Tobacco Documents Online

957 they are indistinguishable from multiple papil- lomas.• " In females, condylomata may regress spontaneously, persist for years, or undergo a ra- pid increase in size during pregnancy. Genital warts of long duration appear to have a low risk for undergoing malignant transformation.' Most show evidence of mild dysplasia but greater de- grees of dysplasia are extremely rare. Approxi- mately 5074 of squamous carcinomas of the vulva have been reported to develop in association with condylomata.' •' Perhaps the best clinical evi- dence of maGgnant potential of genital warts occurred in two rare cases in which carcinoma of the vulva in young teenage girls was preceded by condylomata (reviewed by zur Hausen'). In males, 15074 of squamous carcinomas of the penis have been reported to develop in association with preexisting condylomata.' " Many penile carci- nomas are of the verrucous type with features of giant condylomata acuminatum of Buschke and Lowenstein" Of interest is the observation that circumcision reduces the incidence of penile carci- nomas but not the appearance of penile condylo- mata.' Perineal and perianal condylomata may also undergo malignant transition to squamous cell carcinomas, particularly in homosexual maies."O Of particular importance is the recent observation that papillomas/condylomas of the distal male urethra may be flat or exophytic with some showing atypia.^• •' these lesions are most likely transmitted by sexual contact to the cervix where a productive infection gives rise to cervical dysplasia. Cervical dysplasia (cervical intraepithelial neo- plasia, CIN) has been associated with HPV-6,1 -11!' and -16 (personal communication, zur Hau- sen), and other unknown types, and is the precur- sor of squamous carcinoma." The development of cervical cancer is thought to proceed through a continuum of progressive cellular changes from mild (CIN 1) to moderate (CIN 11) (Figure 34-2) to severe dysplasia and/or carcinoma in situ (CIN III)"" This sequence of events has been docu- mented by a number of studies with transit times from mild dysplasia to carcinoma in situ ranging from 84 to 96 months." Approximately 40074 to 70074 of the dysplasias undergo spontaneous re- gression. Although the etiology of cervical cancer is still unknown, epidemiolojW-studies suggest that both precursor lesions and carcinoma are linked to venereally transmitted agents." For many years, herpesvirus type II was thought to be the most likely etiologic agent based on seroepi- demiologic evidence.1O01Ot However, since 1980, morphologic, immunologic, and molecular viro- logic studies support the view that HPV plays an important role in the development of cervical neo- piasia."" i42"O' HPV is one of the most com- monly sexually transmitted agents, having been identified by the presence of koilocytotic cells in cervical smears of 1076 to 2°70 of all women and S% to 10% of young women screened.'o1.uo-1u E'ig.ee 34-2 Moderate cervical dysplasia showing zone of papillomavirua-auodated hyperplasia occupying the lower half of the thickness of the epitheliutn. The upper half of the epithelium shows cells with vacuoiated cytoplasm and mild nuclear uypia. Papillomavicus an- tigens and DNA were present in this case (hematoxylin and eosin, x I0o). k G

958 CLINICAL SYNDROMES (UNCOMMON) Epidermodysplasia verruciformis (HPV-3, -S, -8, -9, -10, -12, -13, and -I4) or verrucosis gener- alisata"3 is a rare, autosomal recessive, disease characterized by varying degrees of decreased cell- mediated immunity and increased susceptibility to HPV infection manifested clinically by poly- morphogic skin lesions resembling either flat warts or macules indistinguishable from pityriasis rosea." f2 114-11' In approximately 25070 of Cauca- sian patients malignant transformation occurs within the pityriasis rosea-like lesions in sun- exposed areas such as the face and hands. Only lesions containing HPV-S appear to progress to premalignant lesions, histopathologically similar to cervical dysplasia before developing into carci- noma in situ (Bowen's disease) and, eventually invasive squamous carcinoma. HPV-5 can be identified in primary or metastatic malignant le- sions as a replicating, nonintegrated plasmid!' Immunosuppressed patients, particularly those undergoing renal transplantation, also develop pityriasis rosea-like lesions associated with HPV-S, which may undergo malignant transfor- mation."