture system provide an excellent model to study the HPV.induced Carcinogenesis or e.g. the mechanisms whereby virus laIency is malntained and Interrupted. t.IOeider JW, Howett MK, Leure~Dupree A, Zalno R J, Weber JA. Laboratory produ~on in vivo of infectious human papillomavirus type 11. J Virol. 61:590.593, 1~7. 2.Stoler MH, Woli=ky SM, Withbeck A. Broker TFL Chow MR. Infectious cycle of human paptllomavlrus type 11 in human foreskin xenografl~ in nude mice. I Yirot 64:3310-3318, 1990. 3.Stealing J, Stanley M, Gatward G, Mir~on T, Production of human pepillom~vlrue type 16 vidons in keratinocyte cell line. J Vim] 64:6305-6307, 1~0. 4~.sselineau D, Pmnlems M. Reconstruction of simplified skin. Control of fiabrication. Brit J Dermalol 111:219222,1984. 5.Kopan R, Traska G, Fuchs F_ Ret[noids as important regulator= of termina] d~erentalJon: Examining keratin expre.~ion in individual epidermal cells =d vadous stage,= of keratinization. J Cell 8Iol 105:427-440, 19~7. B.Pe~enteau NL Nolte CM, Bilbo P, Rosenberg LM. et.al. Epidermi~ generated in vitro: Practical consideration and applications. J Cell Biochem 45"245-251, 1991. 7.McCance DJ, Kopen R. Fuohs F, Laiminis LA. Human pepillomav]rus type 16 laters human ep~thellal cell dlfferenta~on in v~o. Pros Nat~ Acad Sci 85:7169-7173, 1.o88. BATCo document for Mayo Clinic 28 March 2002
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8.Blonton RA, Pare.~Rey~.' N, Mmdck DT, M~Doug~ -.'K. El~he]bd cegs tmor~dized by human papillomeviru=es have premaJignant ¢hatactlr~-tic= in org-motypic culture. Am J Pathof 138:673-685,1991. 9.Wooo~,K~t~ CD, Cheng S, Simpson S. Hamacher L, Chow LT, Broker TR, DiPeolo JA. Recombinant retroviruses encoding human papillomavirus type 18 E6 and E7 gee~ s~rnul~te cellular prolifend~on ,~nd supress squ~rnous different~tion in human kera~noc'yt~ in vib'o. O~. cogene. 7.619-626, 1992. 10.Bedeg MA, Hudson J.~, Goiub TR, Tu~yk ME, Wilbank GD, Labninis LA. Amplifica~on of human papillomavirus genomea in v~o is dependent on epithelial dlfferenta~on. J Virol 65"2254-2260, 1991. 11oDOllar SC, Wil~on, J~. Demeter LM, Bunnez W, Reichman RC, Broker TR, Chew LT. Production of human papil~om~vlru= and modu~on of ~e infectious progmam in epitheikd ra~ ¢ulture¢ Genes and Development 6:1131-1142, 1992. 12.von Knebet Doeberitz, M; Oltersdorf, T; .Schwar~ E; Glssmann, L Correlation of modified human papfllomavlrus early gene expre=.~ion with alter~l ~rowth properties in C4-1 cervical carcinoma cells. Csncer Res. 48: 3780.3786,1988. l~.Shahabeddin L, Bert~od F, Demour O, Col~ombel C. Chmactedz.a~:~ of skin reconstructed on *, chitosen-cros~ linked collagen- clycosaminog~,can rna~'~ Skin Patma~'~ 3:107-114. 14.Sy~J~en S, ~yd~nanlK. An improved in situ DNA hybridization protocol for detection of humen papiilomavirus DNA sequences in paraffin embedded s~ons. J Vlrof Meth 14:293.304019~6. 15.~'yrJ~nan 6. Viral gen~ detection by in allu hybrld~za~on. In ¢fi'agno~Jc Molecular Pathology. A Praet~c-ai Approtmh. Ed~. C.,~ Hmdngton ~ J. OD. McGee. Oxford Univer=lty Pr~, Oxford. 1992. 16.~d&'lerk S, Hletanen~, $, Syrj~nen. K. Ra~t Culture Studies on HPV ~-po~i~ve cell line derived from VAIN. 8th wodd congres~ of ce~tic~d pa~ology an~ colpo~copy {Chicego, Illinois, MW 12-16, 1993). 3.SIC-=NIRC, ANCE OF HPV, p53, AND RERNOBLASTOMA GENES IN HW-ASSOCIATED CARClNOGi~ESIS 3.1.BACKGROUND New insight Int~ the possible mechanisms of HPV-essociated transfon'nation has recently dedved from the studies on antkonc~enes or tumor suppresscr genes (1,2,3,4,5}. The best known genes of this group are the retlnoblastoma susceptibility gene (Rb) and p53 gene. Inactivation of both alleles of the Rb gene in a single precursor call leads to~e develop .ment of re~noblastoma, a tumor adstng from incompletely diffaremiated retinal cells in young c, hildmn (2).!An altered or delsied Rb gane has also been found in a vadaty of other malignant tumors including osteosarcor~a and small-cetl lung cancer (2). The p53 gene was initialally believed to be a dominant oncogene. However, t.he initial studies were shcwn to be pedormed with a mutated p53, and subsequently it became clear that the wild-tYl~~ p53 acts as a tumor suppressor gene (1,2,3,5,6). Mutated p53 has now been found in an increasing number of different human malignanc~as (3,7). Both the Rb protein and p53 protein are interesting in that they are able to form .s~ble complexes with the ~ansfonllhlg proteins of several DNA tumor viruses. The simian virus 40 (SV40) large T anilgan and adenovirus EiA protein bind the Rb protein, and the SV40 large T antigen and adenovirus EIB protei~ bind to p53 (1). This c~eady Implicates the possibility that cellular tumor suppressor gene products may be inactivated by thel~ binding to these viral oncoproteins. It was also demonstrated recently tl~ HPV 16 E6 protein binds to 1353 gene product (8,9), which usually acts as a negative regulator of cell proliferation (1,3,6). The expression of tumor suppressor genes is changed In several tumors due to genetic alterations, e.g. deletions (10,11) and mutations (2,7,12). In many tumom, the expression of mutant p53 is frequently increased as compared, to the wild-type p53 in normal cells. Due to its longer half-life, the p53 expression detected by immunohistochem~y is usually interpreted as a sign of mutant p53, and not that of the wild-type p53, present in cell cycle Go-Gz phase In proliferating ceils (1,2,3). HPV 16 and 18 E6 protein binding to the wild-type p53 has been found to enhance the degradaffon of the product by the ubiquitin-dependent protease pc~thway (8). "i'he potency to form a comp/ex wi'~h p53 diverges from an HPV-type to another;, HPV 16 E6 is the most potent binder, while HPV 18 E6 is 2 to 3 ~mas weaker (6). BATCo document for Mayo Clinic 28 March 2002
