ARTICLES. Epidemiologic Evidence Showing That Human Papillomavirus Infection Causes Most Cervical Intraepithelial Neoplasia

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1 ARTICLES Epdemologc Evdence Showng That Human Papllomavrus Infecton Causes Most Cervcal Intraepthelal Neoplasa Mark H. Schffman, Hed M. Bauer, Robert N. Hoover, Andrew G. Glass, Dane M. Cadell, Brenda B. Rush, Davd R. Scott, Mark E. Sherman, Robert J. Kurman, Sholom Wacholder, Cyntha K. Stanton, M. Mchele Manos* Background: Expermental studes have provded strong evdence that human papllomavrus (HPV) s the longsought venereal cause of cervcal neoplasa, but the epdemologc evdence has been nconsstent. Purpose: Gven mprovements n HPV testng that have revealed a strong lnk between sexual actvty hstory and cervcal HPV nfecton, we conducted a large case-control study of HPV and cervcal ntraepthelal neoplasa (CIN) to evaluate whether sexual behavor and the other establshed rsk factors for CIN nfluence rsk prmarly va HPV nfecton. Methods: We studed 500 women wth CIN and 500 control recevng cytologc screenng at Kaser Permanente, a large prepad health plan, n Portland, Ore. The establshed epdemologc rsk factors for CIN were assessed by telephone ntervew. We performed HPV testng of cervcovagnal lavage specmens by gene amplfcaton usng polymerase chan reacton wth a consensus prmer to target the LI gene regon of HPV. Uncondtonal logstc regresson analyss was used to estmate relatve rsk of CIN and to adjust the epdemologc assocatons for HPV test results to demonstrate whether the assocatons were medated by HPV. Results: The case demonstrated the typcal epdemologc profle of CIN: They had more sex partners, more cgarette smokng, earler ages at frst sexual ntercourse, and lower socoeconomc status. Statstcal adjustment for HPV nfecton substantally reduced the sze of each of these case-control dfferences. Seventy-sx percent of cases could be attrbuted to HPV nfecton; the results of cytologc revew suggested that the true percentage was even hgher. Once HPV nfecton was taken nto account, an assocaton of party wth rsk of CIN was observed n both HPV-negatve and HPVpostve women. Concluson: The data show that the great majorty of all grades of CIN can be attrbuted to HPV nfecton, partcularly wth the cancer-assocated types of HPV. Implcatons: In lght of ths concluson, the nvestgaton of the natural hstory of HPV has preventve as well as etologc mportance. [J Natl Cancer Inst 85: , 993] The well-establshed assocaton between sexual actvty and the development of.cervcal neoplasa strongly mplcates a sexually transmssble etologc agent (,). Molecular studes have provded strong evdence that human papllomavrus (HPV) may be ths agent (3), but the epdemologc evdence has been weaker (4,5). HPV DNA s dentfed much more frequently n women wth cervcal neoplasa than n women wth normal cervcal cytologc dagnoses. Moreover, statstcal adjustment for HPV nfecton has not explaned the elevated rsk of developng cervcal neoplasa n women wth multple sex partners, suggestng that other venereally transmtted agents play an etologc role (6-9). In addton, the estmated proporton of cervcal neoplasa attrbutable to HPV nfecton n prevous studes has been too low for one to conclude that HPV nfecton causes most cervcal neoplasa. Recently, mproved HPV testng methods revealed for the frst tme a strong lnk between sexual actvty hstory and cervcal HPV nfecton (70). Ths fndng prompted our large case-control study of cervcal ntraepthelal neoplasa (CIN) and HPV nfecton, whch evaluates whether sexual behavor and the other establshed rsk factors for cervcal neoplasa nfluence rsk prmarly va HPV nfecton. *See "Notes" secton followng "References." 958 ARTICLES Journal of the Natonal Cancer Insttute, Vol. 85, No., June 6, 993

