GYNECOLOGIC
ONCOLOGY
46, 6-12 (1992)
Is Human Papillomavirus Associated with Cervical Neoplasia in the Elderly?’ JEANNE MANDELBLATT, STEPHEN MATSEOANE, *Department Pathology,
M.D., M.D.,$
M.P.H.
,**’ RALPH RICHART, M.D.,? LOUYSTHOMAS, M.D.,$ PREM CHAUHAN, PETER KANETSKY, M.P.H.,* MARYANN TRAXLER, M.D.,11 AND PATRICIA LAKIN,
of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021; TDepartment Columbia Presbyterian Hospital, New York, New York; and Departments of $Pathology, OObstetrics and Gynecology, IIMedicine, and #Nursing, Harlem Hospital Center, New York, New York
M.D.*
N.P.# of
Received December 13. 1991
There have been no studiesin the United States of human papillomavirus (HPV) in elderly women. This paper presents cross-sectionaldata on HPV and cervical neoplasiaamong 232 womenage65 or more. HPV deoxyribonucleicacid (DNA) testing wasperformedusinga modifieddot-blot hybridization technique. The prevalenceof HPV DNA positivity was3.5% (95%confidence interval (CI) 0.9%, 6.0%). There were six casesof histologic cervical neoplasia.The crude odds ratio for cervical neoplasia among HPV DNA positiveswas 18.3 (95% CI 2.8, 120.3). The adjustedodds,controlling for age, prior screeninghistory, current sexualactivity, and past contraception use, were 12.2 (95% CI 1.2, 122.9). Ever having had a Papanicolaousmearwas protective, and there was a trend for the odds of having neoplasiato increasewith age. Additional studieswith larger samplesof elderly womenare needed.If confirmed, the resultssuggestthat, independent of past screening, HPV may increase the risk of having cervical neoplasiafor elderly women. 0 1992 Academic Press, Inc.
INTRODUCTION
Human papillomavirus (HPV) has been detected in 17 to 95% of cervical cancers, using a variety of techniques [l-6]. However, evidence of HPV infection has also been noted in 1.3 to 32% of women with normal Papanicolaou smears [2,7-121. Thus, while HPV has been shown to produce in vitro malignant human cell transformation and
MATERIAL
Women with a birth year of 1925 or earlier (hereinafter referred to as “elderly”) who were scheduled for routine appointments to the medical clinic at Harlem Hospital Center between May 15, 1990, and November 15, 1990, were potentially eligible for the study. The clinic is a New York City municipal hospital outpatient clinic providing comprehensive care for over 5000 patients per year with chronic medical conditions. Care is provided regardless of ability to pay. More than 90% of the patients are Black. 6
$4.00
0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Study Setting and Population
’ This work was supported in part by National Institute on Aging Academic Award KOS AGO0471, National Cancer Institute Grant R03 CA51614-01, Enid Ancell Clinical Studies Award, Memorial SloanKettering Cancer Center, and the Society of Memorial Sloan-Kettering Cancer Center (Dr. Mandelblatt). * To whom correspondence and reprint requests should be addressed at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 60, New York, NY 10021.
0090-8258192 Copyright
immortalization [13,14], infection with the virus may not be a sufficient condition for the development of cervical neoplasia [ 151. Possible copromoters include age, immune status, or failure of intracellular control mechanisms [1,2,7,16-181. There are no United States studies of the prevalence or behavior of HPV in cervical lesions in elderly women. The only study of HPV in older women in the United States is a description of HPV in hysterectomy specimens among cytologically normal pre- and postmenopausal women with average ages of 41 and 61 years, respectively, whose surgery was for nonmalignant conditions [19]. This paper presents the results of a cross-sectional study of HPV among Black women, age 6.5 or more, of low socioeconomic class, who participated in a cancer screening program in an urban public hospital. The study describes the HPV types detected in this cohort using a modified dot-blot hybridization technique. The prevalence of HPV deoxyribonucleic acid (DNA) positivity is also estimated and associations between HPV DNA positivity and cervical neoplasia are explored.
PAPILLOMAVIRUS
AND CERVICAL
TABLE 1 Categories for Papanicolaou Smear Reports” Category 1. Negative 2. Benign atypia 3. Significant atypia
4. Suspicious for malignacyh 5. Positive for malignancyh
Comments Includes inflammation, atrophy, and infection Includes cellular changes not associated with inflammatory or reparative processes, but not sufficient quantitatively to be classed as intraepithelial neoplasia Includes intraepithelial neoplasia grades 1 and 2 Includes intraepithelial neoplasia grade 3 and invasive carcinoma
’ Categories determined independently of cellular changes associated with productive HPV infection. * Hereafter defined as “abnormal” or “positive” Papanicolaou smears.
Elderly women who attended the clinic were invited to participate in a breast and cervical cancer screening program approved by the hospital Institutional Review Board. Four hundred ninety-one of the 690 (71%) women who attended consented. Two hundred fifty-nine women were subsequently excluded from the study, including 169 with a history of a hysterectomy and no cervix on examination, 41 who did not have a pelvic examination and Papanicolaou smear (15 gave a history of a smear in the past year and 26 refused), 1 diagnosed with ovarian cancer, and 48 who did not have specimens obtained for HPV DNA testing (4 leaked in transit and 44 could not be obtained due to vaginal stenosis, vaginal friability and/or bleeding, or examiner omission). None of the patients in the latter category had gross cervical lesions. The remaining 232 elderly women with intact cervices and HPV DNA results constitute the final study group.
NEOPLASIA
IN THE ELDERLY
7
sults. An independent pathologist also performed a blind review of all smears interpreted as suspicious or positive for malignancy and a 10% random sample of the remaining slides. A cervicovaginal lavage was performed after the Papanicolaou smear to obtain exfoliated cellular material for HPV DNA testing [20]. The cervix was lavaged with 10 ml of a 0.9% saline solution and the washings were aspirated from the posterior vaginal fornix with a sterile syringe [21]. The aspirate was placed in a sterile tube and centrifuged, and the cellular pellet was suspended in 1 ml of transport media, according to the manufacturer’s instructions (Life Technologies, Inc., Gaithersburg, MD). The samples were tested for the presence and types of HPV DNA with the ViraPap human papillomavirus DNA detection kit (Molecular Diagnostics Division, Life Technologies, Inc., 1989). This is a modified dot-blot hybridization process developed for use as a screening assay to detect HPV DNA types 6, 11, 16, 18, 31, 33, and 35. If the sample was positive, it was analyzed with the ViraType papillomavirus DNA detection kit (Molecular Diagnostics Division, Life Technologies, Inc., 1989). This test uses “‘P-labeled RNA probes to detect HPV DNA types 6/11, 16/18, and 31/33/35. Negative control probes were used to minimize false-positive results. A repeat sample was requested when the results were equivocal. Testing was performed without knowledge of the patient’s clinical or cytologic status. Histologic specimens were obtained by a trained gynecologic oncologist and included colposcopically directed cervical biopsies and endocervical curettage. Interpretation was completed by pathologists unaware of the HPV status. Grading of lesions was done according to the World Health Organization (WHO) criteria for histologic typing of female genital cancers [22]. Interpretations were independent of diagnoses consistent with productive HPV infection, such as koilocytosis and condyloma acuminata.
