The association between human papillomavirus deoxyribonucleic acid status and the results of cytologic rescreening tests in young, sexually active women Anna-Barbara Moscicki, MD," Joel M. Palefsky, MD,b John Gonzales, MD,' and Gary K. Schoolnik, MD d San Francisco and Stanj(lrd, California We examined the utility of cytologic rescreening tests in women who had positive test results for human papillomavirus deoxyribonucleic acid but who were diagnosed as having benign conditions at cytologic testing. One hundred twenty-five Papanicolaou smears from women who were screened for human' papillomavirus deoxyribonucleic acid were sent routinely to a private laboratory for diagnoses. These slides were then reviewed independently by two pathologists who were blinded to the human papillomavirus deoxyribonucleic acid results. The effects of cytologic rescreening in cases of both positive and negative human papillomavirus deoxyribonucleic acid were assessed by calculating z scores. Cervical intraepithelial neoplasia was diagnosed in 40% by pathologist A and in 20% by pathologist B of the human papillomavirus-positive subjects compared with none diagnosed by the private cytology laboratory (z = 3.09, P < 0.005 and z = 1.98, P < 0.05, respectively). No significant differences were found in the human papillomavirus-negative group. We conclude that cytologic rescreening in human papillomavirus deoxyribonucleic acid-positive women who were initially diagnosed as having benign cytologic results will yield a significant proportion of cases of cervical intraepithelial neoplasia. (AM J OBSTET GVNECOL 1991 ;165:67-71.)
Key words: Human papillomavirus, cytology, rescreening The strong association between cervical neoplasia and human papillomavirus (HPV) infection" 2 has stimulated interest in HPV diagnostic tests. Currently, the Papanicolaou smear is used as the primary screening test for cervical disease, including condyloma and intraepithelial neoplasia." However, the Papanicolaou smear appears to be relatively insensitive, detecting only 50% to 80% of the abnormalities found by histologic examination of cervical biopsy specimens obtained by colposcopy. Hi Routine colposcopy remains a time-consuming and cost-ineffective means to identify From the Department oj Pediatrics," the Departments oj Laboratory Medicine and Stomatology," and the Department of Anatomic Patholog),,' Ulliversit~ of California, San Francisco, and the Department oj Im1Jlunology and Medical Microbiology, Stanford University," Supported in part b~ funds provided by the State of California and allocated on tlte recommendations oj tlte University-wide Task Force on Acquired IlIlmunodeficienc~ Syndrome, a Howard Hughes lHediml Institute fellowship award, Hampton Medical School fund to tlte University of California, San Francisro (No. MSC 48), Bureau of Maternal and Child Health and Resource Development Training Grant No. MC/000978, an Acquired Immunodeji(ienc~ S)ndrome Clinical Research Center grant to the University ofCalifiJntia, San Francisco (National Institute of Acquired itnmullodejiciency Syndrome grant No. P01AI21912), and University of California Cancer Research Coordinating Committee. Receivedfor publication August 9, 1990; revised January 11, 1991 ; accepted/anuary 18,1991. Reprint requests: Anna-Barbara Moscichi, MD, 400 Parnassus Ave., Room AC-Ol, Box 0374, Division of Adolescent Medicine, Univenity of CalifiJrllia-San Francisco, San Francisco, CA 94143. 611128066
cervical disease. Therefore tests that identify HPV deoxyribonucleic acid (DNA) have been developed in the hope that these techniques may augment our ability to precisely identify women who are infected with HPV and who may be at risk for cervical intraepithelial neoplasia.' The indications for and clinical utility of these DNA detection tests have not yet been established. However, the correlation between HPV DNA status and cytologic abnormalities is not absolute"; i.e., some women with abnormal Papanicolaou smears will not have detectable HPV DNA and, conversely, many patients with detectable HPV DNA appear to have normal Papanicolaou smears. Therefore, whereas HPV detection tests could augment cytologic testing, they are not equivalent replacements. It has been suggested that HPV DNA testing may be used to identify women at high risk for cervical intraepithelial neoplasia. Previous studies have reported that when cytologic smears from women who are HPV positive and who have "normal" cytologic results are reviewed additional cytologic abnormalities (including intraepithelial neoplasia) can be detected."" These findings seem to indicate that the combined use of HPV DNA testing and the Papanicolaou smear might improve the identification of women with cervical disease. Unfortunately, the results of these studies are difficult to interpret because the smears were reviewed by pathologists who knew the HPV DNA status of the spec-
67
68
Moscicki et al.