• Although warts that are present at the beginning of therapy become aggressive,3 newly acquired warts are usually seen only after the first year of immunosuppression." Squamous cell carcinomas usually do not develop in patients with immunosuppression of short duration, sug- gesting that HP V-S is acquired from some reser- voir which is as yet unknown. Flat warts asso- ciated with HPV-3 have also been reported in renal transplant patients."' IMMUyOLOGIC RESPONSES Available evidence suggests that spontaneous regression or persistence of warts is related to the immunologic responsiveness of the individual (re- viewed by von Krogh"). Studies of HPV infec- tion suggest that stimulation of cellular immunity is associated with regression of lesions and such immunity is HPV type specific. Humoral antibody responses to HPV are almost always type specific and can apparently be used to determine evidence of previous infection." The antibody response can also be used as a prognostic indicator of spon- taneous regression and/or successful therapy. Many of the previous studies of immune re- sponses to HPV infection are, however, difficult to assess in view of our current recognition of the plurality of immunologically distinct HPV. Cell-Mediated Immunity Patients with clinical evidence of altered cell- mediated immunity have an increased incidence of warts that are usually flat." This has been seen in immunosuppressed patients,"3-7" patients with cellular immunodeficiency,'• and patients with weak cutaneous hypersensitivity responses.i° aIl Specific immunologic defects may be involved, however, since not all patients with persistent warts have evidence of nonspecific abnormalities in cell-mediated immunity. There is pathologic evidence of cell-mediated regression of warts, particularly flat warts." A perivascular infiltration of mononuclear leukocytes is usually seen in the upper dermis with epidermal invasion localized to the flat wart. Patients with regressing flat warts will maintain plantar or common warts, suggest- ing that HPV type-specific structural or non- structural proteins are recognized at the level of the cellular immune response.'0 Noteworthy is the observation that spontaneous regression or cure following treatment is frequently associated with enhanced specific cellular immunity as tested by cutaneous hypersensitivity to wart virus-asso- ciated antigens 1::.1u The role of substances associated with cell-mediated responses such as lymphokines is unknown. Humoral Immunity Serologic evidence of wart virus antibodies ha~e been detected by a variety of techniques in various population studies (reviewed by von #Crogh"). The significance of these sudies is unknown because the tests have been performed on dif- ferent preparations of wan virus material. Pa- tients without wart virus antibodies or with 1gM alone are less likely to undergo spontaneous regression or be cured than those with IgM and (gG virus-specific antibodies'•'••i" Furthermore, after spontaneous or therapeutic cure, IgG anti- bodies will remain elevated for up to six months and as long as nine years." IgG antibodies are known to be circulating in half of patients who give no history of having warts."'-"' Patients with chronic andlor multiple warts are much less likely to have IgG against HPV." Of particular interest is that patients with warts that do not con- tain viriotts are less likely to have antibodies."' Regardless, the role of serologic tests in evaluating HPV infections must be performed using HPV type-specific polypeptides as antigens. In such a study PSster and zur Hausen,:' using purified HPV-I polypeptides, demonstrated by radioim- munoassay that 40% of an unselected group of individuals had circulating antibody to HPV-I.

959 LABORATORY DIAGtiOSIS OF iNFECTION The multiplicity of HPV types and the relative lack of cross-reactivity of type-specific reagents has impeded an assessment of the role of HPV types in lesions suspected of a HPV etiology. In the past, evidence for the presence of HPV in a particular lesion was mainty inferential, based on the morphologic similarity of the putative HPV-induced lesion to warts or condylorna acu- minata, lesions known to be caused by HPV. '. Identification of PV particles by electron mi- croscopy" " 103 ` I=s us offered a direct method (Figure 34-3). However, because of the possibility of sampling error, particularly with the mucosal lesions, and the fact that it is a time-consuming, ex- pensive technique, its value is diminished as a prac- tical screening technique.' u Recently, application of immunological and molecular hybridization methods to assay tissues for the presence of PV genus- and HPV type-specific structural antigens and DNA sequences have become possible and are routinely used for diagnostic purposes.'