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Supposing tha~ HPV infecticns do play an etiological role in the p~thogenesis of anogen;-tal carcinomas, then one can easily e~b~te that cenrical ce~cinomas aesocta~d with HPV should contain only the wlid4ype p53 gene (which would be inactivated by its binding to E6 protein), whereas cervical carcinomas without HPV would possess the mutant p53 genes. It has been shown tt~t carcinoma cell lines which are HPV-negatJve have a mutation in e~er p53 or Rb gene. HPV-positive carcinomas 0¢~ the oth~ hand, have been shown to express the wild-type p,53 and pRb (8,12). Thes? data support the hypothesis that Inactivation of the normal function of p&3 or pRb is a c~ step in anogenitzd sq~Jamous cell carcinogenesis. 3.2..aJMS OF THE STUDY The object of th=Ls line of study is to e~aluala the k'Noiveme~ of p53 ~ in the development of gen~ in HPV.negaiJve and :HPV-pos~ve careers. The second aim ls to eluddate the role of pRb inacl~on in genial The hypothesislthat Insctiva~ion of the p53 and pRb tumor supressor gane products is a key genetic event in the Induction of HF~/-associsted maligr~nt transform~on will be tested in the follow-up pstlents with progressed and regressed genitali HPV lesions (Project L5.). The =age of p53 and Rb gene will be assessed aiso in the vaginal and vulvar cell lines grown on the rait culture system. In this model, experimental mutations of the hot spot areas of the p5,3 gene are gpnerated and the morfdogicai alterations are studied on raft cultures. Additional cell lines from the follow-up patJentslwill be established. The first part of ~he study comprises a series of 20 fresh surgical samples of genital carcinomas and precancer lesions, including 8 v~J,: Ivar carcinomas, 1 V1N (vulvar intraepithelisl neoplasia), 5 cervical carcinomas and 6 CIN (cervical IntmepithellaJ neoplasta) olXained from the Department of Gynecology & Obstebtcs, Kuoplo University Hospital. The expres~.:ion of p53 and the presence of HPV DNA was recently analysed in these specimens (Tewahauta et al., sub. mltted). The muttons of both p53 and Rb genes are assessed. The second ma~riai consists of 50 follow-up pstlants (Project 1.5). Half of the patients have had an HPV lesion aasocisted with CIN I;or (3IN II, which showed spontaneous regression during the foltow-up. The other half of the p~ents had CIN 1 or ~31N li which progressed to CIN Iii during the follow-up. The in vitro stu~ utilizes the vulvar e~d vaginal cell lines described in (111.1.3), Experimental mutations are generated in those ceil lines which have no aitera1~ons In the p53 and Rb genes. The genomic DNA of the frozen tissues is isolated by the method of Miller et al. (13). Samples are lysed in 1 ml of 10 mM Tds (.oH 8.3), 400 mM NaCI, 1% SDS, 2 mM EDTA and 0.3 mg/ml proteinase K overnight at 37(3. Proteins are precipitated by ~ck:ling 300 pl of saturated NaCI. After sent(rfugallon, supemaIant is removed and DNA precipitated with ethanol. 3.3.3.Detec=Jon of gene~ mutations in p53 a~d Rb genes Exons 5, 6, 7-8 and 9 of the p53, which are most often alfected by mutations, are emptied separatety with polymerase chain reaction (PCR) method using a pair of pdmers of exon sequences and subjected to SSCP BATCo document for Mayo Clinic 28 March 2002
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analysis. The muta~an analysis for Rb is basically similar, only primem used for PCR ampliflca~on are different including the anaiysi~ of introns, which have been reported to have point mutations in malignant cells as well. For screening .the DNA samples for alterations in the p53 and Rb genes, single-strand conformation polymorphism analys!s of polymerase chain mactlon products (PCR-SSCP) is performed according to the method of Orita et al. (14), with a slight modification. PCRs are performed in 20/~1 reaction volumes containing 200-300 ng of genomic DNA, 8 pmol of each pdmer, 200 pM of each deo~'ynucleotide triphosphate, 0.5 pl of [¢-~2P]dCTP (specific activity, 3000 Cllmmol, 10 mCi/rnl; Amemham, Bucklngh~mshlre, England), 50 mM KCI, 10 mM Tds-HCI (PH 8.8), 1.5 mM MgClz, 0.1% Triton X-100 and 0.75 units of Dyn.aZymeTM DNA polymerese (FinnzTmes, Espoo, Finland). The reactlon mixture is overlaid with 30 pl paraffin oil to pre~vent evapora~on. A~er an initial template denaturation at 95~ for 4.5 mln, ~0 cycles consisting of ~0s at 95~3 (denaturation), 50s at 55=C (annealing) and 1 rain at 72~C (primer extention) are completed. The last extention step is folloived by an additional 5 rain at 72=C to confirm that all products are completed. One/~1 of the ".PcR product is mixed w~ 29 pi of loading buffer (96~ formamlde, 20 mM EDTA, 0.05% bromophenol blue an~ 0.05% xylene cyanol). The mixture is heated at 1000C for 5 rain fo~icwed by cooling on ice. Two to four ~ul of the ~eparation is imr~edlately applied to a neutral 