Materials
Papanicolaou smears were obtained using a narrow cotton-tipped, saline-moistened applicator (calgi-swab) for sampling of the endocervix and an Ayre’s spatula for sampling the endo- and exocervix. Specimens were placed on glass slides and fixed immediately with an aerosol fixative. Several measures were taken to ensure that smears contained sufficient cellular material for cytologic assessment, including staff training, direct observation, and laboratory feedback. Table 1 summarizes the categories used to describe the cytologic findings. Interpretation also included a comment on smear adequacy and an independent assessment of cellular changes characteristic of productive HPV infection [15]. All cytologic interpretations were conducted without knowledge of the HPV DNA hybridization re-
Definitions of Exposure and DiseaseStatus
Exposure to HPV was defined by the HPV DNA test results. Women were identified as being HPV DNA positive if they had results positive for HPV DNA types of high and intermediate oncogenic risk (types 16/18, 31/33/35), low risk (types 6/11), and other (positive, but not reacting with the preceding probes). Women were considered negative if no HPV DNA was detected or if equivocal results were obtained [5,15,23]. Disease status was defined from two perspectives: cytologic and histopathological. For the Papanicolaou smears, disease was defined as positive if the interpretation was suspicious or positive for neoplasia, and negative in all other instances, including negative, benign atypia, or significant atypia reports. If more than one
8
MANDELBLA’TT
smear was obtained, the patient was classified according to the most abnormal [24]. Women were considered positive histologically if they had a pathologic diagnosis of cervical intraepithelial neoplasia (CIN) grade 1 or more. Women with all other diagnoses, including atrophy, metaplasia, koilocytosis, and condyloma, were considered negative.
ET AL.
with equivocal results, 3 had repeat tests (2 were negative and 1 was positive) and 1 refused. Thus, 8 women were classified as HPV DNA positive and 224 as negative, yielding an overall HPV DNA prevalence of 3.5% (8/232) (95% CI 0.9%, 6.0%). The types of HPV DNA detected are summarized in Table 2. The majority are types 31/33/35 (50%), followed by “other” types (37.5%). Only one was type 16/18 (12.5%), and none was 6/11.
Data Analysis
Data were analyzed using the Statistical Program for the Social Sciences-X [25]. Estimated point prevalences of HPV DNA positivity were calculated and 95% confidence intervals (95% CI) were computed [26]. The magnitude of associations was evaluated using odds ratios (OR) and 95% CI [27]. The x2 test with a continuity correction, or, when necessary, a Fisher’s exact test, was used to assess the statistical significance of associations [26]. The kappa statistic [28] with 95% confidence limits [26] was used to assess the interrater reliability of the Papanicolaou smear readings. Adjusted odds ratios derived in a logistic regression model were used to estimate the odds of histologically diagnosed cervical neoplasia, controlling for the simultaneous effects of HPV status and potentially confounding variables [29]. Factors considered potential confounders were age, ever having a prior smear, number of lifetime smears, current sexual activity, past use of any contraceptive technique, parity, number of sexual partners, sexually transmitted disease history, and hysterectomy status [30]. Interactions were not assessed due to small numbers of cases. RESULTS Characteristics
of the Study Group
The average age of the 232 women included in the final study group was 75.2 years (range, 65-96 years), 99.5% were Black, 48% had completed no more than an eighthgrade education, and 90% reported an income of less than $9000 per year. Eighty-four percent of the women gave a history of having at least one prior Papanicolaou smear, only 13% reported having had seven or more tests, and the median time since the last smear was 2 years (range, 1 to 39 years). Forty-four women (19%) gave a history of a hysterectomy in the past; these represented subtotal hysterectomies performed prior to 1960. None of the women reported a history of cervical cancer or hysterectomy for malignant conditions. Only 13 (6%) reported symptoms; 94% were asymptomatic. HPV DNA
Results
Seven women were HPV DNA positive, 4 had equivocal results, and 221 were negative. Among the women
Cytologic
Findings
Papanicolaou smear interpretations. All smears were considered adequate for cytologic assessment. The interrater reliability of the Papanicolaou smear interpretations, as determined by the kappa statistic, was 0.75 (95% CI 0.5, 1.0). There were three smears read as negative by the primary cytologist and positive by the second cytologist; these three women were also HPV DNA positive. Thus, it was decided to use the interpretations of the primary cytologist for the analyses, since this would bias the results of any association between HPV DNA positivity and positive smears toward the null. Overall, 6 women (2.9%) had smears that were interpreted as suspicious or positive for malignancy and 226 women had negative results. HPV DNA prevalence by cytologic diagnosis. HPV DNA was detected in 3.1% (7/226) (95% CI 0.6%, 5.6%) of women with negative Papanicolaou smears and 16.7% (l/6) (95% CI 0, 54.8%) of women with positive smears. This difference did not achieve statistical significance in this small sample (P = 0.19, Fisher’s exact test, twotailed). Association between HPV DNA positivity and cytologic neoplasia. Among HPV DNA-positive women, the odds
of having an abnormal Papanicolaou smear appears elevated, compared to HPV DNA-negative women (OR = 6.3); however, the confidence intervals were very wide (95% CI 0.64-60.9) and included the possibility that there was no increased risk of an abnormal smear among HPV DNA-positive women. Histopathologic
Findings
Diagnoses. Fourteen women were referred for colposcopy: 11 complied and 3 did not (Table 2). These latter 3 women were considered negative in subsequent analyses. Six women were diagnosed with cervical neoplasia-1 with intraepithelial disease grade 1, 1 with CIN 3/CIS, and 4 with invasive disease. HPV DNA prevalence by histologic diagnosis. The prevalence of HPV DNA positivity was significantly higher in women with histologic cervical neoplasia (33.3%, 2/6) (95% CI 0, 79.4%) than in women without
PAPILLOMAVIRUS
AND CERVICAL
NEOPLASIA
9
IN THE ELDERLY
TABLE 2 Final Histopathologic Diagnoses (Years)
Pap smear report
Indication for colposcopy”
Final pathology diagnosis
B.A. R.F. B.E. R.P. H.B. J.S.
72 67 70 75 67 93
Significant atypia Negative Significant atypia Suspicious Negative Positive
16118 Other’ Negative Negative Other’ Negative
HPV HPV P.E. PAP HPV PAP
Negative
31133135
HPV
80 78 74 78 67
Positive Positive Positive Positive Negative
31/33/35 Negative Negative Negative 31/33/35
PAP PAP PAP PAP HPV
R.G.
66
Benign atypia
31/33/35
HPV
C.B.
83
Negative
Othef
HPV
-b CIN, grade 1 -* Normal Atrophic changes ICC, stage unknown Squamous metaplasia ICC, stage la CIN, grade 3/CIS ICC, stage lb ICC, stabe 3b KoiIocytosis/ condyloma Koilocytosis/ condyloma -b
W.P.
67
M.C. A.S. A.K. B.H. M.T.
Age Patient
HPV DNA results
’ Indications for referral included positive HPV DNA results (HPV), abnormal physical examination (P.E), or abnormal pap smear (PAP). ’ Work-up not completed; one patient died and two refused; all three were considered “negative” in subsequentanalyses. ’ Positive for types other than 6/11, 16/18, 31/33/35. ’ Diagnostic work-up completed but staging evaluation and treatment were refused. ’ Symptomatic with vaginal bleeding. Note. CIN, cervical intraepithelial neoplasia; CIS, Carcinoma in situ; ICC, invasive cervical cancer.
neoplasia (2.7%, 6/226) (95% CI 0.34%, 4.97%) (P = 0.015, Fisher’s exact test, two-tailed). Association between HPV DNA posit&&y and histologic diagnoses. The crude odds ratio for cervical neoplasia
among HPV DNA-positive women was 18.3. In this small sample, the confidence interval was very wide (95% CI 2.8, 120.3); however, the lower bound indicates a moderate effect. To minimize the prevalence bias inherent in this cross-sectional study, relative risk was also analyzed separately for the 211 women who reported having one or more prior negative Papanicolaou smears at any time in the past. When this was done, the association between neoplasia and HPV remained significant (P = 0.003, Fisher’s exact test, two-tailed). The odds ratio was somewhat higher (OR = 50.6) in this “incident” group than in the entire group, but the CI was extremely wide (95% CI 5.6, 456).