. 54 #9 Genus-Specific Tests Human and animal papillomaviruses contain regions of conserved polynucleotide sequence homology that can be detected only under non- stringent hybridization conditions such that heteroduplexes with 25076 to 350/o base mismatches remain thermally stable." In addition, papil- lomaviruses from diverse species have cross-reac- tive internal capsid antigens that can be detected using antisera prepared from disrupted papillo- mavirus."" Using these genus-specific reagents, it is now possible to test for the presence of HPV structural antigens and DNA sequences regardless of the HPV type associated with a particular lesion. Productive HPV infections, identified by the presence of genus-specific structural proteins using hyperimmune sera prepared against common anti- gens derived from either HPV or bovine papilloma- virus, can be demonstrated in acetone-fixed frozen sections by immunofluorescence (Figure 34-4) or fotinalin-fixed paraffin-embedded sections rou- tinely processed for pathology using imrnunocyto- chemical techniques (Figure Since 1980, a variety of cutaneous and mucosal lesions have been screened for the presence of the PV com- mon antigen. The frequency of HPV antigenic ex- pression using these techniques was 5007s to 7001o in plantar and common warts•" 40e/o to 60% in ver- rucae (Figure 34-6), multiple papillomas, and con- Finrs 34-3 F.lectron miaoaaph of Fanular layer-keratin junction in same piantar wart as Figures 34-4 and 34-5. PapilLonuvirus particles (50 to $5 nm) in crystalline array fill nucleus of cell immediately prior to undergoing keratiniution. Reprinted by permission from Jenson et al'= ( x 27,000) © ®

1% 960 Flfure 3i-i Acetone-fixed frozen section of plantar wart containing HPV.1. Type- specific structural antigens (arrows) are seen in many nuclei of cells in upper prickle and granular layer of epithelium. Reprinted by permission from Jenson et al.`s (Fluorescent antibody stain, x 400.) E'is.es 344 Formaiin-(lmd paraalnembedded section of same piancar .raet ai in Figure 34-3. Papillomaviru: senus-specific (comaion) struaucal antigens (acrows) stia seen in nudd of koilocytotic Qanular cells. Reprinteri by permission from Jeasoa a al. `s (Peroxidise-aaaperoaidase stain and hernatoxytin, x a00.) I ~~ ~ ~~~~ PP 7a~'~ 5'~

dylomata of the oral cavity;'-" 30474 to 45'1% of single papiAomas;' u approximately 5047s in laryn- geal papillomas;•-"° approximately S0e/i of vulvar condyiomas;10f,10' 4S%a to 6S41a of male urethral papillomas/condylomas^"' and 4047% to 30% of mild dysplasias of the cervix (Figure 34-7).••'-1" leis currently unknown why viral antigens are detected in only 40014 to 70479 of these lesions. Although lack of antigen detection may be due to sampling error, limited sensitivity of the test, or denaturing of viral antigens during tissue processing, it probably is related to a cyclic or periodic expression of struc- tural viral antigens by the viral genome, perhaps modulated by the immune response. In a study of 102 laryngeal papillomas, 48% were positivt for viral antigens.' However, when four or more con- secutive recurrences of the same lesion were ex- amined, at least one was always stained positively. Thus, although a positive immunologic reaction for HPV means that the lesion is associated with the virus, a negative reaction does not necessarily ex- dude an HPV etiology. 961 Figure 3a-6 Verruca from hard palate of oral cavity. Papiilomavirus genus-specific structural antigens are identified by posi- tive PAP reaction (arrows), mainly in koilocytotic cells of granular layer of a single papillary frond. Reprinted by per- mission from Jenson et al.' (Peroxidase- antiperoxidase stain and hematoxylin, x 197.) Nonstringent hybridization techniques have been used to demonstrate HPV sequences in two of four laryngeal papillomas"' and 10 of 16 cer- vical dysplasias (Figures 348A and B)."-10' When correlated with immunocytochemical staining for genus-specific antigens, it was concluded that at least 75% of laryngeal papillomas and 93+ti of cervical dysplasias, respectively, were associated with HPV infections. Type-Specific Tests HPV-1, -2. -3, and -5 type-specific viral anti- gens have been identified by immunofluorescence in frozen sections of cutaneous warts"•"'•"• Although no cross-reactivity was observed among these HPVs using heterologous antisera, the im- munofluorescence technique is of little practical use for diagnostic purposes since tissues for most retrospective studies are preserved in formalin. Recently, HPV-1 type-specific antigens have been identified in formalin-fixed tissue using hyperim- mune rabbit serum and the PAP technique" MASTEER ~ :avp Z. # C'