6% p~lyac~lamide gel containing 10% glycerol. Eisctrophoreeis ls per~rmed at 20 W (6-16h) at morn temperature. The gel is tided o~ ~er paper and exposed to Kodak XAR film at ~ for several hours to 5 d~s, with an Intensifying screen. We h~ve recently adopted a PhestGelei system (Pharmacfa Inc. 8woden) to SSCP. We am nc~v comparing the sensitivity of this method to the radioa~lve ass~ described above. DNA sequencing,', is performed to confirm the results obtained by the PCR-SSCP analysL~ The amplified axons of the p53 and Rb wit! be cloned in pUC19 vectors.and sequenced. PCRs ere done ~ 100 pl reactions containing 500 ng of the genomic DNA, 200/~M of each deoxynucleotlde triphosphate, ~ pmol bf each primer and 1.75 units of DynaZymeTM DNA polyrnerase In the same buffer conditions as dasc~bed above. "l~e PCR protocol is the same as described previously. The PCR products (407 bp containing axons 5-6 and 820 b~p containing exo~.s 7-9) are first purified in 1.5% low.malting-temperature agamse gel. The purified/ragments ar~ cleaved with EcoRI and cloned Into the Smal and E.___~I ~as of pUC19 vectors. The recombinant plesmids me coicr-selected by =~¢omplementation of the 8-galactosidase gone. Structure of the potential recombinant clones ~e checked by @ analysis of minlpreparatlons of the plasmid DNA. DNA of the recombinant plasmid~ Is isolated using Magic Mlnipreps DNA Purification System (Promega, Madison, USA). Sequencing Is perforn~ed by the dideoxy termlnstion matl~od using the AutoReedTM sequencing kit (Pharmecla, Uppsala, Sweden) ar~ the automated A, LF. DNA Sequencer (Pharmacia LKB, Uppsa]a, Sweden). Exons displaying conformationat polym6rphisms are also subjected to direct sequencing using the dideoxy method. 3.4.CURRENT PROGRESS OF THE WORK The status of the p53 gone has been analysed in the first 20 specimens characterized in (111.3.3.1.). No mutations in the p53 were detected in any of the 20 specimens, including three HPV-negative cases. Thus, the occurrence of p53 mutations was not correlated with the HPV status. In addition, no correl~on was found between the p53 overexpression and the state of p53 gene. The p53 expression was detected in 16 of the 20 specimens. An intensely positive staining was obse~ed in three samples, including two HPV-negative carcinomas and one HPV 33- BATCo document for Mayo Clinic 28 March 2002
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posit~e carcinoma. In addition, 13 samples were moden~ly or weakly pS3-positive. Thus, there was a positive immunohistochemical staining in the absence of any p53 mutaiien. 3.5.StGNRCANCE OF." THE STUDY With the present study design, it is possible to evaluate the significance of p53 and pRb tumor supressor genes in the HPV-indu~l carcinogenesis. Our pretlmina~ data suggest thai p53 expression can be found although no mutations am present. Furthermore, HPV type does not have as clear.cut association to p53 protein expression as proposed earlier. Obr preliminary findings have been corrrln~ed by other groups recently. The follow-up patients with known HPV stat~s and clinical behaviour of the lesions offer a unique matedal to study the role of antioncogenes In ceNIcal neoplasis. The studies can be supplemsted by tn vitro studies where cell cultures established from the same patients are grown on the raft culture system. In this model, the effects of experimental mutations on epithelial :morphology are easily assessed. Similar approach can be done with the cell lines described in (111.1.). So far no similar studies have been published. 3.6~UTEP, ATURE CriED 1.Levee, M. The p,53 protein and ~ intera~on~ with 1he onoogene product~ of the ~rn=Jl DNA tumor viruses. Virology 1990; 177:419-426. 2.Levine0 AJ., Momand~ Ji Tumor aupprassor genes: the p5.3 end retinoblastorrm senslt~ genes and gane products. Biochim. Blephy~. Act~ 1990; 1032~, 119-136. 3.Levtne, AJ., Momand, Ji' Finley, CA, The p.53 tumour supp~or gene. Nature 1991; 351: 453-456. 4.Vou=den, KH., Wrede, ~., Crook, T. HPV oncegrotein fun~an: releasing the bra~e= on cell growth controL Paplllom~virus Rap.IS91; 2(1]: 1-3. 5.How~y, PM. Role of thai human psplllomaviru~e~ in human cancer. Cancer Re~, 1991; 51: 5019-5022. 6.F'mlay, CA., Hinds, PW.~ Levlna, AJ. The p53 proto-ormogane can act as ,, ~uppressor of kansformaffon. Cell 1989; 57(7): 10~3.1093. 7.Holletein, M., Sidranskyi O., Vogelsteln, B., l-I~n~, CO. p35 muttons in human can~rs. Science 1991; 253:49-53. 8.scheffner, M.0 Werne~, .BA, Huibregbe, JM., Levlnep AJ., Howely, PM. The E6 oncoprotein enceded by human papillomavims type~-16 and 18 promote~ the deganel~ffon of p53. Cell 1990; 63: 1129-1136. 10.Baker, SJ., Feamn, EFt, Ntgro, JM., Hamilton, SR., Prelslnger, AC., Je~ulo, JM., wn Tuinen, P., Ledbetter, DI-L, Barker, DF., NaJ(emura, Y.° White° FL, V.~elsteln° B. Chromosome 17 de, efforts and 1~3 gene mutatior~ in colorectal carcinoma. Science 1989;, 244, 217-221. 11 .Munroe, DG., P, ovlnski, IB., Bemsteln, A., Benchimol, ,S. Lo~ of a highly conserved domain on p53 as • result of gene deletion dudng Fdend-vtru~-induceid ewthroleukemia. Oncogane 1988; 2, 621-6~4. 12.Ntgro, JM., Baker, SJ.,~;Prelslnger, AC., Je~sup, JM., ~er, R, CleeJy, K.. BIgner, SH., Davidson, H., Baylln, S., Devilee, P., Glever. T., Collins, FS.i Weston, A., Modali, R., I-lan~, CC., Vogefs~ein, B. Mutations in the p53 gene ocsur in diveme human tumo¢ type¢ l~ture 1969;,i342, 705-708 13.Miller, ~A., Byke~, D.Di & Polasky, H.F. (19~). Nucleic Aclds Flas.. 16, 1215. 