The adjusted odds of neoplasia among HPV DNApositive women was 12.2 (95% CI 1.2, 122.9), controlling for age, ever having a Papanicolaou smear, current sexual activity, and past use of any birth control method (Table 3). Past use of any method to prevent pregnancy was also independently associated with cervical neoplasia (OR = 21.8, 95% CI 1.7-280.3). Ever having a Papanicolaou smear was associated with lower odds of having neoplasia,
although the confidence interval included one (P = 0.053), and there was a trend for increasing age to increase the odds of having neoplasia independent of the remaining variables, but in this small sample this did not achieve statistical significance (P = 0.069). DISCUSSION This study, although small, is the first investigation of the types, prevalence, and behavior of cervical HPV DNA in an elderly United States population group. The majority of HPV types detected in this cohort were those considered to have intermediate and high oncogenic potential [5,15,23]. The ranges of prevalence rates of HPV DNA observed among older women in other settings are comparable to those in this study. In Germany, HPV DNA was detected in 3.5% (95% CI 2%, 5%) of cytologically normal elderly women, and in 30% (95% CI 16%, 45%) with cytologic neoplasia [7]. HPV DNA has also been detected in 12% (95% CI l%, 22%) of normal cervices removed during hysterectomy from postmenopausal women [19]. The magnitude of the odds of cervical neoplasia in HPV DNA positive elderly women noted in this investigation is also similar to that noted by other researchers in other countries [2,7]. However, it should
10
MANDELBLA’IT
ET AL.
TABLE 3 Adjusted Odds of Cervical Neuplasia for Selected Factof Variables Age (in l-year intervals) HPV DNA Negative Positive Currently sexually active No Yes Any past contraceptive used No Yes Past screening’ Never Ever
Adjusted odds ratiob 1.14
95% Confidence interval 0.99-1.3
P
value
0.069
1.00” 12.2
1.2-122.9
0.034
1.00 6.1
0.6-62.4
0.128
1.00 21.8
1.7-280.3
0.018
1.00 0.08
0.01-1.03
0.053
a For total n = 232 women. ’ Each variable is adjusted for the remaining variables in a logistic regression model. Parity, sexual partners, number of Papanicolaou smears, sexually transmitted disease (STD) history, and hysterectomy did not enter into the final model. Group means were used for missing values for continuous variables: parity (3 cases missing), number of sexual partners (11 cases missing), and number of smears (55 cases missing). Sexual activity data were missing for 4 cases which were classed as “no.” All data were complete for the categorical variables of STD history, hysterectomy, contraceptive use, and past screening. ’ Referent category for each risk factor. d Includes any technique used in the woman’s lifetime to prevent pregnancy, such as condoms, diaphragm, spermicidal agents, rhythm, and withdrawal. Oral contraceptives or IUDs were only used by two women. ’ Ever screened is defined as having a history of any prior Papanicolaou smear.
be noted that differences across studies with respect to race, setting, selection criteria, control of confounding variables, HPV sampling and testing techniques and types, criteria for the diagnosis of neoplasia, and categorization of koilocytotic changes make direct comparisons difficult. There are several caveats that should be considered when interpreting the findings of this study, including sample size and power, classification errors, and the use of a cross-sectional design. The study had a power of 80% to rule out zero overall prevalence of HPV DNA and to detect a relative risk of neoplasia associated with HPV DNA of 12 or greater; however, due to the low number of diseased women, power was considerably less for detection of increases in risk among neoplasia subcategories. For example, if risk is examined separately for low-grade neoplasia (CIN 1 or 2), the association between HPV and neoplasia remains significant (P = 0.03, Fisher’s exact test, two-tailed); for high grade lesions (CIN 3/CIS or more severe), while HPV was found in 14.3% of cases and only 1.8% of controls, the difference is not statistically significant in this small sample (P = 0.14, Fisher’s exact test, two-tailed). Thus, it will be important to replicate this study in a sample with a larger number of cases of cervical neoplasia. Accurate classification of HPV DNA and cervical lesions remains problematic for studies such as this [30,31].
We have taken several measures to avoid bias associated with misclassification error, including blind determinations of exposure and disease status, assessment of HPV cellular changes independent of cytologic and histologic interpretation of malignancy, and, when necessary, making assumptions which would bias results towards the null. However, exposure misclassification could have arisen using the dot-blot hybridization as a result of sampling or interpretative errors; errors in determination of true disease status could have resulted from difficulties in distinguishing atypical, atrophic, koilocytotic, and malignant changes [15,24,32,33] or from the absence of colposcopic and histologic evaluation of cytologically normal women. HPV DNA sampling error can be introduced when insufficient cellular material is obtained from the lavage or the types of HPV recovered do not represent those infecting the cervix [34]. Cervicovaginal lavage has been demonstrated to provide sufficient cellular material for Southern blot testing in younger populations [21]. This is the first study that we are aware of which uses lavage in an elderly population. Copy numbers of HPV available might have been affected by test order (i.e., sampling before or after the Papanicolaou smear), or physiological factors in elderly women could influence the rate of cervical cellular exfoliation or replication or shedding of viral DNA [7,15,24]. To the extent that insufficient cellular material may have been obtained, the study would
PAPILLOMAVIRUS
AND
CERVICAL
underestimate the true prevalence of HPV infection and bias associations towards the null. In terms of specificity of the lavage, the types of HPV obtained from a cervicovaginal lavage have been found to correlate well with those found in the cervix [21]. Interpretative errors can also occur with all hybridization tests, due to the subjective nature of assessment of HPV DNA reaction strength. For this study, only strongly and moderately positive results were considered positive; other positive results were considered equivocal and classed as negative. Inclusion of equivocal tests in the HPV DNA negative category will be correct to the extent that they represent false positive results and will bias our results towards the null if they represent false negatives [34]. Despite these potential sources of error, the ViraPap dot-blot method has been shown to have a sensitivity of from 90 to 96% and a specificity of from 94 to lOO%, measured against the Southern Blot technique [20,31,34]. Lastly, the impact of the cross-sectional nature of the study on our conclusions must be acknowledged. Since we have ascertained disease and exposure simultaneously, we are unable to make any inferences about causality or temporal relationships. Cross-sectional studies are also prone to prevalence bias [35]. Thus, we may not accurately be portraying the nature of the association between exposure and disease if: (1) cases of longer duration were more likely to be included than those with shorter or more fatal courses; (2) HPV exposure was related to disease duration [36,37]; and/or (3) certain HPV types varied in their ability to produce persistent detectable infection [7,36,38]. Despite these limits, given the consistency with prior research, our results suggest that HPV DNA positivity increases the risk of having cervical neoplasia for elderly Black women. This is the first study to examine an elderly population. Additional studies in other elderly populations and with larger samples of diseased women are needed to understand the true distribution of HPV viral types and their significance in the natural history of cervical neoplasia in the elderly. ACKNOWLEDGMENTS We thank the Harlem Study Team, including Evangelyn Ramsey, N.P., Regina Dunlap, Sook McGrath, N.P., Lillian Jeremiah, N.P., and Charlena Pace, N.P., for assistance in screening; the staffs of the Medical Clinic, Harlem Hospital Center and KytoDiagnostics, for support of the screening program; Ms. Sheryl Yarbrough, Coordinator, Harlem Hospital Center Colposcopy Clinic, for assistance in clinical follow-up; Mr. Jian Tang, Division of Cancer Control, Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, for assistance with computer programming; and Ms. Claudette Green, Division of Cancer Control, Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, for assistance with manuscript preparation.