1 962 Figure 34-7 Mild cervical dysplasia with prominent epithelial spikes containing numerous cells with intranuclear genus-specific papiilomavirus antigens (arrows). Papillomavirus DNA was also recovered from this case. (Peroxidase-antiperoxidase stain without counterstain, x 197.) Reprinted by permission from Lancaster et al.s' HPV-1 antigens were found in S407o of plantar warts and 11 07e of common warts. Moreover, the presence of HPV-1 viral antigens corresponded to the presence of HPV-I specific nucleotide se- quences determined by molecular hybridization studies. Preliminary classifications of HPV DNA in biopsy specimens are made on the basis of the hybridization spectrum of viral DNA fragments generated by appropriate restriction endonu- cleases and by assessing the relatedness of viral se- quences as judged by hybridization efficiency under relaxed and stringent conditions.'••" When warts and/or mucosal papillomas undergo malig- nant transformation, late gene expression (struc- tural viral proteins) is no longer detected, but the PV genome continues to replicate as a plasmid, al- though at a lower copy number. Detection of HPV DNA associated with the transformed state is best accomplished under stringent conditions that are at least ten times more sensitive then non- stringent conditions, a difference which appears to be particularly critical in PV systems. At least 16 different HPV types have been char- acterized by molecular hybridization studies (Table 34-1). Besides being preferentially asso- ciated with specific clinical lesions, subsets of HPV appear to be primarily cutaneotropic or mu- cosotropic (HPV-6, -1 l, and -16) and noncarcino- genic or potentially carcinogenic (HPV-S, -6, -11. -16). Therefore, most studies on squamous cell carcinoma of ttie skin have been probed with HPV-S DNAs, whereas most studies of squatnous cell carcinoma of the laryngeal and anogenical areas have been probed with HPV-6,"'6 -11,'• and, more recently, HPV-16 (zur Hausen, per- sonal communication). HPV-S has been detected in both primary and metastatic squamous carcinomas of patients with EV•= and in the primary carcinomas of patients undergoing immunosuppressive therapy."• Dem- onstration of HPV in metastatic lesions is of par- ticular importance since HPV DNA sequences found in the primary lesions could possibly be a contaminant from an adjacent benign skin lesion. HPV-Il has been found in half of laryngeal papillomas, approximately one fourth of genital warts, cervical dysplasias, and four of 24 cervical cancers (two in situ; two invasive)!' HPV-6 se- quences have been found in the majority of typical condylomata acuminata (venereal warts) and condylomata acamittata of Lowenstein and Bushke," an ag=ressive infiltrating squsmous lesion that usually does not metastasize." Other evidence that HPVs are associated with malignant tumors has been the finding of an untyped DNA isolated from a puient with EV in four squamous cell carcinomas of the vulva and cervix." Re- cently, HPV-16 has been found in 609e of squa- mous cell carcinomas of the cervix from German

A --IWE #LttMBER - ..v -C=- 13 Kr 19 23 30 Figures 344A&B Aucoradiograms representing hybrid- ization of in vitro 'lP-labeled bovine paptllomavirus type 1(BPV-1) DNA to human papillomavirus type I (HPV-1) DNA and DNAs isolated from five cervical biopsies. HPV-1 DNA (10 ng) (lane C) and tissue DNAs were digested with either BamHl (panel A) or Hindlil (panel B) and electrophoresed in 0.8m+ agarose gels. The separated DNA fragments were transferred to nitrocel- lulose membranes and subsequently hybridized to 3jP-BPV-1 DNA. Bands I and 2 represent the migration of circular and linearized HPV.I DNA. respectively. In both panels A and B. four of the five cases tested con- tained DNA sequences to which the BPV-i probe hy- bridized. These sequences comigrated with either open circular and: or linear forms of HPV-1 DNA. Arrows indicate migration of low molecular weight DNA. pre- sent in case 19 (seen in Figure 34-7), that specifically hybridized to the BPV- I DNA probe. (Performed under relaxed or nonstringent conditions to detect PV genus- specific polynucleotide sequences). Reprinted by permis- sion from Lancaster et al." patients but in only 10*l0 of cervical dysplasias (zur Hausen, personal communication). TREATMENT Bunney,' in her assessment of a 10-year pro- gram evaluating wart cures in approximately 14,000 patients, concluded that 709s to 8044 of paiients could have been cured of warts by simple methods applied either at home or in the clinic. Whether warts are treated or left alone is usually a prerogative of the individual patient. Although most warts are usually treated for cosrttetic tYSsons, others are treated because they cause discomfort or are truly disabling. Since warts frequently undergo spontaneous regtession, veatmett should not result in permanent damage such as excessive scarring or disfigurement. 