2 Subjects and Methods Study Subjects We selected 500 case wth CIN and 500 control from a cohort of 46 women ncluded n an ongong natural hstory study of HPV nfecton at the Kaser Permanente prepad health plan n Portland, Ore. Ths cohort, establshed between Aprl, 989, and November, 990, was chosen to be representatve of all women presentng for routne cervcal cylologc (Pap smear) screenng at Kaser Permanente, whch serves about one fourth of the adult female populaton of Portland. The 500 case were selected from among the 673 women found at enrollment to have cytologc evdence of CIN or condylomatous atypa [a dagnoss now combned wth CIN n the Bethesda cytologc classfcaton system (//)]. (We excluded from consderaton as case the 89 women [3%] of the cohort who had a medcal hstory of CIN but were currently cytologcally negatve, often because of ablatve treatment.) The 500 control were randomly selected from the 7654 women wth normal cytologc dagnoses and no known hstory of CIN. We selected the 000 on the bass of the orgnal enrollment cytologc dagnoses, regardless of subsequent cytologc revew or hstopathologc confrmaton. The case group ncluded all 8 women n the cohort wth enrollment cytologc dagnoses of CIN or greater (3 wth CIN 3 or cytologc changes suspcous for cancer, 7 wth CIN, and 3 wth CIN ) and 39 women chosen randomly from the 49 wth condylomatous atypa. Among the control, the enrollment cytologc dagnoses of 43 were negatve, and 87 (7.4%) were classfed as havng "bengn reactve atypa"; there was no statstcally sgnfcant dfference between these two control subgroups wth regard to HPV test results or demographc characterstcs. Telephone Intervew and Cytologc Samplng All were contacted by telephone. If oral nformed consent was obtaned, traned ntervewers admnstered a 0-mnute telephone questonnare that measured the establshed epdemologc rsk factors for CIN except det. For HPV testng, a 0-mL cervcovagnal lavage was obtaned from the study partcpants as part of the cervcal cytologc screenng procedure, wth approval from the Kaser Permanente Insttutonal Revew Board. The lavage conssted of a drected rnse of the cervcal os (usng sterle physologc salne admnstered va a 0-mL syrnge wth a 3'/-nch flexble ntracatheter extender), recollected after poolng n the posteror vagnal fornx (). The lavage was obtaned after the routne one-slde cervcal smear, consstng of a cervcal scrape performed wth an Ayre spatula and a cytobrush sample of the endocervx. HPV test data and at least some questonnare data were obtaned from 438 (88%) of the case and 44 (85%) of the control. Four n the case group and 0 n the control group were nelgble for study because of a prevous hysterectomy or past CIN determned after ther selecton. Of those elgble, 7 case and 30 control had cervcal specmens that were nadequate for HPV testng, manly because there were no apparent cervcal cells, as assessed by the appearance of the samples and the lack of human DNA (B-globn) amplfcaton. An addtonal 4 case and 36 control refused the ntervew or were lost to follow-up. In the results presented below, all avalable data are ncluded; the Fndngs were unchanged when the analyss was restrcted to partcpants wth complete data. Laboratory Analyss The cervcovagnal lavage specmens were refrgerated wthn hour of collecton and transported to a central laboratory for processng. A ml alquot was removed and frozen (at -70 C and kept on dry ce durng shpment) untl preparaton for HPV determnaton by polymerase chan reacton (PCR)-based analyss, whch was performed wthout knowledge of medcal hstory, cytologc dagnoss, or other data. Of the ml alquot, 50 (L was prepared for PCR analyss (3). Negatve controls contanng only human DNA (from the K cell lne; the Amercan Type Culture Collecton, Rockvlle, Md.) were nterspersed to montor potental contamnaton durng sample preparaton. Gene amplfcaton methods ncludng PCR prmers, buffer, reacton components, and cyclng parameters have been prevously descrbed n detal (4). Duplcate alquots of 5 u.l (0.05% of the orgnal 0-mL lavage) were tested usng a consensus prmer PCR method targetng the LI gene regon of HPV (5,6). The amplfcaton of a human B-globn gene fragment was used as an nternal control for sample ntegrty. The PCR products were analyzed usng dot blot hybrdzaton as prevously descrbed (4), wth mnor modfcatons ncludng the use of a