14.O~ta, M, SuzuU, Y., S~ly~, T. & Haya.~hl, ~_ (lSe~). Ger~rn~, S, a74-s'~. 4.ONCOGENES AND GROWll-I FACTORS IN HIW'-ASSOCIATED GENITAL LESIONS AND CANCER 4.1.BACXGROUND Viral oncogenes, E6 and E7 proteins am present in HPV-tnduced neoplasms, frequently integrated Into the host cell genome. E6 and E7 are capable of im .mortalizing prima~ human squamous cells and E7 is sufficient to transform established NIH3T3 rodent fibroblast cell line. Other genes of HPVs have effect on transformation: disruption of E1 and E20RFs leads to increased expression of E6/E70RFs enhancing the transformation rate of BATCo document for Mayo Clinic 28 March 2002
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61 pdmary human kerat~es (MOnger et aL 199"2). Atthough HPV-DNA is found in approximstaly 85% of cervical cancers, additional faCtom am needed for malignant transform~orL V~ra] proteins can interact with cellular proteins. E7 protein of the high-risk HPV-types inactivates pRb by forming a complex v,~th it. pRb is a negative growth regulator at G1/S boundary and its fun~on is related to the d~rse of phospho~jletion. Similarly, E6 protein binds to p53 turnout suppre~r gene leading to its inactivation and degradation. Expression of ~p53 in HPV-assoclated cervical carcinomas and cancer-derived cell lines has been reported to be low or totally absent, p53 is also inactivated by mutatidns, resulting in inactive protein accumulated in the cell. This mutated p53 is ~ndlly detected by immunohistochemical staining in contrast to the wgd-type p53 protein expression which is ~upposed to be non-detectable by this means (MOnger et al. 1992). Recently, however, controversial results h~e been presented while demonstrating p&3 expression in HPV-positive carcinomas, although muttons are rare In ~esions containing HPV DNA (BOrmsan et al. 1991). Cellular proto-o~cogenes are also involved in HPV-induced transformation. In transfection essays, HPV 16, 18, 31 and 33 were sh~vn to co-operate with EJ-ras~ to transform BRK cells (pdmary baby mt kidney cells) and with v-fce tO transform BMK cells (baby mouse kidney cells), but not BRK cells (Metlashewskl 1989). c-mvc over- expression, amplifJcatl0n and rearrangements are found in several late stage cervical carcinomas and HPV-16 is oRan Integrated near the rnyc locus in many cewical carcinoma cell lines (Arands et al. 1~0). Canfcat cancem., and cell lines are found to express high numbers of EGFR (epidermal growth factor receptor), especially the HPV-positive ones which seem to express more EGFR than HPV-negative cells. It is thought tha~ growth stimulation by E6/E7 proteins Is mediated by enhanced EGFR expression and it seems that E50RF is also involved. Binding of EGF or TGF-= (transforming growth factor,,) to EGFR stimulates the proliferation of normal cells. When EGF Is remove~., from the squamous cells cultured on raft systems, a normal stratification and differentiation follows with rnalura virbses synthezised in differentiated epithel~ ceils (Vambutes et aL 1993). TGFB, on the other hand, is a potent negative growth regulator of many epithelial cells, the effect being mediated by transcdptlonal repression of c-mvc. S.~me HPV-positive cancer cell lines are refractory to this Inhibition, however, and HPV 16 and HPV 18 E7 was show.h to revert the effects of TGFB on c-mv~ transcription (Braun et al. 1990). Although certalr~ HPV types seem to be l~m~t~ partiolpating in this process, It seems likely that this oncegenic virus alone m~ht be necessary but (alone) not sufficient to induce a full-blown malignant transformatloR Undoubtedly, a variety;of oncogenes and antl-oncogenas as well as different growth factors and their receptors are Involved in the multist~p process of squamous cell cardnogenasis in the genital tract. A substantial amount of Information still remains to be elucidated to fully understand the role of each of these factors In this complex Interplay. 4.2.AIMS OF "rilE STUDY As a part of our iPapillomavirus programme, a line of research to assess the role of oncogenes and growth factors (and their receptors) In Hi=V-associated genital carcinogenesis has been on progress since 1990 (Aria Tervahauta, M.Sc., Research Assistant of the Academy of Finland). The first phase of the project (aiming to her Ph.D. Thesis) is focused on the following issues: 1. To assess the role of immunoh|stochemist~y, In situ bybridiz~on and PCR techniques in detecting c- mvc proto-oncogene expression in genital cancer as we~l as to establish optimal fix~ion methods for mRNA in situ hybridization. 2. To study the expression of p53 gen~ product In HPV-positlve and HPV-negative cervical cancers and precancer lesions e.g. in reiat~on to the cell cycle as marked by Ki-b'7 proliferat~n antigen expression, using an Immunohistochemicat double staining. O~ BATCo document for Mayo Clinic 28 March 2002