8 C 963 CASE NUMBER ~r 13 15 19 23 30 In general, warts that respond most favorably to treatment are usually single, of short duration, and found in children.' Home treatment of warts includes topical application of salicylic acid and formalin or glutaraldehyde; podophyllin or strong acids or bases should be used under supervision. Cryotherapy is the most universally popular treat- ment for warts, although surgical intervention may be indicated in some cases. Perhaps the best biological reason for not routinely recommending surgical intervention is that surgical extirpation of the wart is not associated with enhancement of the immunologic response such as is seen following multiple topical or freeze-thawing treatments that appear to facilitate recognition of HPV-asso- ciated antigens by the immunologic system.'='-"_ Quick removal of the bulk of wart virus antigens without concomitant exposure to the immune sys- tem is probably the main reason for the high re- currence rates of warts following surgery. Other treatments, such as interferon and retinoic acid, appear promising but are experimental at this time. The duration of treatment and criteria for cure of warts are important factors for clinical man- agement of these lesions. Bunney' suggests that 12 weeks after beginning treatment is an adequate length of time to expect warts to disappear. Since failure to completely eradicate the initial lesion is the main reason for recurrence, a valid criterion for cure is the appearance of a normal skin after disappearance of the wart(s). Similar criteria have not been developed for treatment and cure of HPV- associated iesions arising in mucosal epithelium. HITRS T E Ik ip

964 VACCINATION Vaccination offers potential prevention and/or cure for cutaneous warts and HPV-associated pa- pillomas of the tnucosal surfaces. It is apparent that effective vaccination will utilize either type- specific structural or nonstructural antigens and that the lesions to be targeted will be those of the anogenital area that are sexually transmitted and are at risk of malignant transformation.' Future vaccines will depend on the progress made in the molecular virology of HPV and the development of bacterial expression vectors capable of synthe- sizing highly purified, immunogenic structural and nonstructural HPV proteins. Otherwise, the use of an autogenous vaccine derived from tlte pa- tient's own lesion is the only other alternative for vaccination because of the possibility of poten- tially harmful biological contaminants associated with heterologous vaccines. Most cutaneous warts either spontaneously regress or can be cured by simple treatment.' Ap- proximately 20074 to 3Q07o of these lesions will be recalcitrant to therapy. Aithough presumably associated with general or specific immunologic defects, half of these recalcitrant warts may dis- appear after vaccination of patients with formalitt- inactivated HPV'• all patients cured developed a positive IgG response and delayed hypersensitivity reaction to the HPV preparation. Vaccination of persons at high risk for develop- ing or transmitting anogenital HPV-associated lesions appears to be indicated for several reasons. First, many of these are sexually transmitted and reinfection by consorts must occur frequently. Second. S°1o to 154% of squamous carcinomas of the vulva and penis appear to arise in preexisting condylomata, usually of long duration. Third, successful autogenous vaccination appears to be higher for venereal warts (up to 80410) than cutan- eous wans.+" Fourth, recombinant DNA tech- nology should be able to provide the necessary biological materials in the near future for vaccina- tion. Evaluation of the efficacy of vaccination for lesions of the aaogenital area, however, will de- pend on well-controlled epidemioiogic studies and clinical ttiaLt.rith evaluation of humoral (possibly secretory ISA) and cell-mediated responses to type-specific antigens. Regardless, preventive measures to avoid transmission of these viruses is much preferable to the best cure. SiJMIri.