nonradoactve detecton method (7). Blots were ndvdually hybrdzed wth the generc probe, desgned to detect most known HPV types, as well as probes specfc for HPV types 67, 6, 8, 6, 3, 33, 35, 39, 40, 4, 45, 5, 5, 53, 54, 55, 57, and 59. In addton, we probed for prelmnarly dentfed types known as PAP88, PAP55, PAP38A, PAP5, PAP9, and W3B. Recombnant plasmds were provded by Dr. G. Orth (Insttut Pasteur, Pars, France) for HPVs 33, 39, 4, 54, and 55; Dr. K. Shah (the Johns Hopkns Unversty, Baltmore, Md.) for HPV 45; Dr. S. Slversten (Columba Unversty, New York, N.Y.) for HPV 5; Dr. W. Lancaster (Wayne State Medcal School, Detrot, Mch.) for HPV 5; and Dr. R. Ostrow (Unversty of Mnnesota, Mnneapols) for HPV 6. On the bass of prevous work (8), HPV types were combned nto three groups dfferng n ther assocaton wth nvasve cervcal cancer: hgh-rsk HPV types (6 or 8), ntermedate-rsk types (3, 33, 35, 39, 45, 5, or 5), and low-rsk types (6/, 4, or unknown and all other types). Multple nfectons, whch accounted for 35% of nfectons, were assgned herarchcally to the hghest rsk category applcable. Statstcal Analyss Uncondtonal logstc regresson analyss was used to compute odds ratos as estmates of the relatve rsk () of CIN, wth 95% confdence ntervals (CIs). CIs excludng ndcated statstcally sgnfcant assocaton. We estmated the assocaton of each epdemologc factor wth and wthout adjustment for HPV test results. If a strong assocaton (ndcated by an elevated ) dsappeared wth adjustment for HPV ( approached ), we nferred that HPV nfecton medated the assocaton between that epdemologc factor and the rsk of CIN (9). We calculated the attrbutable proporton, the fracton of dsease attrbutable to HPV, by the followng formula: attrbutable proporton = % HPV postvty n case X ( - [/]). Because of the age dfference between case (medan age, 8 years) and control (medan age, 34 years), all estmates were adjusted for age n sextles (6-4, 5-8, 9-34, 35-39, 40-46, and 47+ years) or tertles (6-8, 9-39, and 40+ years) n analyses wth few. Cytology Revew and Case Defnton The orgnal cytologc dagnoses were used to defne case and control n the man analyss, but the results of possble msclassfcaton of case-control status were addressed by ancllary analyses based on cytology revew and hstopathologc confrmaton. Two hundred ffty cervcal smears were selected for cytology revew, ncludng 00 smears from borderlne lesons. All avalable sldes classfed as bengn reactve atypa (borderlne controls) were revewed as well as a sample of condylomatous atypas (borderlne cases). In addton, 5 randomly selected negatve dagnoses and 5 CIN 3 were chosen for revew. When the three revewers (M. E. Sherman, D. R. Scott, R.. Kurman) dd not agree, the results of the prmary outsde revewer (M. E. Sherman) were used. Possble msclassfcaton n case-control status was manly n the condylomatous atypa category; 6% were revewed as negatve. None of the cases orgnally dagnosed as CIN were called negatve on revew. Only two control smears (one bengn reactve atypa and one negatve) were judged to be defntely msclassfed. Colposcopcally drected bopses, performed accordng to standard referral protocol on 363 of the 500 case, confrmed that the case seres was a typcal unselected U.S. seres wth regard to severty of dsease. Almost all of the unbopsed case (7/37; 85%) had a cervcal cytologc dagnoss of condylomatous atypa. Of the case women Journal of the Natonal Cancer Insttute, Vol. 85, No., June 6, 993 ARTICLES 959