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IN THE
11
ELDERLY
REFERENCES 1. Meanwell, C., Cox, M., Blackledge, G., and Maitland, N. HPV 16 DNA in normal and malignant cervical epithelium: Implications for the aetiology and behaviour of cervical neoplasia, Lancetl, 703707 (1987). 2. Reeves, W. C., Brinton, L. A., Garcia, M., et al. Human papillomavirus infection and cervical cancer in Latin America, N. Engl. J. Med. 320, 1437-1441 (1989). 3. Reid, R., Stanhope, C. R., Herschman, B. R., Booth, E., Phibbs, G. D., and Smith, J. P. Genital warts and cervical cancer. I. Evidence of an association between subclinical papillomaviral infection and cervical malignancy, Cancer 50, 377-387 (1982). 4. Jenkins, D. J., Tay, S. K., Campion, M. J., McCance, D. J., Clarkson, P. K., and Singer, A. Histological and immunocytochemical study of cervical intraepithelial neoplasia associated with HPV 16 and HPV 18 infections, J. Clin. Puthol. 39, 1177-1180 (1986). 5. Nelson, J. H., Averette, H. E., and Richart, R. Cervical intraepithelial neoplasia and early invasive cervical carcinoma, Ca Cancer J. Clin. 39, 157-178 (1989). 6. Rosenfeld, W. D., Vermund, S. H., Wentz, S. J., and Burk, R. D. High prevalence rate of human papillomavirus infection and association with abnormal papanicolaou smears in sexually active adolescents, Am. J. Dis. Child. 143, 1443-1447 (1989). 7. devilliers, E. M., Wagner, D., Schneider, A., et al. Human papillomavirus infections in women with and without abnormal cervical cytology, Lancet 2, 703-705 (1987). 8. Reid, R., Laverty, C. R., Coppleson, M., et al. Noncondylomatous cervical wart virus infection, Obstet. Gynecol. 55, 476-483 (1980). 9. Meisels, A. and Fortin, R. Condylomatous lesions of the cervix and vagina: Cytologic patterns, Acta Cytol. (Baltimore) 20, 505-509 (1976). 10. Grubb, G. S. Human papillomavirus and cervical neoplasia: Epidemiological considerations, Int. 1. Epidemiol. 15, l-7 (1986). 11. Schneider, A., Sawada, E., Gissman, L., and Shah, K. Human papillomaviruses in women with a history of abnormal Papanicolaou smears and in their male partners, Obstet. Gynecol. 69. 554-562 (1987). 12. Kjaer, S. K., Engholm, G., Teisen, C., et al. Risk factors for cervical human papillomavirus and herpes simplex virus infections in Greenland and Denmark: A population-based study, Am. J. Epidemiol.131, 669682 (1990). 13. Kreider, J. W., Howert, M. K. Wolfe, S. A., et al. Morphological transformation in vivo of human uterine cervix with papillomavirus from condylomata acuminata, Nature 317, 639-641 (1985). 14. Pirisi, L., Yasumoto, S., Feller, M., Doniger, J., and DiPaolo, J. A. Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA, J. Virol. 61, 1061-1066 (1987). 15. Koss, L. Cytologic and histologic manifestations of human papillomavirus infection of the female genital tract and their clinical significance, Cancer 60, 1942-1950 (1987). 16. Schwartz, E., and Schneider-Gadicke, A. Expresssion of human papillomavirus type 18 DNA cells DNA tumor viruses,
in cervical
carcinoma
cell lines, cancer
Cold Spring Harbor Laboratory Press, New York, Vol. 4, pp. 581-587 (1986). 17. Shokri-Tabibzadeh, S., Koss, L., Molnar, J. and Romney, S. Association of human papillomavirus with neoplastic processes in genital tract of four women with impaired immunity, Gynecol. Oncol. 12, S129-S140 (1981). 18. zur Hausen, H. Intracellular surveillance of persisting viral infections, Lancet 2, 489-491 (1986).
12
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19. Nuovo, G. J., Cottral, S. and Richart, R. M. Occult human papillomavirus infection of the uterine cervix in postmenopausal women, Am. J. Obstet. Gynecol. 2, 340-344 (1989). 20. Kiviat, N. B., Koutsky, L. A., Critchlow, C. W., et al. Comparison of southern transfer hybridization for detection of cervical human papillomavirus infection with types 6, 11, 16, 18, 31, 33, and 35 Am. J. Clin. Puthol. 94, 561-565 (1990). 21. Burk, R. D., Kadish, A. S., Calderin, S., and Romney, S. L. Human papillomavirus infection of the cervix detected by cervicovaginal lavage and molecular hybridization: Correlation with biopsy results and Papanicolaou smear, Am. J. Obstet. Gynecol. 982988 (May 1986). 22. World Health Organization. Histologic typing of female genital tract tumors, in Diagnostic cytology and Its histopathologic bases (L. G. Koss, Ed.), Lippincott, Philadelphia, 3rd ed. Vol 1, pp. 184-190 (1979). 23. Syrjanen, K. Parkinen, S. Mantyjami, R., er al. Human papillomavirus (HPV) type as an important determinant of the natural history of HPV infections in uterine cervix, Eur. J. Epidemiol. 1, 180-187 (1985). 24. Koss, L. G. Diagnostic cytology and its histopathologic bases, Lippincott, Philadelphia, 3rd ed., Vol 1, pp. 184-190 (1979). 25. SPSS-X user’s Guide, SPSS, Inc., Chicago, 3rd ed. (1988). 26. Fleiss, J.L. Statistical methods for rates andproportions, Wiley, New York, 2nd ed., pp. U-18, 212-221 (1981). 27. Breslow, N. E., and Day, N. E. Statistical methods in cancer research, Vol. 1, The Analysis of Case-Control Studies, International Agency for Research on Cancer, Oxford Univ. Press, Lyon, pp. 69-73, 28.
(1980).
Northern Carolina and Chapel Hill, PWS-KENT Publishing Co., Boston, pp. 341-386 (1988). 30. Munoz, N., Bosch, X., and Kaldor, J. M. Does human papillomavirus cause cervical cancer? The state of the epidemiological evidence, Br. J. Cancer 57, l-5 (1988). 31. Koutsky, L. A., Galloway, D. A., and Holmes, K. K. Epidemiology of genital human papillomavirus infection, Epidemiol. Rev. 10,122163 (1988).
Mandelblatt, J., Gopaul, I., and Wistreich, M. Gynecological care of elderly women: Another look at Papanicolaou smear testing, J. Am. Med. Assoc. 256, 367 (1986). 33. Frost, J. K. Gynecologic and obstetric clinical cytopathology, No32.
vak’s gynecologic relations (E. R.
Meas.
Kleinbaum, regression
20, 37-46
(1960).
D. G., Kupper, analysis
and other
multivariable
K. E. Applied methods, University of
pathology
with clinical
and endocrine
(1989).
Kelsey, J. L. Thompson, W. D., and Evans, A. S. Methods in observational epidemiology, Oxford Univ. Press, New York, pp. 187-211, 285-308 (1986). 36. Lorincz, A. T., Schiffman, M. H., Jaffurs, W. J. Marlow, J. Quinn, A. P. and Temple, G. F. Temporal associations of human papillomavirus infection with cervical cytologic abnormalities, Am. J. Obstet. Gynecol. 3, 645-651 (1990). 37. Lorincz, A. T., Temple, G. F., Kurman, R. J., Jenson, A. B., and Lancaster, W. D. Oncogenic association of specific human papillomavirus types with cervical neoplasia, J. Natl. Cancer Inst. 79,67135.
677 (1987). 38.
L. L., and Muller,
and obstetric
Novak and J. D. Woodruff, Eds.), Saunders, Philadelphia, pp. 68%781 (1979). 34. Lancaster, W. D., and Norrild, B. Diagnosis of HPV by DNA hybridization techniques, in Human papillomavirus and cervical cancer, (N. Munoz, F. X. Bosch, and 0. M. Jensen, Eds.), IARC, World Health Organization, Oxford Univ. Press, Lyon, p. 103
Cohen, J. A coefficient of agreement for nominal scales, Educ. Psychol.
29.
133-134
ET AL.
Ferenczy, A., Mitao, M., Nagai, N., Silverstein, S. J., and Crum, C.P. Latent papillomavirus and recurring genital warts, N. Engl. J. Med. 313, 784-788 (1985).