+1RY HPVs are a beterotenous group of oncogenic viruses that replicate as true plasmids in selected benign (wares/papillomas). dysplastic (cervical dysplasias/?bowenoid papulosis), and malignant (anogenital carcinomas) lesions of squamous epi- thelium. Spontaneous regression or successful topicallcryosurgical treatment of the benign lesions are largely dependent on either naturally acquired or iatrogenically-related stimulation of HPV type-specific cellular immunity. Malignant transformation of warts and papillomas appears to relate to a variety of factors: 1) infection by certain HPV types (HPV-S, -6, -11, and -16), 2) decreased cellular immunity to HPV-associated antigens, and 3) interaction with physical carcin- ogens such as sunlight or x-rays. Research on HPV has benefited more from re- combinant DNA technology than any other area in virology, mainly because HPV cannot be grown in culture or transmitted to other animal species. Future use of recombinant DNA technology in HPV research should: 1) allow many more HPVs to be identified and characterized; 2) permit the products of early and late gene regions to be syn- thesized in bacterial expression vectors and used as immunogens for diagnostic and vaccination purposes, and 3) provide a substantial amount of information about mechanisms involved in carcinogenesis. REFERENCES i. Rowson ICEK, Mahy BWJ: Human papova ( wart) virus. Bacterrol Rev 1967;31:114-131. 2. Bunney MH: Viral Wans: Their Biology and Treatmettt. New York, Oxford University Press. 1982. 3. Praetorius-Clausen F: Rare oral viral disorders (moUuscum contaposurn, localized keratoacan- thotna, verrucae, condyioma actuninatum and focal epithelial hyperplasia). Ora/ Surs Oral Med O.al Pathol 1972;34:60~b1n. 4. Jenson AB, Lancaster WD, Hartman DP. Shaffer EL Jr: Frequency and distribution of papillomavi- rus structural antigens in verrucae, multiple papillomas, and condylomsta of the oral cavity. ,4 m I Pathol 1962a:147:212-2I a. S. Cook TA, Cohn AM, &unschrvit JP, et at: Laryngeal papilloma: Etiolopc and therapeutic considerations. Am I Otol 1973;32:649-653. 6. Lack EE, Jenson AB, Smith HG, a al: Itttmuno- peroxida>ee loaliation of human pzpillomavirus in laryngeal papillomas. tnnrvirolotr 196Q:11: 148-154. 7. zur Hausen H: Human pspillomaviruses and their possible role in squamous cell carcinomas. Cttrs Top MteroDicl hnmuttol 2977t78:I-30. . i. Kurtnaa RJ. Jenson AB, Lmeasta WD: Papillo- mavirus infection of the cavitt.lI. Rdadonship to irttraepitlsdial neoplasia based on the preseace of speci6c viral structural proteia:. Am JSrrj Aathol 1963a:7:39-52. 9. Kutman RJ, Jmsac A8. Sinelair C, et al: Detec- tion of human pepillomaviruses by immuno- cytochemisay, in Detsiis RA (.d): Advasica in €

AL , Immunocyrochemi.rtry. New York, Masson, 1983b. 10. Orth G, Breitburd F, Favre M, et ai: papillomavi- ruses: Possible role in human cancer. Cold Sprtnt Harbor Conf Cell Prollf 1977a;4:1043-1068. 11. Orth G. Jablonska S, Breitburd F, et a1: The human papiliomavtruses. Bull Cancer 1978a;6S: 151-164. 12. Butel J: Studies with human papillomavirus modeled after known papovavirus systems. J Natl Cancer Inst 1972:48:285-299. 13. Koller LD, Olson C: Attempted transmission of warts from man, cattle and horses and of deer fibroma to selected hosts. J invest Derrrtatol 1972:58:366-368. 14. Melnick JL, Allison AC, Butel JS, et ai: Papova- viridae Intervirol 1974;3:106-120. 15. Jenson AB, Rosenthal JD, Olson C. et al: Irttmu- nologic relatedness of papillomaviruses from different species. I Nat/ Canrer Inst 1980;64: 495-500. 16. Law M-F, Lancaster WD, Howley PM: Conserved nucleotide sequences among the;enomes of papiI- lomavirttses. I Viro! 1979;32:199-207. 17. Crawford LV: Nucleic acids of tumor viruses. Adv Virus Res 1969;14:89-132. 18. Lancaster WD, Meinke W: Persistence of viral DNA in human cell cultures infected with human papilloma virus. Nature (London) 1975;256: 434-436. 19. La Pona RF, Taichman LB: Human papilloma viral DNA replicates as a stable episome in cultured epidermal keratinocytes. Proc Nat! Acad Sci USA 1982;79:3393-3397. 20. Lancaster WD, Olson C: Papovavirus infections of vertebrate animals. Comp Dwtn Viral Dis 1981;II1:69-98. 21. Bett WR: Wart I bid thee begone. Practitioner 1951;166:77-8p. 22. Clarke GHV: The charming of warts. J Invest Dermato! 1965;14:15-21. 23. Rolleston JD: Dermatology and folklore. Br J Dermatol 1940;43:43. 24. Licht C de F: Om Vonters Smitsomhed. Uteskr Laej S Raekke 1894; I:368-369. 25. Ciuffo G: Imnesto posidvo con 5luato di verruca colgare. G Itol Mat Vener 1907;45:12-17. 26. Serra A: Ricerche istologiche e spetimeatali sui condiloma capo e la verruca voi8sre. Coatributo all'etiologia, patotanesi, Ntrabilita. G Itd Mol Vener 1908:49:11-42. 