3 from whom bopsy specmens were taken, 63% had a hstopathologc leson dagnosed as defnte CIN (ncludng 8% hgh-grade, wth two nvasve cancers). As a fnal aspect of case defnton, apparently ncdent (.e., newly dagnosed) cases dd not dffer from known prevalent (prevously dagnosed) cases wth regard to HPV test results or epdemologc rsk profles (data not shown). Thus, for ease of presentaton, data on ajl case were combned for the man analyss. Addtonal HPV Testng After the HPV data were unmasked and after the data were analyzed, we dentfed HPV type PAP5 as HPV 58, a type that has been assocated wth cervcal cancer. In addton, we hybrdzed all amplfed specmens wth probes specfc for HPVs and 68, two other cancer-assocated types. These refnements n the HPV-testng protocol would have changed the herarchcal HPV group assgnments, from the type 6 group to the type 3 group, for 46 (seven control and 39 case ). Because these test results changed after the data were unmasked, we have chosen to present the orgnal, masked results. The conclusons were absolutely unchanged when the data were reanalyzed usng the revsed HPV results. Results HPV Data HPV DNA was detected much more frequently n case than n control (Table ). The s for CIN were very hgh for all three HPV type groups but were notceably hgher for the cancer-assocated HPV types (type 3 and type 6 groups). Adjustment for the epdemologc rsk factors left the estmates n Table unchanged. The analyss of ndvdual HPV types was lmted by small numbers; nevertheless, the type-specfc estmates confrmed the a pror groupng of types nto low, medum, and hgh rsk, wth the slght exceptons for women wth multple HPV types noted n the "Addtonal HPV Testng" secton. Wthn the type 6 group, the unknown types of HPV (3 case, 9 control ) had the weakest assocaton wth rsk of CIN ( = 4). Women wth multple HPV types detected (35% of postves) were more lkely to be n the case group ( = 36) than women wth sngle HPV types ( = 6). Man Analyss Women n the case group demonstrated the typcal epdemologc profle of CIN ( # n Table ). On average, relatve to control, those n the case group had more sex partners, earler ages at frst ntercourse, less educaton and lower famly ncomes, and smoked cgarettes more often. They were margnally more lkely than control to have used oral contraceptves. All of these characterstcs were assocated wth HPV nfecton. Several of the epdemologc rsk factors could be explaned, even pror to consderaton of the HPV test results, as correlates of sexual behavor, partcularly as correlates of ncreasng lfetme numbers of sex partners ( # n Table ). For example, the assocatons of smokng and early age at frst ntercourse wth rsk of CIN were explaned by takng lfetme numbers of sex partners nto account. However, sexual behavor dd not explan the assocatons of rsk wth educaton and ncome. Adjustment for the HPV test results ( #3 n Table ) explaned most of the rsk of CIN assocated wth ncreasng lfetme numbers of sex partners and correlates of sexual behavor, as well as the assocatons of rsk wth educaton and ncome. Adjustment for HPV status generally resulted n very modest resdual assocatons that were not statstcally sgnfcant. As a notable excepton, adjustment for HPV nfecton uncovered a moderately strong assocaton between ncreasng party and rsk of CIN. Among HPV-postve women, there was no evdence that sexual actvty, educaton, ncome, or smokng remaned assocated wth rsk of CIN (Table 3). Some persstently elevated s were seen n the HPV-negatve stratum, most notably for sexual behavor and low ncome. Increasng party appeared to be a rsk factor for CIN n both the HPVnegatve and HPV-postve groups. Ancllary Analyses We repeated the analyses shown n Tables and 3, ncludng only the 309 case wthout a documented hstory of CIN. The results were smlar; n fact, the assocatons wth sexual behavor and low ncome were even better explaned by HPV nfecton when usng the more restrctve case defnton. For example, restrcted to case wth no hstory of CIN, an age-adjusted of.6 for an ncome of less than $0000 (compared wth $50000 or more) became. when HPV test results were taken nto account. We also repeated the man analyses restrctng the case group to the most strngent case defnton possble, namely, Table. Relatve rsks of CIN accordng to HPV test results* HPV test resultt No. Case % Control No. % 95% CI Negatve Types 6,, 4, or other or unknown type Types 3, 33, 35, 39, 45, 5. or 5 Types 6 or 8 Total *CN ncluded a cytologc dagnoss of condylomatous atypa or CIN 3. thpv typng results grouped herarchcally accordng to rsk when multple types were present: 6 group > 3 group > 6 group. 960 ARTICLES Journal of the Natonal Cancer Insttute, Vol. 85, No., June 6, 993