July 1991 Am J Obstet Gynecol
Table I. Population characteristics Age (yr, mean ± SE) Age at initial sexual intercourse (mean ± SE) No. of lifetime sexual partners 0-3 4-10 2: 11
No. of sexual partners in past 2 mo (mean ± SE) Race-ethnicity (%) White Black Hispanic Other Reason for visit (%) Family planning Sexually transmitted disease related
17.1 ± 1.5 15.5 ± 1.5
65% 25%
10%
1.2 ± 0.7
71% 5%
10%
14% 90% 10%
imens at the time the reviews were conducted, possibly biasing the outcome. We describe a study that examines the association between HPV DNA status and diagnostic interpretations on review of cytologic results by two independent pathologists who were without knowledge of the HPV DNA results. Methods
This study was part of a large epidemiologic study examining the prevalence of HPV DNA in sexually active adolescents. 10 All female adolescents (aged 13 to 19 years) who were attending a private, suburban family planning clinic and having a pelvic examination for any reason were asked to enter this study according to the guidelines set by the Committee for Human Research to screen for the presence of cervical HPV DNA. The refusal rate was < 10%. Populations attending the clinic were predominantly middle-class and the racialethnic distribution of the clinic was 60% white, 20% black, 20% Hispanic, and 10% other. All female adolescents who entered the study were asked to complete a self-administered questionnaire before examination; this included demographic and sexual behavior information. Pelvic examination included a Papanicolaou smear obtained with a Dacron swab and spatula followed by an endocervical and exocervical sample for HPV DNA with a Dacron swab. Slides for cytologic testing were immediately fixed in alcohol and were sent to a contracted private cytology laboratory for initial review per routine protocol. Slides from 134 consecutive subjects during the second month through the fourth month of the study were identified and released for review by two independent pathologists. Although the two pathologists were without knowledge of the HPV DNA status of each specimen and were not apprised of the specific purpose of the review, they were asked to carefully review the slides for the epidemiology study.
Cytology. Papanicolaou smears were prepared by standard methods at the private cytology laboratory and were then screened according to the following routine protocol. All smears were first screened by a cytotechnician. Those smears found to have abnormal findings including inflammation, atypia, and changes suggestive of condyloma and neoplasia, as well as 10% of the benign smears, were then reviewed by a supervising pathologist. Cytologic diagnoses from the private laboratory were categorized into one of the following: benign, atypia, or cervical intraepithelial neoplasia with or without koilocytosis. The term cervical intraepithelial neoplasia is used for easier reference in this article although it is understood that cytologic testing cannot determine the site of origin of neoplastic cells. Pathologist A then reviewed each slide independently without knowledge of either the HPV DNA status or the private laboratory's cytologic diagnosis at X 10 magnification with higher magnification used for the examination of questionable or abnormal areas. For comparison, the second pathologist (pathologist B) used a method more common to cytology laboratories; that is, a cytotechnician screened the smears first and then pathologist B verified the diagnosis of all the smears. The diagnoses reported by the reviewing pathologists were categorized as described here. Standard criteria were used to diagnose neoplasia and koilocytosis. II Atypia was diagnosed when rare abnormal cells (neoplastic or koilocytes) were found or if some but not all of the features of cervical intraepithelial neoplasia or koilocytosis were found on Papanicolaou smears. Since cytologic correlates for condyloma are controversial, cells suggestive of condyloma (koilocytes) but without the nuclear features of neoplasia also were categorized as atypia. Slides that appeared to have been air-dried or inadequately stained were excluded from the analysis. The presence or absence of endocervical cells was noted by the reviewing pathologists. HPV DNA hybridization. HPV DNA was identified with a commercially available ribonucleic acid-DNA dot blot hybridization technique (ViraPap).12 Cervical swabs for HPV were immediately placed into 1 ml of the sample transport medium and refrigerated at 4° C for no longer than 2 weeks before processing. Samples were processed with the ViraPap method according to the manufacturer's recommendation. 12 This method allows for identification of HPV types 6, 11, 16, 18, 31, 33, and 35. Unused portions of the samples that were positive for the HPV probe mixture were then studied under similar conditions to determine the HPV type more precisely. Samples were filtered onto three separate membranes, and each membrane was then hybridized with one of three separate radioactively labeled probe mixtures: type 6111, type 16/18, or type 31133/35.1l