27. Wile UJ, Kingrey LB: Tbe etiology of common warts: Prdiminary report of an atperitnental study. JAMA 1919;73:974-973. 28. Strauss MJ, Bunting H, Melnick JL: Virus-like particles and inclusion bodies in skin papillomas. I Invest Denrtatol 1949;15:433-444. 29. Meinick JL, Bunting H, Banfield WO, et al: Elec- tron microscopy of viruses of human papilloma, moiluacuta contaposum. and vaocinia, including obsavations on the formation of virus within the cell. Ann NY Acad Sef 19S2:S4:1214-1225. 30. Bunting H: Close-packe+d array of virus-like par- ticles within cells of a human skin papilloma. Proc Soc Esp Bio! Med 19S3:a4:327-332. 31. Ahneida JD, Howatson AF, Wiiliams MG: Elec- tron microscope study of human warts: Sites of 965 virus production and nature of the inclusion bodies. I Invest Dermatol 1962:38:337-345. 32. Melnick JL: Papova virus group. Science 1962; 135:1128-1130. 33. Favre M, Orth G, Croissant O, et al: Human papillomavirus DNA: Physical map. Proc Nar! Acad Sci USA 1973b;72:4810-4814. 34. Gissmann L, zur Hausen H: Human papillomavi- ruses: Physical mapping and 8enetic heterogeneity. Proc Nat! Acad Sci USA 1976;73:1310-l313. 35. Gisstnantt L. Pfister H, zur Hausen H: Human pap- iDomavirtues (HPV): Characterization of four different isolates. Virology 1977a:76:J69-380. 36. Orth G, Favre M, Croissant 0: Characterization of a new type of human papiliomavirus that causes skin warts. I Virol 1977b;24:106-120. 37. Orth G, Jablonska S, Favre M, et al: Character- ization of two types of human papillomaviruses in lesions of epidermodysplatia verruciformis. Proc Natt.lcad Sci 1978b;7S:l337-1541. 38. Kluj A, Finch JT: Structure of viruses of the papilloma-polyoma type. I. Human wart virus. J Mo! Bio! 1963;11:403-423. 39. Viac J, Thivolet J, Chardonnet Y: Specific im- munity in patients suffirrina from recurring warts before and after repetitive intradermal tests with human papillornavirus. Br J Desmatol 1977b; 97:365-370. 40. Crawford LV, Crawford EM: A comparative study of polyoma and papilloma viruses. Virology 1963;21:238-263. 41. Crawford LV: A study of human papil)oma viruses DNA. J Mo! Bio! 1965;13:362-372. 42. Gissmann L, zur Hausen H: Physical characteriza- tion of the deoxyribonucleic acids of different human papilloma viruses (HPV). Med Microbiol Immunol 1978;166:3-11. 43. Howley PM. Law M-P, Heilman C, et al: Molecular characterization of papillomavirus genomes. Cold Spring Harbor ConJ Cell Prol 1980;7:233-247. 44. Howley PM: The human papillornaviruses. Arch Pathol Lab Med 1982; 106:429-432. 45. Lancaster WD, Olson C: Animal Papillomavi- ruses. Microbiol Rev 1982;46:I9I-207. 46. Morrison JM, Keir HM, Suback-Sharp H, et al: Nearest neighbour base sequence analysis of the deoxyribonucleic acid of a further three matn- maliatt viruses; simian vinn 40, human papillottta virus and adenovirus type 2. J Gen Virol 1967;1:I01-106. 47. Gisstnann L, zur Hausen H: Inverted repetitive se- quences in human papiUoma virus I (HPV-1) DNA. Virology 1977b;83:271-276. 48. Favre M. Breitburd F, Croissant O, et al: Struc- tural polypeptides of rabbit, bovine, and human papitloma viruses. J Vlrol 197Sa;1S:1239-1247. 49. Orth G, Breitburd F. Favre M: Evidence for aati- genic determinants shared by the structurai poiy- peptides of (Shope) rabbit pepillomavirus aud human type I. Virology 1979;91:243-235. S0. PSstet H, Gissmann L, zur Hausen H: Pntial characterization of the proteins of human pspillo- maviruses (HPV.I) 1-3. Vbotosy 1977;83:131-137. 51. Pass F. Maizel JV: Wart-aasociated antijets. 11. Human immunity to viral structural proteins. Jltt- vest Dermarol 1973:60:307-3! 1.

966 52. Spira G. Estes MK, Dreesman GR, et al: Papova- virus structrual polypeptides; Comparison of human and rabbit papilloma viruses with Simian virus 40. IRtervirolojy 1974;3:220-231. 53. Favre M. Breitburd F. Croissant O. et ai: Chromatin-llke structures obtained after alkaline disruption of bovine and human papillomavtruses. J Virol 1977;21:1205-1209. 54. Coggin JR, zur Hausen H: Workshop on papillo- maviruses and cancer. Cancer Res 1979:39: 545-546. 55. Gissmann L, de Villiers E-M. zur Hausen H: Analysis of human warts (condylom.ata acu- minata) and other genital tumors for human papillomavirus type 6 DNA. Int J Cancer 1982a;29:143-146. 56. Gissmann L. Diehl V. Schultz-Coulton H-J, et al: Molecular cloning and characterization of human papillomavirus DNA derived from laryngeal papilloma. ! Virol 1982b:44:393-400. 57. Heilman CA. Law M-F, Israel MA. et al: Cloning of human papillomavirus 8enomic DNAs and analysis of homologous polynucleotide sequences. J Virol 1980:36:393-407. 