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3. To evaluate ithe eventual prognostic value of EGFR, c-erb.__B-2 proto-oncogene and estrogen receptor expression by lmmunphistocher~stry in HPV-associated ge~ltai lesions. 4. To analyse t.he biologic sign~cance of transforming growth factor B (TGFI~ expression by immunohis- tochemist~ in genital ~HPV lesions. Paraffin-embedded sections are deparaffinized in xyisne and rehydrmed with graded ethanol Frozen sections are fixed in acetone for 10 rain at 4°C. Prlma~y antibody (p53, EGF, EGF-R, c-erbB-2, TGF or K~-67) is applied to the sections at a appr~oriate dilution and incubated in a moist chamber overnight at 40. After washing in 0.05 mol/L phosphate-buffered s~llne, biotinylated goat anti-mouse IgG is applied and the sections are incubated for 30 rain at recto temperature. A~ter a thorough washing in phosphate-buffered saline, peroxldaseconjugated strept~Nidin (STRAVIGEN BSA-PO~ W.~t, BioGenex Lab, Dublin, CA) is applied and the sections are again incubated for 30 min. LooaJizalion of zmtlge~ is visualized by incubating the sect~ns for 4 mln In 0.05% mel/L Tris-HCI Coil 7.6) containing both 0.02% (w/v) 3,3'~iaminobenzidine tetrahydrochlodde and 0.03% (v/v) hydrogen peroxide. 4.3.2.1n situ ~ ~ bic~nylmB:l probes Formalin-fixed, iparaffin-embedded sections are deparatfinized in xylane, rehydrated and o~gested with protelnase K solution. ~/hole genomic DNA probes for HPV 6, 11, 16, 18, 31 and 33 are labeled by nick-transla~on (BRLNIck Translation .Kit, BRL Life Technologies, Inc., Gaithemburg, MD, USA) using b~o-11-dUTP (Sigma Cl~ernlcal Co., St. Louis, Me, U ,.S~). Hybridization is castled out as explained above. Specimens are incubated with strepta~idlm a~a]lna phosphatese ~omplex and developed with (NBT) and (BCIP). Frozen sections; are fixed in 4% paraformaidehyde, Paraffin-embedded sectfons are first deparaffinized and a/r~ded. All buffers u~cl are made In DEPC~eated water. Slides are treated with protelnase K and acetylated. Frozen CaSId and Hel~ cell slides can be used directly without any pretreatmen¢. The hybddizatk~n Is performed in 6xSSPE, 50% delon~ze~d formamide, 10% dextrane sulphate, 1 mg/ml yeast t-RNA and approximately 500.000 cpm of the probe on the sli~e" As a negative control, one addltlonai section of each lesion is treated similarly by omitting the probe in the hybridization mixture. For RNA-DNA hybridization, the probe is applied on the slide, covered with ccverslip and denatur~ted at 100=C for 5 mtn. For RNA-RNA Wbridlzatlon, the probe is denaturated at 100~ for 5 rain before application !on the slide. Slides are sealed with rubber cement and hybridized overnight at 45"C in humidi- fied chamber. Slides aie washed, dehydrated, air-dried and immersed In Kodak NTB-2 emulsion diluted 1:1 with 0.6 mM ammonium acetate. After autoradlography (6-7 days) slides are developed with Kodak D-19 developer for 5 min,flxed with Kodak A-30(X) fixer and pisnisher, stained with water-soluble carboifuchsin, dehydreded and mounted. 4.3.4.Po~ chain reaction (PCR) 4.3.4.1 .DNA-PCR ~ DNA isol~on for PCR is made from both frozen and formalin-fixed, paraffin embedded ~esue. TechnP.~-.al details O of me PCR are simliar to those described above. For paraffin.embedded samples and frozen tissue DNA, 40 and O 35 cycles are used, respectively. After PCR, 10 ~1 of the reaction mL'~lure is electrophoresed on a 3% NuSleve age- ~ rose gel (FMC BioProducts. Rockland, ME, USA) and DNA visualized in the gel by ethidium bromide stoning. (=,.q BATCo document for Mayo Clinic 28 March 2002
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1.3.4.2.RNA-PCR For nucleic acid~ isolation, five 5-.¢¢n sections are cut from paraffin blocks. Paz-~ffin sections am incubated twice in xylene at 55=C for 5 mln, centrifuged and washed twice with absolute ethanol Samples are than incubated in 20 mM Tds, 20 mM EDTA pH 8, 2% SDS and 500/~g/ml protetnase K at 6(T'C for 624 hours. Nucleic acids are purified twice with phenol and precipitated twice at 4"C for 5 min with 0,6 x volume of isopropanol, 0,3 M NaAc pH 5,3 and 20 ~j of glycogen as a carrier. Pellet is diluted in c~istilled water. Reverse transcription and PCR are made with RNA- PCR kit (Perkin Elmer Cetus, USA) according to manufacturer's procedures. PCR program is: 95=0 2 rain for I cycle and 35 cycles of 95°(~ for 30 s, 55'~3 for 2 rain and 72~ for 30 s. 1.4.CURRENT PROCESS OF THE STUDY A preliminanj .s~.des of 10 invasive carcinomas (including 2 vulvar ones) was studied for overexpression of c-myc by in situ hyb~dIz~on with 3sS-tabelisd RNA probes and immunohistochamistn]. DNA amplification and transcription of c-myclwere studied by PCR (with ~-giobin standard). In this work, the effect of formalin fixation on the detectability of c.myc expression was analysed as compared with the frozen sections. Half of the tumors showed c-myc mRNA overexp::ression by ISH pefl'on'ned on frozen sections, an¢~ two of the samples also demonstrated overaxpression of c-rnyc protein by ISH. The data suggest that ISH with RNA probes is a useful method for detecting the ~anscdptlon of acilvated oncogenes in malignant tissues, especially when applied on frozen sections. To assess the role of p53 in HPV-associated genital carcinogenesis, the expression of p5,3 protein was studied immunohistochemically in 22 genital carcinomas and precancer lesions (8 vuiva~ ca, 1 VIN, 5 cewicaJ ca, 8 CIN) using monoclonal anti.body PAb 1801. Presence of HPV DNA was demonstrated by PCR using HPV consensus primers, and amplified;HPV.DNA was dtgesled v,~th the restriction enzymes giving distinct i:~tems for vadous HPV- types