4 Table. Estmated relatve rsks of CIN for the establshed epdemologc rsk factors, showng the effects of adjustment for HPV test results Rsk factor case control #* #t #3 (95% CI)+ Lfetme sex partners Age at frst ntercourse Educaton Postgraduate College graduate Some college Hgh school graduate <Hgh school graduate (0.5-.). (0.6-.).8 (-3.4).8 (-3.3). (0.6-.0) (0.6-.8).4 (0.8-.5) 0.8 (0.4-.6) (0.5-.9) 0.8 (0.4-.6). (0.5-.8) Famly ncome per annum >$50000 $ $ $ <$0000 Smokng Never Past Current Oral contraceptve use Never used Past use Tmng of use unknown Current use Party 0 lve brths % _ (0.4-.4) (0.6-.8).4 (0.8-.4).6 (0.9-.8) (0.6-.6). (0.8-.8).5 (0.8-.7).4 (0.7-.7).3 (0.6-.8).4 (0.8-.4).9 (.3.3).5 (.3-4.8).8 (.3-5.9) * estmates adjusted for age n sextles. t estmates adjusted for age n sextles, lfetme number of sex partners, age at frst ntercourse, educaton, ncome, smokng, oral contraceptve use, and number of lve brths, categorzed as shown n the table. t estmates adjusted for age n sextles and HPV test results categorzed as shown n Table. 95% confdence ntervals exclude, analogous to statstcal sgnfcance at the P<.05 level. newly dagnosed, bopsy-confrmed CIN excludng condylomatous atypa. Despte the ncreased mprecson of the estmates because of smaller numbers, the results were fundamentally the same as those n Tables and 3. In addton, we analyzed the data pertanng to condylomatous atypa, CIN, and CIN -3 as three dstnct case groups (Table 4). All three dsease categores were strongly assocated wth both ncreasng lfetme numbers of sex partners and HPV postvty, partcularly postvty to types 6 and 8. Multvarate analyses ncludng both lfetme numbers of sex partners and HPV test results ponted to HPV nfecton as the prmary rsk factor for newly dagnosed condylomatous atypa, CIN, and CIN -3, although the analyss of CIN -3 was mprecse because of small numbers. In a smlar grade-specfc analyss (separatng condylomatous atypa, CIN, and CIN -3), HPV-postve current smokers were almost three tmes as lkely to have CIN -3 than HPV-postve women who had never smoked ( =.7; 95% CI =.6.5). Proporton of Cases Attrbutable to HPV Infecton We estmated the proporton of cases attrbutable to HPV nfecton, for all grades of dsease combned and for the three grade categores separately. For the entre case group, the attrbutable proporton was 0.76, ndcatng that 76% of all cases of CIN n the study populaton could be attrbuted to HPV nfecton. The comparable fgures for condylomatous atypa, CIN, and CIN -3 were 0.70, 0.90, and 0.88, respectvely. The results of cytologc revew tended to ncrease the proporton of cases of condylomatous atypa attrbutable to HPV nfecton, because HPV prevalence ncreased wth Journal of the Natonal Cancer Insttute, Vol. 85, No., June 6, 993 ARTICLES 96

5 Table 3. Estmated relatve rsks of CIN for selected epdemologc rsk factors n HPV-negatve and HPV-postve women Rsk factor Lfetme number of sex partners Age at frst ntercourse Educaton Postgraduate College graduate Some college Hgh school graduate <Hgh school graduate Famly ncome per annum 3"$50000 $ $ $ <$0000 Smokng Never Past Current Oral contraceptve use Never used Past use Tmng of use unknown Current use Party 0 lve brths 3 4+ * estmates adjusted for age n sextles. n HPVnegatve women (95% CI)* -.4 ( ). (0.6-.6).5 (.-5.4).0 ( ). (0.6-.4) 0.8 (0.4-.7).4 (.5.) 0.8 (0.3-.0).4 (0.7-3.) 0.9 (0.4-.).7 (0.6-5.) (0.4-.5). (0.6-.6).4 (.-5.).4 (.5.5) (0.5-.7).4 (0.7-.5).0 (0.94.6).4 (-5.8). ( ).8 ( ). (0.94.9) 3.5 (.4-8.6).9 (.8.0) n HPVpostve women (95% CD* (0..4) 0.6 (0.-.4) 0.8 (0.-3.) ( ) ( ).3 ( ) 0.9 (0.3-.) 0.8 (0.-3.0) 0.6 (0.-.8) 0.6 (0.-.0) 0.8 (0.-.9) 0.4 (0..) 0.8 (0.3-.4) 0.6 (0.-.5) 0.8 (0.3-.0). (0.5-.7). (0.7-.) 0.7 (0.