58. Jenson AB, Sommer S. Paylin8-WriBht C. et al: Human papillomavirus: Frequency and distribu- tion in plantar and common warts. Lab Invest 1982c;47:491-497. 59. de Villiers E-M, Gissmann L. ztu Hausen H: Molecular cloning of viral DNA from human genital warts. I Viro! 1981;40:932-935. 60. Green M. Brackmann KH, Sanders PR, et al: Isolation of a human papillomavirus from a pa- tient with epidermodysplasia verruaformis: Pres- ence of related viral DNA genomes in human uroQenital tumors. Proc Nati Acad Sci USA 1982; 79:4437-4441. 61. Lancaster WD, Kurman RJ, Sanz L. et al: Human papilloma virus: Detection of viral DNA sequences and evidence for molecular heterogeneity in meta- plasias and dyspiasias of the uterine cervix. Inter- virology 1983:20:202-212. 62. Ostrow RS, Bender M. Niimura M. et al: Human papillomavirus DNA in cutaneous primary and metastasized squamous cell carcinomas from pa- tients with epidetmodysplasia verruciformis. Proc Nat! Acad Sci USA 1962;79:1634-1638. 63. Hirt B: Selective exuaction of potyonta DNA from infected mouse cell cultures. I Mo( Biol 1967;26:365-369. 64. Chad AL, Giumann L, Me:er B, a al: Molecular donin= and partial nudeotide sequence of human p.piiloma virus type Ia. Vlro/oLy 1952;118: 2Si-739. 65. Soutbatn EM: Detection of speci5c sequences amon~ DNA fragments separated by gel electro- pbocsut. ! Mo! Biof 1975;93:303-317. 66. Howley PM, israel MA, La.v M-P, a al: A rapid method for detecting tlfe mapping homology be- tween lteeteroiorous DNAs. I Biot Chent 1979; 254:48764a33. 67. Getb LD, Kohne DE, Martin MA: Quantituion of simian virus 40 sequences in African aeat rttonkey, mouse and virtts-transfotmed cell genoma. I Mol Qiot 1971:57:129-243. 6i. Massiag AM. Epstein WL: Natural history of warts. A two-yeu study. Arch Dermatol 1963; 87:306-310. 69. Quick CA. Foucar E. Dehner LP: Frequency and significance of epthelial atypia in laryngeal papd- lomatosis. Laryngoscope 1979;89:530-560. 70. FriedberQ SA. Stagman R, Hass GM: Papillary le- sions of the larynx in adults: a patholo8tc study. Ann Otol Rhinol Larynjo! 1971:80:683-692. 71. Pfister H. zur Hausen H: Seroepidemtolo8ic studies of human papilloma virus HPV-1 infec- tions. 1978;21:161-163. 72. Summers L, Booth DR: Intraoral condylotna acu- minatum. Oral Surg Oral Med Oral Parho! 197438:273-278. 73. Morison WL: Viral warts, herpes simplex and herpes zoster in patients with secondary immune deficiencies and neoplasms. Br J Dermarol )975c: 92:625-630. 74. Reid TMS, Fraser NG, Kernohan IR: Generalized warts and immune deficiency. Br J Dermaro! 1976; 95:559-564. 75. Spencer ES, Andersen HK: Clinically evident, non-temnal infections with herpes virus and the wart virus in immunosuppressed renal allograft recipients. Br Mcd J 1970:3:2S1-2i4. 76. von Krogh G: Warts: Immunolo8ic factors of prognostic significance. Int I Dermarol 1979; 18: 195-204. 77. Orid JD: Natural history of genital warts. Br J Vener Dis 1971a:46:37-42. 78. Jenson AB, Openshaw H, Hooks JL, et al: Herpes and other virally-induced oral diseases, in Slavkin HC, Cohen DW (eds): Current Advances in Oral Biology. Bristol, Distributions System. 1979. 79. Grussendorf El, zur Hausen H: Localization of viral DNA replication in sections of human warts by nucleic acid hybridization with complementary RNA of human papillomavirus type 1. Arch Der- rnato! Res 1979;264:55-63. 80. Berman A, Berman JE: Efflorescence of new warts: A si8tt of involution: Histopathological findings. Arch Derntatoi 1977;113:1219-1221. 81. Berman A, Winkelmann RK: Flat warts undergo- ing involution: Histopathological findings. Arch Dermotol 1977; 113:1219-1221. 82. Tagami H, Ogino A. Ra.kipwa M, a al: ReRes- sion of plane warts followinj spontaneous in!!am- mation. A histopatholopcal study. Br I Dermatol 1974;90:147-154. 83. Ricciuti L, Lancaster WD, Scofield H, a al: Iden- tification of papillomavinu structural antt;ens in single papillomuous lesions of the oral cavity. Submitted for publication. $4. Gissm^^ L, Woinik L, Ikenbas H, a al: Human papillomavirtn types 6 and 11 DNA sequences in nnital and larytskai papillomas and in some csr- virai ¢anoars. Prot 1Votl Aced Sd USA 19e3;W. 560-363. 83. Galloway TC, Soper GR, Elxa J: Carcinoma of the latynx afia itradiation for papiilomaa. Arch OtolCrynfaJ i960:72:289-294. d6. Putney FJ: Borderline maliptant lesions of the larynx. Aidr Orolaytrtot 1933;61:3i 1-333. S7. waislt TE, Bamer PR: Epidetmoid carcinoma of the iarYnx occurrint in 2 chiidrm with papilloma of the larynx. LaryRta*cbpe 196Q;60:1110-1124. 4.