ONCOLOGY
46, 6-12 (1992)
Is Human Papillomavirus Associated with Cervical Neoplasia in the Elderly?’ JEANNE MANDELBLATT, STEPHEN MATSEOANE, *Department Pathology,
M.D., M.D.,$
M.P.H.
,**’ RALPH RICHART, M.D.,? LOUYSTHOMAS, M.D.,$ PREM CHAUHAN, PETER KANETSKY, M.P.H.,* MARYANN TRAXLER, M.D.,11 AND PATRICIA LAKIN,
of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021; TDepartment Columbia Presbyterian Hospital, New York, New York; and Departments of $Pathology, OObstetrics and Gynecology, IIMedicine, and #Nursing, Harlem Hospital Center, New York, New York
M.D.*
N.P.# of
Received December 13. 1991
There have been no studiesin the United States of human papillomavirus (HPV) in elderly women. This paper presents cross-sectionaldata on HPV and cervical neoplasiaamong 232 womenage65 or more. HPV deoxyribonucleicacid (DNA) testing wasperformedusinga modifieddot-blot hybridization technique. The prevalenceof HPV DNA positivity was3.5% (95%confidence interval (CI) 0.9%, 6.0%). There were six casesof histologic cervical neoplasia.The crude odds ratio for cervical neoplasia among HPV DNA positiveswas 18.3 (95% CI 2.8, 120.3). The adjustedodds,controlling for age, prior screeninghistory, current sexualactivity, and past contraception use, were 12.2 (95% CI 1.2, 122.9). Ever having had a Papanicolaousmearwas protective, and there was a trend for the odds of having neoplasiato increasewith age. Additional studieswith larger samplesof elderly womenare needed.If confirmed, the resultssuggestthat, independent of past screening, HPV may increase the risk of having cervical neoplasiafor elderly women. 0 1992 Academic Press, Inc.
INTRODUCTION
Human papillomavirus (HPV) has been detected in 17 to 95% of cervical cancers, using a variety of techniques [l-6]. However, evidence of HPV infection has also been noted in 1.3 to 32% of women with normal Papanicolaou smears [2,7-121. Thus, while HPV has been shown to produce in vitro malignant human cell transformation and
MATERIAL
Women with a birth year of 1925 or earlier (hereinafter referred to as “elderly”) who were scheduled for routine appointments to the medical clinic at Harlem Hospital Center between May 15, 1990, and November 15, 1990, were potentially eligible for the study. The clinic is a New York City municipal hospital outpatient clinic providing comprehensive care for over 5000 patients per year with chronic medical conditions. Care is provided regardless of ability to pay. More than 90% of the patients are Black. 6
$4.00
0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Study Setting and Population
’ This work was supported in part by National Institute on Aging Academic Award KOS AGO0471, National Cancer Institute Grant R03 CA51614-01, Enid Ancell Clinical Studies Award, Memorial SloanKettering Cancer Center, and the Society of Memorial Sloan-Kettering Cancer Center (Dr. Mandelblatt). * To whom correspondence and reprint requests should be addressed at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 60, New York, NY 10021.
0090-8258192 Copyright
immortalization [13,14], infection with the virus may not be a sufficient condition for the development of cervical neoplasia [ 151. Possible copromoters include age, immune status, or failure of intracellular control mechanisms [1,2,7,16-181. There are no United States studies of the prevalence or behavior of HPV in cervical lesions in elderly women. The only study of HPV in older women in the United States is a description of HPV in hysterectomy specimens among cytologically normal pre- and postmenopausal women with average ages of 41 and 61 years, respectively, whose surgery was for nonmalignant conditions [19]. This paper presents the results of a cross-sectional study of HPV among Black women, age 6.5 or more, of low socioeconomic class, who participated in a cancer screening program in an urban public hospital. The study describes the HPV types detected in this cohort using a modified dot-blot hybridization technique. The prevalence of HPV deoxyribonucleic acid (DNA) positivity is also estimated and associations between HPV DNA positivity and cervical neoplasia are explored.
PAPILLOMAVIRUS
AND CERVICAL
TABLE 1 Categories for Papanicolaou Smear Reports” Category 1. Negative 2. Benign atypia 3. Significant atypia
4. Suspicious for malignacyh 5. Positive for malignancyh
Comments Includes inflammation, atrophy, and infection Includes cellular changes not associated with inflammatory or reparative processes, but not sufficient quantitatively to be classed as intraepithelial neoplasia Includes intraepithelial neoplasia grades 1 and 2 Includes intraepithelial neoplasia grade 3 and invasive carcinoma
’ Categories determined independently of cellular changes associated with productive HPV infection. * Hereafter defined as “abnormal” or “positive” Papanicolaou smears.
Elderly women who attended the clinic were invited to participate in a breast and cervical cancer screening program approved by the hospital Institutional Review Board. Four hundred ninety-one of the 690 (71%) women who attended consented. Two hundred fifty-nine women were subsequently excluded from the study, including 169 with a history of a hysterectomy and no cervix on examination, 41 who did not have a pelvic examination and Papanicolaou smear (15 gave a history of a smear in the past year and 26 refused), 1 diagnosed with ovarian cancer, and 48 who did not have specimens obtained for HPV DNA testing (4 leaked in transit and 44 could not be obtained due to vaginal stenosis, vaginal friability and/or bleeding, or examiner omission). None of the patients in the latter category had gross cervical lesions. The remaining 232 elderly women with intact cervices and HPV DNA results constitute the final study group.
NEOPLASIA
IN THE ELDERLY
7
sults. An independent pathologist also performed a blind review of all smears interpreted as suspicious or positive for malignancy and a 10% random sample of the remaining slides. A cervicovaginal lavage was performed after the Papanicolaou smear to obtain exfoliated cellular material for HPV DNA testing [20]. The cervix was lavaged with 10 ml of a 0.9% saline solution and the washings were aspirated from the posterior vaginal fornix with a sterile syringe [21]. The aspirate was placed in a sterile tube and centrifuged, and the cellular pellet was suspended in 1 ml of transport media, according to the manufacturer’s instructions (Life Technologies, Inc., Gaithersburg, MD). The samples were tested for the presence and types of HPV DNA with the ViraPap human papillomavirus DNA detection kit (Molecular Diagnostics Division, Life Technologies, Inc., 1989). This is a modified dot-blot hybridization process developed for use as a screening assay to detect HPV DNA types 6, 11, 16, 18, 31, 33, and 35. If the sample was positive, it was analyzed with the ViraType papillomavirus DNA detection kit (Molecular Diagnostics Division, Life Technologies, Inc., 1989). This test uses “‘P-labeled RNA probes to detect HPV DNA types 6/11, 16/18, and 31/33/35. Negative control probes were used to minimize false-positive results. A repeat sample was requested when the results were equivocal. Testing was performed without knowledge of the patient’s clinical or cytologic status. Histologic specimens were obtained by a trained gynecologic oncologist and included colposcopically directed cervical biopsies and endocervical curettage. Interpretation was completed by pathologists unaware of the HPV status. Grading of lesions was done according to the World Health Organization (WHO) criteria for histologic typing of female genital cancers [22]. Interpretations were independent of diagnoses consistent with productive HPV infection, such as koilocytosis and condyloma acuminata.