-.7) 0.5 (0..8) 0.5 (0..0). (0.5-.).7 ( ).5 (0.6-4.).7 ( ) ncreasng certanty of cytopathologc confrmaton. Specfcally, cases of condylomatous atypa judged on revew to be negatve, equvocal, or defnte were 5%, 70%, and 9% HPV postve, respectvely. Thus, the total proporton of cases attrbutable to HPV nfecton (as well as the estmates relatng HPV to rsk of CIN) would be even greater f cytopathologc msclassfcaton of condylomatous atypa (false-postve borderlne cases) were elmnated. Dscusson The assocaton between HPV nfecton and CIN now satsfes all of the accepted crtera for assessng causalty from epdemologc studes (0,7). These crtera nclude strength and consstency of the epdemologc assocaton, tme sequence, specfcty of the assocaton, and coherence wth other known facts (bologc and epdemologc) about the rsk factor and dsease. Accordngly, the relatonshp between HPV and CIN s strong, and the assocaton has been verfed n numerous case-control studes conducted throughout the world (). The cohort studes wth longest follow-up show an elevated rsk of development of apparently new CIN wthn the frst few years (3 and Schffman MH, Manos MM, Hoover RN, et al.: unpublshed data). HPV nfecton appears to cause a specfc set of mucocutaneous tumors, ncludng anogental tumors and, to a lesser extent, some subtypes of head and neck neoplasa (4). Bologc plausblty has been proven by abundant expermental data demonstratng the oncogenc potental of HPV (5,6). Wth the present nvestgaton, a causal role for HPV nfecton was shown to be thoroughly consstent wth longstandng epdemologc knowledge of cervcal neoplasa. In partcular, HPV nfecton was shown to explan the sexual rsk factors for CIN, as well as most other behavoral and demographc determnants. As the one excepton, ncreasng party appeared to be an HPV-ndependent rsk factor for CIN, a fndng that s consstent wth some prevous epdemologc work (7) but requres confrmaton n addtonal studes. A major dffculty n all studes of CIN s precse defnton of the dsease. We examned rsk n relaton to the severty (grade) of CIN, the relablty of the cytologc dagnoss, the correlaton of cytologc wth hstopathologc dagnoss, and our best estmate of the duraton of the condton (newly dagnosed versus prevously dagnosed dsease). HPV nfecton was strongly assocated wth the entre spectrum of CIN and largely accounted for the establshed behavoral rsk factors for CIN. Some rsk factors appeared to persst partally, especally among HPV-negatve women, suggestng that a mnorty of CIN cases arse wthout HPV, perhaps wth a dstnct epdemology nvolvng other venereal agents. However, the resdual assocatons could also result n part from error n the HPV measurements (resdual confoundng). Despte the mproved HPV test used n ths nvestgaton, HPV measurement error remans because of problems wth the collecton of optmal cervcal specmens, dffcultes n testng for such a large famly of ncompletely characterzed vruses, and the nherent lmtatons of a sngle HPV test n defnng nfecton (8). If HPV causes most CIN, nfecton (even as assessed by the one-tme detecton of HPV DNA) s stll much more common than the prevalence of CIN (5). The remanng etologc questons relate, therefore, to cofactors for HPV persstence and progresson to CIN among women exposed to HPV nfecton. In ths context, low- and hgh-grade CIN must be consdered separately. Wth the potental excepton of party, we uncovered no cofactors for low-grade CIN among HPV-postve women. It s possble that all gental HPV nfectons mght be suffcent to cause small and transent foc of low-grade CIN that dsappear under mmune survellance nfluenced by both envronmental and host factors. Transent nfectons,.e., transent foc of CIN, could easly be mssed n the 96 ARTICLES Journal of the Natonal Cancer Insttute, Vol. 85, No., June 6, 993

6 Table 4. Estmated relatve rsks of condylomatous atypa, CIN, and CIN -3 assocated wth lfetme numbers of sex partners and HPV test results Condylomatous atypa CIN CIN -3 Rsk factor control case * (95% CI)t case * (95% Cl)t case * (95% CI)t Lfetme number of sex partners HPV test result Negatve Types 6,, 4, or other or unknown types Types 3, 33, 35, 39, 45. 