in gel etectroph ..0resis. Altogether, 7/% of the specimens showed p53 expression (67% of precancar lesions and 83% of cercinom~). HPV DNA was detected in 86% of specimens. All HPV-negetive samples showed p53 expression. Of the 19 HPV-positive lesions, 5 were p53-negatJve, three of these being HPV16 positive CIN-lesiofls. HPV6 and 11-posith, e I~ions showed a weak p53 expression more frequently than HPV-negative cases and HPV33 lesions. The results ind.icete that p53 expression is detectable, but it is less frequent and less intense in HPV DNA- positive genital precan~er lesions and carcinomas (particularly those with HPV16 DNA) as compared with HPV DNA- negative lesions. p53 protein expr~"~sion was studied with two antibodies (CM1 polycionai- and PAb 1801 rno~ocionat Abe), and ~ proliferation antigen (monoclonai Ab) using an immunohistochemioai (IHC) double-staining In 77 HPV-posittve cervical lesions and in 15 HPV-negative cases, p53 protein expression was detected in 39.1% of the specimens. Of the p53-pesitive cases, 80.6% were HPV-positive samples, Including 43.5% of HPV16- and 30% of HPV18-positive biopsies. Ki-67 proliferation marker was found in 91/92 specimens, most intensely in those infected by HPV 16. p53 was more abundant in progressed or pemistent lesions, but no differences were found between HPV-positive and HPV-negetive samples. The positive IHC double-staining for both p53 and KI-67 proliferation antigen in the same basal (and .oarabassf~ ceils indicates that these two normal cell-cycle proteins are being expressed while the cells a~e entedrg .'rom the G] to the S phase ~f the cell cycle. Since the latter property ts only a~dbuted to the wild- type p53 (~ut not to mutated p53), the p53 protein detected in HPV lesions by IHC IS I~kely to be the wild-type p.53 0 0 O~ BATCo document for Mayo Clinic 28 March 2002
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rather than mutaIe~ p53. Accordingly. the concept on HPV inactivat~ng the wild-type p53 prote~n should be reexamined, and other mechanisms for HPV-medi~ed carcinogenesis should be considered as well lesions To assess the value of EGF-R, c-erbB-2 and estrogen receptor expression to the clinical course of HPV infection 81 cervical biopsies were studied Immunohlstochemlcelly. Of the specimens 78% contained HPV DNA and CIN grade varied from NCIN to carcinoma In situ. EGF-R expression was found in all specimens, usually in basal and parabasal cells. In addition, ahomogenous staining was observed in dysplastic lesions which indicate a raised level of EGF-R in these cells. No clear-cut corrsia~on was found to dysplasia or HPV type, except 77% of the HPV 18 positive samples Showed homogenous staining In the lesions, while the percentage with other HPV-types vaded from 7% to 40%. c-erbB-2 expression was found in 31% of the specimens while epithelial ER expression was seen In or~ In 13% of the samples. The expression had no correlation to the HPV status or dysplasia. 4.5.SIGNIFICANCE O~ THE STUDY As repeatedly emphasized, HPV life-cycle within the Infected keratlnocyte is intimately linked with and regulated by a large number of~ different up- and dowrH'egul~ng factom, belonging to the complex machinenj of the signal transducgon pathway~ within the normal cell Ample evidence is available indicating that HPV is capable of dlsturbi~lg the normal intraceliular microenvlronment, leading to the loss of normal cell growth control, eventually resulting In immortalized and transformed cell phenotype, wherefrom a clone of frankly ma~gnant cells occasionally evolves with a clinical malignancy ~ an end-polnt. It is essential to elucidate the mechanisms by which these highly divergent factors interact with HPV in such a process. AJbeIt at its Infancy as yet, evenj piece of work on these lines will help exp~ning, step by ~ep, more of these complex phenomena. The prospectively collected samples with well cheraofedzed disease! hlsto~y is par~culady vakrable for this kind of study, while providing the immediate biological relevance [or lrrelevaq.ce) for any new l~nding and phenomena eventually disclosed by these assays. Matlashewski G. The ce! biology of human papillomevlrue "¢andormed cells. AntManeer Rea 9: 1447-1556, 1989. MOnger k, Scheffner M, ~.Huibregtse JM, Howley PM. Intera~t~n= of HPV E6 and E7 on¢oprotein~ wifft tumour suppressor gena p~. Cancer Smv~e 12: 197-217, 1992. I~n'esan AC, Hovlg E,'Smith-S~ansen B, Malkln D, Lystad S, Andersen T, Nesland J, Isselbacher K, Friend S. Constant denaturant gel electropl~oresis (CDGE) as a rapid bcreening technique for p53 mutations. Pro~ N~ff Acid Sol USA 88: 8405. 8409, 1991. Arend= MJ, Wyl]ie AH. B)rd CC. Papillomavirusas and human cancer. Human Pathology 21: 666-6~8, Vambutas A, DI Lorenzo:TP, Steinberg BM. La~'nge,q papgloma cell~ h~ve high leveb of epiderma] growth fa~tor receptor and reepond to epidermaJ gr~o~ fagtor by a d~ In epithetlel dlfferanffatJon. Cancer Rea 53: 910-91,% 1993. Braun I., Dt3mt M, Mikumo R, Gmppuso P. Differant~l response of nontumodganl¢ and tumorigenic human papillomavims type 16-positive epP~hellal cells to trandcrming growth factor I~1. Cancer Ras 50: 7324-7332, 1990. 5.ANTISENSE OUGONUCLEOTIDES; AN EXPERIMENTAL APROACH FOR HPV THERAPY 5.1.BACKGROUND With the advent of more and more genes as being responsible for human disease, an urgent need has aroused to search for therapeutic tools capable of regulating such disease genes. Control of expression of selected genes may be achieved using ant~sense technology. Antisense molecules (DNA, RNA or short sequenses of i BATCo document for Mayo Clinic 28 March 2002