Cytologic rescreening in HPV-positive women
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69
Table II. Comparison of cytologic diagnoses among pathologists A and B and the private cytologic laboratory for HPV-positive and HPV-negative subjects Primtate cytologic laboratory: HPV /)()sitivf Benign Pathologist A Benign Atypia Cervical intraepithelial neoplasia Pathologist B Benign Atypia Cervical intraepithelial neoplasia
9 :)
I
Atypical
I
Cervical intraepitltelia/ /teoll/asia
0 I
4
"
13 3 2
0 :3 :3
Data analysis. For each of the reviewers, X" tests were performed to examine the relationship between HPV DNA status and diagnosis. Interrater reliability is reported for agreement between the two reviewing pathologists. The effect of rescreening was assessed by calculating z scores. This test examined whether rescreening of HPV-positive subjects and HPV-negative subjects resulted in a significant difference in the proportion of subjects with a diagnosis of cervical intraepithelial neoplasia by the private cytology laboratory (first screen), when compared with diagnoses assigned by pathologist A or B (rescreens). Because the private cytology laboratory did not diagnose any cases of cervical intraepithelial neoplasia, a second variable for cytologic diagnosis was created for comparison. This variable compared only two categories: benign and any abnormality (atypia or cervical intraepithelial neoplasia). The z test was also performed to examine the difference in the proportion of subjects with any abnormality. Results
Population characteristics. Table I describes the demographic characteristics of the subjects including sexual behaviors and reasons for visit. Twenty-four subjects (19%) had positive HPV DNA results: 5 were positive for group 6111, 11 for group 16118,4 for group 31/33/35, and 1 for both group 16118 and group 31/33/35. Three subjects had undetermined types with the described dot blot method. Interrater reliability between the pathologists. The overall interrater reliability between the two pathologists with three cytologic categories (benign, atypia, and cervical intraepithelial neoplasia with or without koilocytosis) was 79% (K = 0.28, P = 0.001). When categories were subdivided into benign and any cytologic abnormality, the interrater reliability was 86% (K = 0.51, P = 0.001). For the latter analysis, the interrater reliability was 75% (K = 0.51, P < 0.002) in the HPVpositive group and 89% (K = 0.208, P < 0.03) in the HPV-negative group.
Privatate cytologic laboratory: HPV negative Benign
I
Atypical
0 0 0
92 6
I 0
0
2
0 0 0
93 4 I
I
Cervical intraepithelia/ neoplasia 0 0 0 0 0 0
Comparison of cytologic diagnosis and HPV DNA status. There was a significant association between abnormal cytologic findings and positive HPV DNA status for the private cytology laboratory, pathologist A, and pathologist B. The private cytology laboratory found that none of the patients had cervical intraepithelial neoplasia; however, 6 of 24 HPV-positive suqjects had atypia, compared with 3 of 101 HPV-negative subjects (X 2 = 10.98, P < 0.0009). Pathologist A diagnosed abnormal cytologic findings in 15 of 24 women in the HPV -positive group (9 with cervical intraepithelial neoplasia and 6 with atypia) compared with 8 of 101 women in the HPV -negative group (2 with cervical intraepithelial neoplasia and 6 with atypia) (X" = 42.2, P < 0.0001). A similar association was found for pathologist B; 11 of 24 HPV-positive women had abnormal cytologic findings (5 with cervical intraepithelial neoplasia and 6 with atypia) and 7 of 101 HPV -negative women had abnormal cytologic findings (2 with cervical intraepithelial neoplasia and 5 with atypia) (X 2 = 24.59, P < 0.0001). Comparison of cytologic findings among the private laboratory and pathologists A and B within HPVpositive and HPV-negative groups. Table II compares the cytologic diagnoses of the private cytology laboratory with those of pathologist A and pathologist B by HPV DNA ,[dIllS. Of the cervical intraepithelial neoplasia cases di,H~ll()sed, only 2 had grade 2; the rest had grade I. h< ,I h (~l'(" of grade 2 were assigned the same diagnosis l)v IJd~h pathologist A and pathologist B; both of these \\'Onllll were HPV positive. Pathologist A diagnosed 38% of the HPV-positive patients as having cervical intraepithelial neoplasia, compared with none diagnosed by the private cytology laboratory (z = 3.09, P < 0.005); pathologist A diagnosed any cytologic abnormality in 63% of the HPV-positive patients, compared with 25% by the private cytology laboratory (z = 2.39, P < 0.03). In contrast, there was no significant difference in the proportion of abnormal cytologic smears diagnosed in the HPV-negative group. Specifically, 2% of HPV-negative subjects were diagnosed to
70
Moscicki et al.