5, or 5 Types 6 or *.8* 7.7* 6.0* 37.0* (0.6-.7).6 (-.6) 6.4 ( ) (-4) 33.0 ( ) * 4.7* * 0.0* 40.0*.5 ( ).7 ( ) 4.0 ( ) 30.0 ( ) 00.0 ( ) * 7.5* 7.0* 33.0* 90.0*.3 ( ). ( ) 0.0 ( ).0 ( ) 80.0 ( ) estmates adjusted for age n tertles. t estmates mutually adjusted for age, HPV test results, and lfetme numbers of sex partners. *95% confdence ntervals exclude, analogous to statstcal sgnfcance at the P<.05 level. References examnaton of annual or less frequent cervcal smears; thus, the determnants of HPV persstence mght be the rsk factors for the clncal dagnoss of low-grade CIN. Future (/) HAIS RW, BRINTON LA, COWDELL RH, ET AL: Characterstcs of epdemologc studes of low-grade CIN mght, therefore, women wth dysplasa or carcnoma n stu of the cervx uter. Br J focus on evolvng measurements of HPV mmunty, n the Cancer 4: , 980 () BRINTON LA, HOOVER RN: Epdemology of gynecologc cancers. In context of frequently repeated cytologc samplng and HPV Prncples and Practce of Gynecologc Oncology (Hoskns WJ, Perez testng. CA, Young RC, eds). Phladelpha: Lppncott, 99, pp 3-6 Our ablty to study the development of hgh-grade CIN (3) ZUR HAUSEN H: Papllomavruses n human cancers. Mol Carcnog :47-50, 988 was lmted by small numbers of cases. Nevertheless, the (4) MUNOZ N, BOSCH X, KALDOR JM: Does human papllomavrus cause results of the present nvestgaton confrm that the cervcal cancer? The state of the epdemologcal evdence. Br J Cancer development of hgh-grade CIN s lnked to certan types of 57:5, 988 (5) SCHIFFMAN MH: Recent progress n defnng the epdemology of HPV. In addton, the data suggest that smokng mght be a human papllomavrus nfecton and cervcal neoplasa. J Natl Cancer cofactor for the development of hgh-grade CIN, concordant Insl 84: , 99 wth prevous studes of cervcal cancer and hgh-grade CIN (6) REEVES WC, BRINTON LA, GARCIA M, ET AL: Human papllomavrus nfecton and cervcal cancer n Latn Amerca. N Engl J Med (). Fnally, the data do not rule out the exstence of another 30:437-44, 989 sexually transmtted agent as a cofactor n the development (7) VILLA LL, FRANCO EL: Epdemologc correlates of cervcal neoplasa of hgh-grade CIN (). It s worth consderng that the and rsk of human papllomavrus nfecton n asymptomatc women n Brazl. J Nat] Cancer Inst 8:33-340, 989 cofactors for CIN progresson could be dfferent from the (8) KJAER SK, ENOHOLM G, THSEN C, ET AL: Rsk factors for cervcal establshed epdemologc rsk factors for cervcal neoplasa, human papllomavrus and herpes smplex vrus nfectons n Greenland whch are manly explaned by HPV. and Denmark: A populaton-based study. Am J Epdemol 3:669-68, 990 In addton to searchng for HPV cofactors, t wll be (9) FRANCO EL: The sexually transmtted dsease model for cervcal cancer: mportant to examne the geographc dversty of HPV and Incoherent epdemologc fndngs and the role of msclassfcaton of to learn whether the same types of HPV nfecton are human papllomavrus nfecton. Epdemology :98-06, 99 (0) LEY C, BAUER HM, REJNOOLD A, ET AL: Determnants of gental responsble for most cervcal neoplasa worldwde. Such human papllomavrus nfecton n young women. J Natl Cancer Inst nternatonal efforts are already under way. It also wll be 83: , 99 mportant to study the cases of cervcal neoplasa not related (//) Natonal Cancer Insttute Workshop: The 988 Bethesda System for reportng cervcal/vagnal cytologcal dagnoses. JAMA 6:93934, to HPV nfecton. Defnng such cases convncngly n 989 epdemologc projects wll requre near-perfect HPV tests () VERMUND SH, ScHrFFMAN MH, GOLDBERO GL, ET AL: Molecular that may not yet exst. However, the exstence of HPVnegatve cervcal neoplasa has been supported by recent 60: , 989 dagnoss of gental human papllomavrus nfecton: Comparson of two methods used to collect exfolated cervcal cells. Am J Obstet Gynecol molecular bologc (6) and epdemologc (9) studes. A (3) SCHIFFMAN MH, BAUER HM, LORINCZ AT, ET AL: Comparson of crucal queston s whether HPV-negatve CIN gves rse to Southern blot hybrdzaton and polymerase chan reacton methods for the detecton of human papllomavrus DNA. 