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oligodsoxynucleotides) may be formulated as compismenta~y to a spesific gene or mRNA, and thus are pote~at blockers of transiatior~ while fon'ning a hybrid duplex by hydrogen-bonded base pa~dng with the target nucleo~de sequence (1-4). Hence, oligonucleotides may represent a future source of biotechnologlcally dedved compounds of therapeutic importa~." ce in several diseases, including cancer ~ viral infections. The viruses function through the transfer of genetic information from DNA to mRNA, and further 1o proteins. These genetic processes can be intervened using ol~go~ucleotldes. The most widei~ used approach to interrupt the flow of genetic Information is to use oligonuclsotides as inh~l~itom of translation..., with the complementary or "antisense" base sequence targeted to a specific "sense" sequence in the mRNA. Thus, expression of spec~c proteins can be regulated or inhibited (2). The preferred target ~ this antfsense Inhib.:~on is the 5'-initiation codorl. In genera], ~1 oligonucleotlde of 15-20 bases is used for the antlsense inhib~on. The tn vitro applicetlorts being successful so far include inh~"oitlon of expression of several oncoganes (c-myc, N~myc, myb) and HW (1-4). Anttsense ollgonucleotldes have been suggested to be applicable In the treatment of various viral infec~ons and vtrus-assoc~ed =cancer, including those due to HPV ~fact]ons. The protein-coding sequenees of HPVs are classified as early (E) ~nd late (L) genes, E6-E7 genes coolng for the major transforming proteins (5-7). E6-L=7 genes of the high-risk HPV t~pes immortalize human foreskin or cervical keratinocytas (8). In organotyplc (re/t) cu~res, these immodalized ce~ resemble Ir~aapitheiisl neoplasias In their growth characteristics (9). Aitough HPV E6-ET- immortalized human ~lis am initially nontumodgenic in nude mice, long-time in vitro culti~-~tion may lead to malignant clones (10). ~ccordingly, the development of apesi~c means to Inhibit these transforming functions of the HPV E6 and E7 genes might provide a solid basis for a new therapeutic strategy against e.g. cervical cancer. One of the majo~ problems in prac~cal appllcat]ons of the antisense oltgonucleottdes is the fact that unmodified eilgonucleotldas have .~ooor biologIca~ sta~T~ty due to degradation by nucisases. To circumvent these obstacles, a vadety of modified an~i more stable oligocucleotide analogues have been developed including phosphorotioate ollgonucisottdes ($-ollg." os). Even these may be degr .aded. mistlvely quickly, however (¢,11). Another problem Is the poor cellular uptake df oligonuisotldes due to their size and negative charge (4,11). In addition, the cell type, compo~en~ of the media, length of ollgonucleot~as as well as the presense of ~ked groups affect the cellular uptake of oligonucisot~es C12). For these masons, a wide variety of methods have been developed to Improve the cellular uptake of ollgpnucleotldes` These methods include the use of ceisium phosphatase, ratroviruses and ~ectroperat~ (13). However, all these modifications suffer some Inherent problems rela~ed to either cellular toxity, poor reproducibility, Inconvenience, or inefficiency of oligonur.Je~e delivery. One of the most promising approaches is the use of liposornes as a carder system (13,14). Especially the use of c~oni~ liposomes has been effective in transporting o~gonucle0tldes into the cells. Negatively cha~ed ollgonucleotides interact spontaneously with positively • ; ..~ c~tlonl¢ Ilposo~nes. Ca~lonlc Ilposome-medlated DNA delivery may alternatively occur via fusion with the plasma membrane o~" 1he cellular endocytosis palhway (13,14). 5~?.AIMS OF THE STUDY Antisense ofigonucleotides are finding increasing use as research tools to elucidate the function of specific genes. They also ho~d great promise as potential chemotherapeutic agents for specifically inh~iting the expression C) of undesirable genes such as oncogenes and viral genes. HPVs responsible for local (i.e., non-systemic) infections C~ in the squamous epithelium are an att/active t~rget for the therapeutic applications of oligonucleotides, especially O~ because no specific chemotherapy exists for HPV Infections. Furthermore, the role of E6 and E7 genes in malignant -~ transfom~icn can be furth~ assessed w~th this approach. CO BATCo document for Mayo Clinic 28 March 2002