Materials
Papanicolaou smears were obtained using a narrow cotton-tipped, saline-moistened applicator (calgi-swab) for sampling of the endocervix and an Ayre’s spatula for sampling the endo- and exocervix. Specimens were placed on glass slides and fixed immediately with an aerosol fixative. Several measures were taken to ensure that smears contained sufficient cellular material for cytologic assessment, including staff training, direct observation, and laboratory feedback. Table 1 summarizes the categories used to describe the cytologic findings. Interpretation also included a comment on smear adequacy and an independent assessment of cellular changes characteristic of productive HPV infection [15]. All cytologic interpretations were conducted without knowledge of the HPV DNA hybridization re-
Definitions of Exposure and DiseaseStatus
Exposure to HPV was defined by the HPV DNA test results. Women were identified as being HPV DNA positive if they had results positive for HPV DNA types of high and intermediate oncogenic risk (types 16/18, 31/33/35), low risk (types 6/11), and other (positive, but not reacting with the preceding probes). Women were considered negative if no HPV DNA was detected or if equivocal results were obtained [5,15,23]. Disease status was defined from two perspectives: cytologic and histopathological. For the Papanicolaou smears, disease was defined as positive if the interpretation was suspicious or positive for neoplasia, and negative in all other instances, including negative, benign atypia, or significant atypia reports. If more than one
8
MANDELBLA’TT
smear was obtained, the patient was classified according to the most abnormal [24]. Women were considered positive histologically if they had a pathologic diagnosis of cervical intraepithelial neoplasia (CIN) grade 1 or more. Women with all other diagnoses, including atrophy, metaplasia, koilocytosis, and condyloma, were considered negative.
ET AL.
with equivocal results, 3 had repeat tests (2 were negative and 1 was positive) and 1 refused. Thus, 8 women were classified as HPV DNA positive and 224 as negative, yielding an overall HPV DNA prevalence of 3.5% (8/232) (95% CI 0.9%, 6.0%). The types of HPV DNA detected are summarized in Table 2. The majority are types 31/33/35 (50%), followed by “other” types (37.5%). Only one was type 16/18 (12.5%), and none was 6/11.
Data Analysis
Data were analyzed using the Statistical Program for the Social Sciences-X [25]. Estimated point prevalences of HPV DNA positivity were calculated and 95% confidence intervals (95% CI) were computed [26]. The magnitude of associations was evaluated using odds ratios (OR) and 95% CI [27]. The x2 test with a continuity correction, or, when necessary, a Fisher’s exact test, was used to assess the statistical significance of associations [26]. The kappa statistic [28] with 95% confidence limits [26] was used to assess the interrater reliability of the Papanicolaou smear readings. Adjusted odds ratios derived in a logistic regression model were used to estimate the odds of histologically diagnosed cervical neoplasia, controlling for the simultaneous effects of HPV status and potentially confounding variables [29]. Factors considered potential confounders were age, ever having a prior smear, number of lifetime smears, current sexual activity, past use of any contraceptive technique, parity, number of sexual partners, sexually transmitted disease history, and hysterectomy status [30]. Interactions were not assessed due to small numbers of cases. RESULTS Characteristics
of the Study Group
The average age of the 232 women included in the final study group was 75.2 years (range, 65-96 years), 99.5% were Black, 48% had completed no more than an eighthgrade education, and 90% reported an income of less than $9000 per year. Eighty-four percent of the women gave a history of having at least one prior Papanicolaou smear, only 13% reported having had seven or more tests, and the median time since the last smear was 2 years (range, 1 to 39 years). Forty-four women (19%) gave a history of a hysterectomy in the past; these represented subtotal hysterectomies performed prior to 1960. None of the women reported a history of cervical cancer or hysterectomy for malignant conditions. Only 13 (6%) reported symptoms; 94% were asymptomatic. HPV DNA
Results
Seven women were HPV DNA positive, 4 had equivocal results, and 221 were negative. Among the women
Cytologic
Findings
Papanicolaou smear interpretations. All smears were considered adequate for cytologic assessment. The interrater reliability of the Papanicolaou smear interpretations, as determined by the kappa statistic, was 0.75 (95% CI 0.5, 1.0). There were three smears read as negative by the primary cytologist and positive by the second cytologist; these three women were also HPV DNA positive. Thus, it was decided to use the interpretations of the primary cytologist for the analyses, since this would bias the results of any association between HPV DNA positivity and positive smears toward the null. Overall, 6 women (2.9%) had smears that were interpreted as suspicious or positive for malignancy and 226 women had negative results. HPV DNA prevalence by cytologic diagnosis. HPV DNA was detected in 3.1% (7/226) (95% CI 0.6%, 5.6%) of women with negative Papanicolaou smears and 16.7% (l/6) (95% CI 0, 54.8%) of women with positive smears. This difference did not achieve statistical significance in this small sample (P = 0.19, Fisher’s exact test, twotailed). Association between HPV DNA positivity and cytologic neoplasia. Among HPV DNA-positive women, the odds
of having an abnormal Papanicolaou smear appears elevated, compared to HPV DNA-negative women (OR = 6.3); however, the confidence intervals were very wide (95% CI 0.64-60.9) and included the possibility that there was no increased risk of an abnormal smear among HPV DNA-positive women. Histopathologic
Findings
Diagnoses. Fourteen women were referred for colposcopy: 11 complied and 3 did not (Table 2). These latter 3 women were considered negative in subsequent analyses. Six women were diagnosed with cervical neoplasia-1 with intraepithelial disease grade 1, 1 with CIN 3/CIS, and 4 with invasive disease. HPV DNA prevalence by histologic diagnosis. The prevalence of HPV DNA positivity was significantly higher in women with histologic cervical neoplasia (33.3%, 2/6) (95% CI 0, 79.4%) than in women without
PAPILLOMAVIRUS
AND CERVICAL
NEOPLASIA
9
IN THE ELDERLY
TABLE 2 Final Histopathologic Diagnoses (Years)
Pap smear report
Indication for colposcopy”
Final pathology diagnosis
B.A. R.F. B.E. R.P. H.B. J.S.
72 67 70 75 67 93
Significant atypia Negative Significant atypia Suspicious Negative Positive
16118 Other’ Negative Negative Other’ Negative
HPV HPV P.E. PAP HPV PAP
Negative
31133135
HPV
80 78 74 78 67
Positive Positive Positive Positive Negative
31/33/35 Negative Negative Negative 31/33/35
PAP PAP PAP PAP HPV
R.G.
66
Benign atypia
31/33/35
HPV
C.B.
83
Negative
Othef
HPV
-b CIN, grade 1 -* Normal Atrophic changes ICC, stage unknown Squamous metaplasia ICC, stage la CIN, grade 3/CIS ICC, stage lb ICC, stabe 3b KoiIocytosis/ condyloma Koilocytosis/ condyloma -b
W.P.
67
M.C. A.S. A.K. B.H. M.T.
Age Patient
HPV DNA results
’ Indications for referral included positive HPV DNA results (HPV), abnormal physical examination (P.E), or abnormal pap smear (PAP). ’ Work-up not completed; one patient died and two refused; all three were considered “negative” in subsequentanalyses. ’ Positive for types other than 6/11, 16/18, 31/33/35. ’ Diagnostic work-up completed but staging evaluation and treatment were refused. ’ Symptomatic with vaginal bleeding. Note. CIN, cervical intraepithelial neoplasia; CIS, Carcinoma in situ; ICC, invasive cervical cancer.
neoplasia (2.7%, 6/226) (95% CI 0.34%, 4.97%) (P = 0.015, Fisher’s exact test, two-tailed). Association between HPV DNA posit&&y and histologic diagnoses. The crude odds ratio for cervical neoplasia
among HPV DNA-positive women was 18.3. In this small sample, the confidence interval was very wide (95% CI 2.8, 120.3); however, the lower bound indicates a moderate effect. To minimize the prevalence bias inherent in this cross-sectional study, relative risk was also analyzed separately for the 211 women who reported having one or more prior negative Papanicolaou smears at any time in the past. When this was done, the association between neoplasia and HPV remained significant (P = 0.003, Fisher’s exact test, two-tailed). The odds ratio was somewhat higher (OR = 50.6) in this “incident” group than in the entire group, but the CI was extremely wide (95% CI 5.6, 456).