have cervical intraepithelial neoplasia by pathologist A and none were diagnosed by the private cytology laboratory (z = 0.714, difference not significant). Similarly, no difference was found for diagnosing "any cytologic abnormality" in the HPV-negative group (z = 0.15; difference not significant). In addition, 5 of the 6 subjects who were HPV positive and were diagnosed as having atypia by the private cytology laboratory had cervical intraepithelial neoplasia. Two of the subjects with atypia and who were HPV negative had cervical intraepithelial neoplasia. Pathologist B also found significantly more subjects with cervical intraepithelial neoplasia on rescreening the HPV-positive group than were diagnosed by the private cytology laboratory, 21 % by pathologist B versus 0% by the private cytology laboratory (z = 1.98, P < 0.(5). However, there was no difference between the private cytology laboratory and pathologist B in the proportion of subjects with a diagnosis of cervical intraepithelial neoplasia among the HPV-negative group (z = 0.714, difference not significant). Pathologist B diagnosed any cytologic abnormality in 46% of HPVpositive subjects, compared with 25% diagnosed by the private cytology laboratory (z = 1.2; difference not significant). There was also no significant difference found between the proportion of those subjects with a diagnosis of any cytologic abnormality between pathologist B and the private cytology laboratory among the HPVnegative group (z = 0.15, difference not significant). When readings between the two groups were compared by each HPV category, the significant differences found between the two readings remained similar for each HPV group (data not shown). In addition, the presence or absence of endocervical cells did not influence the differences in diagnosis on reviews. Comment
This study demonstrates that a significant proportion of HPV-positive women in our study were diagnosed as having cervical intraepithelial neoplasia when their Papanicolaou smears were rescreened. In contrast, cytologic rescreening from HPV-negative women did not result in a significant proportion of additional abnormal diagnoses. This association was less clear for diagnosing the category any cytologic abnormality because pathologist A was more likely than the private cytology laboratory to identify atypia on smears from HPV DNA-positive women, whereas no such association was found for pathologist B. However, the clinical implications for atypia are controversial and this cytologic category is considered less predictive of a significant lesion. Our findings suggest that the reported insensitivity of the Papanicolaou smear is due in part to screening error. Currently, the Papanicolaou smear is the primary
July 1991 Am J Obstet Gynecol
screening tool to identify women with cervical disease, specifically intraepithelialneoplasia. Although this kind of cytologic screening remains the most cost-effective method, numerous studies have identified the limitations of the Papanicolaou smear, including substantially lower sensitivity rates than those of colposcopy and histology. 16 These limitations have been attributed to sampling error, difficulty in cytologic interpretation, and fatigue (in both cytotechnicians and pathologists) resulting in screening error. II. I., Although most laboratories set high standards for quality assurance, previous studies have documented that reviews of cytologic smears will result in a fivefold increase in the number of significant abnormalities as compared with the results from the initial routine screening. 16 Our study both confirms and extends these findings. We showed that additional abnormalities were found when cytologic smears were reviewed. However, when the rescreening results were analyzed by HPV status, the majority of additional significant abnormalities were present in the HPV DNA-positive group even though the reviewers were blinded to the HPV DNA status. This finding suggests that it would be more efficacious to review smears from this positive population. Although our findings apppear to have important implications for the cytologic identification of cervical intraepithelial neoplasia, these results must be interpreted with caution. First, the clinical significance of the reviews is unknown because we did not perform colposcopic examinations and obtain histologic confirmations. It is possible that some of our reviews reflect erroneous diagnoses; however, this is unlikely since the positive predictive value of the Papanicolaou smear to identify disease is considered to be relatively high." Our pathologists used strict criteria to diagnose disease. In addition, statistical analysis of our data indicated that the association between HPV DNA status and abnormal reviews was greater than chance alone. Second, our findings may not necessarily be generalizable because the quality of screening can vary considerably among laboratories. 16 As a result, it is possible that our findings simply reflected a higher-quality screening process. However, we feel that our results were not biased by this possibility for several reasons. First, it should be stressed that, although the reviewers were without knowledge of the HPV status, they were aware that they were reviewing slides for a study that possibly reflected an increased "effort per slide" and were reviewing slides without the conventional pressures associated with rapid bulk screening. This situation may reflect a similar situation found when slides are reviewed for routine quality assurance measures. In addition, if the reviewers were significantly "better" at screening, it would have been more likely that additional abnormalities would have been found propor-