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The purpose ~ this study ls to investigate the inhibition of E6 and E-7 gane expression of the oncogenic HPV tTpes 16 and 18 by!antisense oligonucleot~des. The first step is to find out the most effective oligonucieotide sequence and its size for maximal inhibition of E6 and E7 gene expression. In add~on, the basic mechanisms of the cellular uptake arid intrscailulsr degrada~on of the ollganucleotides are studied. For this purpose, CaSki ceils (with some 600 copies of HPV 16) and HeLa ceils (2050 copies of HPV 18) are treated with modified and unmodified oligonucieotldes of different size. Based on these results, the experiments will be directed to the cell lkles described in (111~1.). The effect of inhibition of E6 and E7 genes on epithelial morphology will be studied on the ra~ cuitum system. The final aim will be to design an effective and clinically feasible local therapy against HPV- Induced epithelial lesions, especially to prevent the progression of the pcecancer lesions in the genital tract. 5.3.MA~ AND METHODS The first experiments are being carried out w~ the CaSki and HeLa cells, two human cervical cancer cell lines containing HPV 16 an~l 18 DNA, respectively. Antisense ollgonuclsotides ere targeted to the initiation region of the E6 and E7 genes of ~PV 16 or 18. We are using unmodified and moditied phosphorotloate 12- and 23-mar, which are synthasised on ~ automatic DNA synthesizer. The cellular uptake of ollgonuclsotidas with or without cationic lipesome~ are studied. For this purpose, different lip~ome preparations are used. Proliferation of the CaSki cells ls monitored by the ~ assay, which measures the activity of mitochonddal dehydroganases of viable cells. Morphology of the c~tls is analysed after oilgonucleotlde and llposome treatment. The cellular uptake of the oligonucleotldas into the ceils ls assessed using fiuorescein.lsothiocyanate (FITC).iebailed ollgonucleotidas and confocai laser scanning microscope {CLSM) as well as 32p-labelled oIIgonucieotIdes by liquid scintillation in a ~- emission counter. Th(i stability of unmodified and rnod~ed oligonucieotides is investigated by using PhastGel Syster~ with polyecryiamide gels. The effects of antlsanse oligonucleotides on HPV E6 and E7 mRNA are measured by Northern blot an .a.ly~."."is. The expression levels am also assessed by in ~ hybrldlsation and RNA-PCR. The levels of E6 and E7 proteins:= are analyzed by Western blotting or lmmunoh~ochemlsW. s.4.~ PROGRESS. OF THE WORK The effect of mc~dified and unmodified antisense ol~gonucleottdes on CaSki cell prol'dera~on has now been studied. Unmodified o~onuclsotldes did not Inhibit prollferat~n of the CaSki cells, neither did the phosphorothIoate modified 23-mar. Instea~d, phosphorothioate 12-mer retarded cell proliferation and this effect was potentiated with the liposomes (DDAB:DOP~. Thls etfect was regarded specific, because modified phosphorothioate random 12-mar did not ~ any effect on'these celts. Also the cel~u~ uptake of oligonucles~les was significantly enhanced with the canonic liposomes as lassessed on CLSM. The cellular pen~,ation of oligonucieotldes without liposomes was significantJy lower thar~ with liposomes. In the absence of liposomes, the oligos appeared to fuse Into the cell membrane, whereas th.e oligos with liposomes were also found within the cytoplasm. Cationic liposomes were toxic to the cells, however~ The toxicity was dose-dependsnt. Currently, we axe studing the effect of antlsense oljgonuleotide on E6 or E7 gene expression in these cells. The most optimal antisense oligonucleotides are selected on the basis of our previous work. 5.5.SIGNIFICANCE OF THE STUDY The aigniticance of this study lies in the fact that it is one of the first attempts made towards the new theapeutic approach against HPV infection. The fJmt step will be to show that antisense oligonucieotidas work in vitro. Moreover, in vitro studies can also be used to understand the mechanisms by which E6 and E7 genes are involved C BATCo document for Mayo Clinic 28 March 2002
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in malignant tra~sforma~ "on. The possibil~ that oligonuc~eotides can be used topically for HPV infections on the genital mucosa overcomes saveral problems associaIed with oligonucleotide therapeutics for other purposes. 1.Ste~n, C. A., and J. S, Cohen. 1988. ONgonucleotide= e.s inhlb~ of g~a ~pr~Mn: A r~, ~r R~ ~: ~. 2,Zo~ G. 1~. Oiigonu~de ~u~ ~ ~ chemurgic agen~ A R~. P~ ~s. 5: ~. 3.He~ene C.. ~d J.~. To,me. 1~. S~¢ r~n ~ gene ~r~ by ~se~e, ~e~a ~d ~nse nuc~c ectds. Biochim. Blop~s. Ad¢ 1~: ~1~. 4.C~ke S. T. 1~ ~er~¢ ~plic~s ~ o~nuole~d~: A ~i~. Biat~hno~. 1~ ~. 5.~ ~ J. 1~. P~lom~i~ and c~r. In: ~d~en ~ J., Gi~mann L ~d K~ LG. (Erie.). Papillom~i~ ~d hum~ d~e=~. He[del~: Spfinger-Vedsg 1~7. ~. 6.~en ~ J. I~ Mol~cul~ bio~ of hum~ ~itlom~i~ ~s & ~e. 1: 1~16. 7.Chug F. 1~. ~le ~ PapBlom~ms.: A ~. J. Clin. P~I, ~: ~6. 8.Muir, ~, W. ~ Phel~, V. Bubb. P. M. How~, ~d R ~hl~. 1~. ~e ~ ~d ~ ~n~ ~ hum~ papillom~i~ ~pe 16 ~g~er me n~ ~d s~deM for ~o~n ~ ~m~ hum~ keynote. J. ~;~. ~: ~17~1. 9.W~, ~ D.. S. Wa~goner, W. Bam~, M. H, ~let, ~ J. ~ D]Paolo. 1~. Hum~ ¢e~ ~ for~kln ep~ell~ 10.P~, G., M. L., D~ ~rg~ ~d V Defendl. 1~1. ~ol~on ~ in ~o ~o~on ~ ~dgen~is ~ H~ 16 ~d 11.~eln, C. ~, J, L Tonkln~n, ~d L Y~ub~. 1~ P~phor~]o~ oligode~nu~d~ - ~en~ inhib~om of gene ~r~on ? A r~. Phenol. ~. ~: ~. l~Jm~kl, J. W., ~d ~. ~ Co~n, 1~1. Cellul~ up~e ~ =~nse oligonucl~Sd~. A ~. A~, ~g Dell, Rev. 6: ~2~. 13,Feigner, P. L,T. ~ G~, M. Holm, ~ Rom~ H. W. Ch~ M. We~ J. P. N~rop, G. M. ~go~, ~d M. D~ielsen. 1~. U~f~on: A hlgh~ ~cle~ llpt~l~ D~~ pr~ure. P~. N~. Acad. ~, ~ ~: 741~7417. 14,~leW, ~ R,, ~ ~m~, ~ ~ D~hilo. 1~ U~ del~e~ ~ a n~ apwo~¢h to ~s~ ~e ol~onue)~d~, ln: R P ~ck~n, ~d J, G. =~ (E~).~ne ~laffon: ~oI~ ~ ~ ~ ~d D~ ~v~ Prm, Md., N~ Yo~. 147-1~ 0 0 O~ BATCo document for Mayo Clinic 28 March 2002

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