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7 vruses. Cancer Cells 7:09-4, 989 (6) TING Y, MANOS MM: Dejecton and typng of gental human papllomavruses. In PCR Protocols: A Gude to Methods and Applcatons (Inns M, Gelfand D, Snnsky J, et al, eds). San Dego: Academc Press, 990, pp (7) BAUER HM, GREER CE, MANOS MM: Determnaton of gental human papllomavrus nfecton usng PCR. In Dagnostc Molecular Pathology: A Practcal Approach (Herrngton CS, McGee JO, eds). Oxford: Oxford Unv Press, 99, pp 35 (8) LORINCZ AT, REID R, JENSON AB, ET AL: Human papllomavrus nfecton of the cervx: Relatve rsk assocatons of ffteen common anogental types. Obstet Gynecol 79:38-337, 99 (9) SCHATZKIN A, FREEDMAN L, SCHIFFMAN MH, ET AI.: Valdaton of ntermedate end ponts n cancer research. J Natl Cancer Inst 8:746-75, 990 (0) HILL AB: Envronment and dsease: Assocaton or causaton? Proc R Soc Med 58:95-300, 965 () SUSSER M: Causal Thnkng n the Health Scences: Concepts and Strateges n Epdemology. New York: Oxford Unv Press, 973 () MUNOZ N: HPV and cervcal cancer Revew of case-control and cohort studes. In Epdemology of Cervcal Cancer and Human Papllomavruses (Munoz N, Bosch FX, Shah KV, et al, eds). ARC Scentfc Publ No. 9, Lyon: IARC, 99, pp 56 (3) KOUTSKY LA, HOLMES KK, CRITCHLOW CW, ET AL. A cohort study of the rsk of cervcal ntraepthelal neoplasa grade or 3 n relaton to papllomavrus nfecton. N Engl J Med 37:7-78, 99 (4) PALEFSKY J, BERUNE K, SEID MM, ET AL: Heterogenety of HPV types detected n nvasve oral cancer. J Dent Res 70s:73, 99 (5) MCCANCE DJ, KOPAN R, FUCHS E, ET AL: Human papllomavrus type 6 alters human epthelal cell dfferentaton n vtro. Proc Natl Acad Sc U S A 85: , 988 (6) HOWLEY PM: Role of the human papllomavruses n human cancer. Cancer Res 5:509s-50s, 99 (7) BRINTON LA, REEVES WC, BRENES MM, ET AL: Party as a nsk factor for cervcal cancer. Am J Epdemol 30:486-^96, 989 (8) SCHIFFMAN MH: Valdaton of hybrdzaton assays: Correlaton of flter n stu, dot blot, and PCR wth Southern blot. In Epdemology of Cervcal Cancer and Human Papllomavruses (Munoz N, Bosch FX, Shah KV, et al, eds). IARC Scentfc Publ No. 9. Lyon: IARC, 99, pp (9) HIOGINS GD, DAVY M, RODER D, ET AL: Increased age and mortalty assocated wth cervcal carcnomas negatve for human papllomavrus RNA. Lancet 338:90-93, 99 Notes Afflatons of authors: M. H. Schffman, R. N. Hoover, S. Wacholder, Epdemology and Bostatstcs Program, Dvson of Cancer Etology, Natonal Cancer Insttute, Bethesda, Md. H. M. Bauer, M. M. Manos, Cetus Corporaton, Emeryvlle, Calf. A. G. Glass, B. B. Rush, D. R. Scott, Kaser Permanente, Portland, Ore. D. M. Cadell, C. K. Stanton, Westat, Inc., Rockvlle, Md. M. E. Sherman, R. J. Kurman, Department of Pathology, The Johns Hopkns Medcal Insttutons, Baltmore, Md. Present address: M. M. Manos, The Johns Hopkns School of Publc Health, Baltmore. Correspondence to: Mark H. Schffman, M.D., M.P.H., Natonal Insttutes of Health, Executve Plaza North, Rm. 443, Bethesda, MD 089. We acknowledge the substantal ntellectual contrbutons to ths work by Julan Peto, Valere Beral, Aleen Lazns, and Patrca Hartge. The bologc specmens were obtaned by the physcans and nurse practtoners of the Department of Obstetrcs and Gynecology and the Department of Internal Medcne, Kaser Permanente, Northwest Regon. Patrca Lawler coordnated specmen collecton n the clncs. The ntervewers were Lelan Wlson, Conne Blalock, Peggy Brune, Rose Brght, and Jean Jennngs. Davd Tabor and Davd Garner of Westat, Inc., and Jule Buckland of Informaton Management Systems provded computer programmng support. Manuscrpt receved November 8, 99; revsed March, 993; accepted March 7, 993. How n the world do you fnd out about the earth? The Earth Scence Data Drectory (ESDD) s compled and produced by the US Geologcal Survey, an agency of the Department of the Interor and the Federal Government's largest earth-scence research agency. References n the ESDD nclude nformaton about Data bases concerned wth the geologc, hydrologc, cartographc, and bologc scences Data that supports the protecton and management of natural resources Geographc, socologc, economc, and demographc data sets To secure nformaton about becomng an ESDD user, wrte or call ESDD Project Manager U.S. Geologcal Survey 80 Natonal Center Reston, Vrgna 09 (703) or FTS ARTICLES Journal of the Natonal Cancer Insttute, Vol. 85, No.. June 6, 993