The adjusted odds of neoplasia among HPV DNApositive women was 12.2 (95% CI 1.2, 122.9), controlling for age, ever having a Papanicolaou smear, current sexual activity, and past use of any birth control method (Table 3). Past use of any method to prevent pregnancy was also independently associated with cervical neoplasia (OR = 21.8, 95% CI 1.7-280.3). Ever having a Papanicolaou smear was associated with lower odds of having neoplasia,
although the confidence interval included one (P = 0.053), and there was a trend for increasing age to increase the odds of having neoplasia independent of the remaining variables, but in this small sample this did not achieve statistical significance (P = 0.069). DISCUSSION This study, although small, is the first investigation of the types, prevalence, and behavior of cervical HPV DNA in an elderly United States population group. The majority of HPV types detected in this cohort were those considered to have intermediate and high oncogenic potential [5,15,23]. The ranges of prevalence rates of HPV DNA observed among older women in other settings are comparable to those in this study. In Germany, HPV DNA was detected in 3.5% (95% CI 2%, 5%) of cytologically normal elderly women, and in 30% (95% CI 16%, 45%) with cytologic neoplasia [7]. HPV DNA has also been detected in 12% (95% CI l%, 22%) of normal cervices removed during hysterectomy from postmenopausal women [19]. The magnitude of the odds of cervical neoplasia in HPV DNA positive elderly women noted in this investigation is also similar to that noted by other researchers in other countries [2,7]. However, it should
10
MANDELBLA’IT
ET AL.
TABLE 3 Adjusted Odds of Cervical Neuplasia for Selected Factof Variables Age (in l-year intervals) HPV DNA Negative Positive Currently sexually active No Yes Any past contraceptive used No Yes Past screening’ Never Ever
Adjusted odds ratiob 1.14
95% Confidence interval 0.99-1.3
P
value
0.069
1.00” 12.2
1.2-122.9
0.034
1.00 6.1
0.6-62.4
0.128
1.00 21.8
1.7-280.3
0.018
1.00 0.08
0.01-1.03
0.053
a For total n = 232 women. ’ Each variable is adjusted for the remaining variables in a logistic regression model. Parity, sexual partners, number of Papanicolaou smears, sexually transmitted disease (STD) history, and hysterectomy did not enter into the final model. Group means were used for missing values for continuous variables: parity (3 cases missing), number of sexual partners (11 cases missing), and number of smears (55 cases missing). Sexual activity data were missing for 4 cases which were classed as “no.” All data were complete for the categorical variables of STD history, hysterectomy, contraceptive use, and past screening. ’ Referent category for each risk factor. d Includes any technique used in the woman’s lifetime to prevent pregnancy, such as condoms, diaphragm, spermicidal agents, rhythm, and withdrawal. Oral contraceptives or IUDs were only used by two women. ’ Ever screened is defined as having a history of any prior Papanicolaou smear.
be noted that differences across studies with respect to race, setting, selection criteria, control of confounding variables, HPV sampling and testing techniques and types, criteria for the diagnosis of neoplasia, and categorization of koilocytotic changes make direct comparisons difficult. There are several caveats that should be considered when interpreting the findings of this study, including sample size and power, classification errors, and the use of a cross-sectional design. The study had a power of 80% to rule out zero overall prevalence of HPV DNA and to detect a relative risk of neoplasia associated with HPV DNA of 12 or greater; however, due to the low number of diseased women, power was considerably less for detection of increases in risk among neoplasia subcategories. For example, if risk is examined separately for low-grade neoplasia (CIN 1 or 2), the association between HPV and neoplasia remains significant (P = 0.03, Fisher’s exact test, two-tailed); for high grade lesions (CIN 3/CIS or more severe), while HPV was found in 14.3% of cases and only 1.8% of controls, the difference is not statistically significant in this small sample (P = 0.14, Fisher’s exact test, two-tailed). Thus, it will be important to replicate this study in a sample with a larger number of cases of cervical neoplasia. Accurate classification of HPV DNA and cervical lesions remains problematic for studies such as this [30,31].
We have taken several measures to avoid bias associated with misclassification error, including blind determinations of exposure and disease status, assessment of HPV cellular changes independent of cytologic and histologic interpretation of malignancy, and, when necessary, making assumptions which would bias results towards the null. However, exposure misclassification could have arisen using the dot-blot hybridization as a result of sampling or interpretative errors; errors in determination of true disease status could have resulted from difficulties in distinguishing atypical, atrophic, koilocytotic, and malignant changes [15,24,32,33] or from the absence of colposcopic and histologic evaluation of cytologically normal women. HPV DNA sampling error can be introduced when insufficient cellular material is obtained from the lavage or the types of HPV recovered do not represent those infecting the cervix [34]. Cervicovaginal lavage has been demonstrated to provide sufficient cellular material for Southern blot testing in younger populations [21]. This is the first study that we are aware of which uses lavage in an elderly population. Copy numbers of HPV available might have been affected by test order (i.e., sampling before or after the Papanicolaou smear), or physiological factors in elderly women could influence the rate of cervical cellular exfoliation or replication or shedding of viral DNA [7,15,24]. To the extent that insufficient cellular material may have been obtained, the study would
PAPILLOMAVIRUS
AND
CERVICAL
underestimate the true prevalence of HPV infection and bias associations towards the null. In terms of specificity of the lavage, the types of HPV obtained from a cervicovaginal lavage have been found to correlate well with those found in the cervix [21]. Interpretative errors can also occur with all hybridization tests, due to the subjective nature of assessment of HPV DNA reaction strength. For this study, only strongly and moderately positive results were considered positive; other positive results were considered equivocal and classed as negative. Inclusion of equivocal tests in the HPV DNA negative category will be correct to the extent that they represent false positive results and will bias our results towards the null if they represent false negatives [34]. Despite these potential sources of error, the ViraPap dot-blot method has been shown to have a sensitivity of from 90 to 96% and a specificity of from 94 to lOO%, measured against the Southern Blot technique [20,31,34]. Lastly, the impact of the cross-sectional nature of the study on our conclusions must be acknowledged. Since we have ascertained disease and exposure simultaneously, we are unable to make any inferences about causality or temporal relationships. Cross-sectional studies are also prone to prevalence bias [35]. Thus, we may not accurately be portraying the nature of the association between exposure and disease if: (1) cases of longer duration were more likely to be included than those with shorter or more fatal courses; (2) HPV exposure was related to disease duration [36,37]; and/or (3) certain HPV types varied in their ability to produce persistent detectable infection [7,36,38]. Despite these limits, given the consistency with prior research, our results suggest that HPV DNA positivity increases the risk of having cervical neoplasia for elderly Black women. This is the first study to examine an elderly population. Additional studies in other elderly populations and with larger samples of diseased women are needed to understand the true distribution of HPV viral types and their significance in the natural history of cervical neoplasia in the elderly. ACKNOWLEDGMENTS We thank the Harlem Study Team, including Evangelyn Ramsey, N.P., Regina Dunlap, Sook McGrath, N.P., Lillian Jeremiah, N.P., and Charlena Pace, N.P., for assistance in screening; the staffs of the Medical Clinic, Harlem Hospital Center and KytoDiagnostics, for support of the screening program; Ms. Sheryl Yarbrough, Coordinator, Harlem Hospital Center Colposcopy Clinic, for assistance in clinical follow-up; Mr. Jian Tang, Division of Cancer Control, Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, for assistance with computer programming; and Ms. Claudette Green, Division of Cancer Control, Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, for assistance with manuscript preparation.
NEOPLASIA
IN THE
11
ELDERLY
REFERENCES 1. Meanwell, C., Cox, M., Blackledge, G., and Maitland, N. HPV 16 DNA in normal and malignant cervical epithelium: Implications for the aetiology and behaviour of cervical neoplasia, Lancetl, 703707 (1987). 2. Reeves, W. C., Brinton, L. A., Garcia, M., et al. Human papillomavirus infection and cervical cancer in Latin America, N. Engl. J. Med. 320, 1437-1441 (1989). 3. Reid, R., Stanhope, C. R., Herschman, B. R., Booth, E., Phibbs, G. D., and Smith, J. P. Genital warts and cervical cancer. I. Evidence of an association between subclinical papillomaviral infection and cervical malignancy, Cancer 50, 377-387 (1982). 4. Jenkins, D. J., Tay, S. K., Campion, M. J., McCance, D. J., Clarkson, P. K., and Singer, A. Histological and immunocytochemical study of cervical intraepithelial neoplasia associated with HPV 16 and HPV 18 infections, J. Clin. Puthol. 39, 1177-1180 (1986). 5. Nelson, J. H., Averette, H. E., and Richart, R. Cervical intraepithelial neoplasia and early invasive cervical carcinoma, Ca Cancer J. Clin. 39, 157-178 (1989). 6. Rosenfeld, W. D., Vermund, S. H., Wentz, S. J., and Burk, R. D. High prevalence rate of human papillomavirus infection and association with abnormal papanicolaou smears in sexually active adolescents, Am. J. Dis. Child. 143, 1443-1447 (1989). 7. devilliers, E. M., Wagner, D., Schneider, A., et al. Human papillomavirus infections in women with and without abnormal cervical cytology, Lancet 2, 703-705 (1987). 8. Reid, R., Laverty, C. R., Coppleson, M., et al. Noncondylomatous cervical wart virus infection, Obstet. Gynecol. 55, 476-483 (1980). 9. Meisels, A. and Fortin, R. Condylomatous lesions of the cervix and vagina: Cytologic patterns, Acta Cytol. (Baltimore) 20, 505-509 (1976). 10. Grubb, G. S. Human papillomavirus and cervical neoplasia: Epidemiological considerations, Int. 1. Epidemiol. 15, l-7 (1986). 11. Schneider, A., Sawada, E., Gissman, L., and Shah, K. Human papillomaviruses in women with a history of abnormal Papanicolaou smears and in their male partners, Obstet. Gynecol. 69. 554-562 (1987). 12. Kjaer, S. K., Engholm, G., Teisen, C., et al. Risk factors for cervical human papillomavirus and herpes simplex virus infections in Greenland and Denmark: A population-based study, Am. J. Epidemiol.131, 669682 (1990). 13. Kreider, J. W., Howert, M. K. Wolfe, S. A., et al. Morphological transformation in vivo of human uterine cervix with papillomavirus from condylomata acuminata, Nature 317, 639-641 (1985). 14. Pirisi, L., Yasumoto, S., Feller, M., Doniger, J., and DiPaolo, J. A. Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA, J. Virol. 61, 1061-1066 (1987). 15. Koss, L. Cytologic and histologic manifestations of human papillomavirus infection of the female genital tract and their clinical significance, Cancer 60, 1942-1950 (1987). 16. Schwartz, E., and Schneider-Gadicke, A. Expresssion of human papillomavirus type 18 DNA cells DNA tumor viruses,
in cervical
carcinoma
cell lines, cancer
Cold Spring Harbor Laboratory Press, New York, Vol. 4, pp. 581-587 (1986). 17. Shokri-Tabibzadeh, S., Koss, L., Molnar, J. and Romney, S. Association of human papillomavirus with neoplastic processes in genital tract of four women with impaired immunity, Gynecol. Oncol. 12, S129-S140 (1981). 18. zur Hausen, H. Intracellular surveillance of persisting viral infections, Lancet 2, 489-491 (1986).
12
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19. Nuovo, G. J., Cottral, S. and Richart, R. M. Occult human papillomavirus infection of the uterine cervix in postmenopausal women, Am. J. Obstet. Gynecol. 2, 340-344 (1989). 20. Kiviat, N. B., Koutsky, L. A., Critchlow, C. W., et al. Comparison of southern transfer hybridization for detection of cervical human papillomavirus infection with types 6, 11, 16, 18, 31, 33, and 35 Am. J. Clin. Puthol. 94, 561-565 (1990). 21. Burk, R. D., Kadish, A. S., Calderin, S., and Romney, S. L. Human papillomavirus infection of the cervix detected by cervicovaginal lavage and molecular hybridization: Correlation with biopsy results and Papanicolaou smear, Am. J. Obstet. Gynecol. 982988 (May 1986). 22. World Health Organization. Histologic typing of female genital tract tumors, in Diagnostic cytology and Its histopathologic bases (L. G. Koss, Ed.), Lippincott, Philadelphia, 3rd ed. Vol 1, pp. 184-190 (1979). 23. Syrjanen, K. Parkinen, S. Mantyjami, R., er al. Human papillomavirus (HPV) type as an important determinant of the natural history of HPV infections in uterine cervix, Eur. J. Epidemiol. 1, 180-187 (1985). 24. Koss, L. G. Diagnostic cytology and its histopathologic bases, Lippincott, Philadelphia, 3rd ed., Vol 1, pp. 184-190 (1979). 25. SPSS-X user’s Guide, SPSS, Inc., Chicago, 3rd ed. (1988). 26. Fleiss, J.L. Statistical methods for rates andproportions, Wiley, New York, 2nd ed., pp. U-18, 212-221 (1981). 27. Breslow, N. E., and Day, N. E. Statistical methods in cancer research, Vol. 1, The Analysis of Case-Control Studies, International Agency for Research on Cancer, Oxford Univ. Press, Lyon, pp. 69-73, 28.
(1980).
Northern Carolina and Chapel Hill, PWS-KENT Publishing Co., Boston, pp. 341-386 (1988). 30. Munoz, N., Bosch, X., and Kaldor, J. M. Does human papillomavirus cause cervical cancer? The state of the epidemiological evidence, Br. J. Cancer 57, l-5 (1988). 31. Koutsky, L. A., Galloway, D. A., and Holmes, K. K. Epidemiology of genital human papillomavirus infection, Epidemiol. Rev. 10,122163 (1988).
Mandelblatt, J., Gopaul, I., and Wistreich, M. Gynecological care of elderly women: Another look at Papanicolaou smear testing, J. Am. Med. Assoc. 256, 367 (1986). 33. Frost, J. K. Gynecologic and obstetric clinical cytopathology, No32.
vak’s gynecologic relations (E. R.
Meas.
Kleinbaum, regression
20, 37-46
(1960).
D. G., Kupper, analysis
and other
multivariable
K. E. Applied methods, University of
pathology
with clinical
and endocrine
(1989).
Kelsey, J. L. Thompson, W. D., and Evans, A. S. Methods in observational epidemiology, Oxford Univ. Press, New York, pp. 187-211, 285-308 (1986). 36. Lorincz, A. T., Schiffman, M. H., Jaffurs, W. J. Marlow, J. Quinn, A. P. and Temple, G. F. Temporal associations of human papillomavirus infection with cervical cytologic abnormalities, Am. J. Obstet. Gynecol. 3, 645-651 (1990). 37. Lorincz, A. T., Temple, G. F., Kurman, R. J., Jenson, A. B., and Lancaster, W. D. Oncogenic association of specific human papillomavirus types with cervical neoplasia, J. Natl. Cancer Inst. 79,67135.
677 (1987). 38.
L. L., and Muller,
and obstetric
Novak and J. D. Woodruff, Eds.), Saunders, Philadelphia, pp. 68%781 (1979). 34. Lancaster, W. D., and Norrild, B. Diagnosis of HPV by DNA hybridization techniques, in Human papillomavirus and cervical cancer, (N. Munoz, F. X. Bosch, and 0. M. Jensen, Eds.), IARC, World Health Organization, Oxford Univ. Press, Lyon, p. 103
Cohen, J. A coefficient of agreement for nominal scales, Educ. Psychol.
29.
133-134
ET AL.
Ferenczy, A., Mitao, M., Nagai, N., Silverstein, S. J., and Crum, C.P. Latent papillomavirus and recurring genital warts, N. Engl. J. Med. 313, 784-788 (1985).