Volume 165 :'>lumber I
tionately in both groups. Finally, the percent agreement between the pathologists reflected "differences in opinion" similar to those found in other studies. I7 To help remove the "type-of-reader" bias, we selected two types of review: One involved an independent pathologist and the other involved a screening cyto-technicianpathologist team. The two groups occasionally disagreed; however, both were significantly more likely to find additional abnormalities in the HPV-positive group and not in the HPV-negative group. Another factor that influences the generalizability of our findings is the population examined. Our population consisted of sexually active young women with a high prevalence of HPV and cervical intraepithelial neoplasia (14% had cervical intraepithelial neoplasia on the reviewed smears). The prevalence of these factors may affect the outcome in such studies. Last, our abnormal smears with cervical intraepitheIial neoplasia were associated with a high rate of DNA positivity (86%). This rate is higher than that reported in other studies,I8. 19 which may reflect the high prevalence of these specific HPV types in our population and the association between these types and abnormal cytologic findings. In summary, reviewing cytologic results in patients who are HPV DNA positive increased the number of diagnosed cases of cervical intraepithelial neoplasia. This suggests that in a population at high risk for HPV and cervical intraepithelial neoplasia but with normal cytologic findings, routine screening for HPV DNA would be beneficial if the cytologic findings from HPV DNA-positive women are reviewed. Longitudinal studies that include colposcopy and histology are currently under way to confirm these findings. We thank Dr. Barbara Winkler for her assistance in the reviews of the Papanicolaou smears. We also thank Roy Rodriguez for his assistance in manuscript and figure preparation. REFERENCES I. Broker TR, Botchan M. Papillomaviruses: retrospectives and prospectives cancer cells. Cold Spring Harbor Symp Quant Bioi 1986;4: 17-36. 2. Durst M, Gissman L, Ikenberg H, Zur Hausen H. A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc Nat! Acad Sci USA 1983;80:3812-5.
Cytologic rescreening in HPV-positive women
71
3. Meisels A, Fortin R, Roy M. Condylomatous lesions of the cervix: cytologic, colposcopic and histopathologic study. Acta Cytol 1977;21:379-90. 4. Shulman]], Ley ton M, Hamilton R. The papanicolaou smear: an insensitive case-finding procedure. AM] OBSTET GV:\ECOL 1974;120:446-51. 5. Waeckerlin RW, Potter N], Cheatham GR]r. Correlation of cytologic, colposcopic, and histologic studies with immunohistochemical studies of human papillomavirus structural antigens in an unselected patient population. AM] OBSTET GVI\:ECOL 1988; 158: 1394-402. 6. Ritter DB, Kadish AS, Vermund SH, Romney SL, Villari D, Burk RD. Detection of human papillomavirus deoxyribonucleic acid in exfoliated cervicovaginal cells as a predictor of cervical neoplasia in a high-risk population. AM ] OBSTET GV:'>IEcoLl988;159:1517-25. 7. Roman A, Fife KH. Human papillomaviruses: are we ready to type? Clin Microbiol Rev 1989;2:166-90. 8. Martinez], Smith R, Farmer M, et al. High prevalence of genital tract papillomavirus infection in female adolescents. Pediatrics 1988;82:604-8. 9. Schneider A, Meinhardt G, De-Villiers EM, Gissmann L. Sensitivity of the cytologic diagnosis of cervical condyloma in comparison with HPV-DNA hybridization studies. Diagn Cytopathol 1987;3:250-5. 10. Moscicki AB, Palefsky], Gonzales], Schoolnik GK. Human papillomavirus infection in sexually active adolescent females: Prevalence and risk factors. Pediatr Res 1990; 28:507-13. II. Ferenzy A, Winkler B. Cervical intra-epithelial neoplasia and condyloma. In: Kurman RJ, ed. Blaustein's pathology of the female genital tract. 3rd ed. New York: SpringerVerlag, 1987:177-217. 12. Product information. ViraPap human papillomavirus (HPV) DNA detection kit. Gaithersburg, Maryland: Life Technologies, Bethesda Research Laboratories, 1989. 13. ViraType HPV DNA typing kit manuals. Gaithersburg, Maryland: Life Technologies, Bethesda Research Laboratories, 1989. 14. Genack L, Schumann GB. Chain of events in papanicolaou smear testing: impact on quality assurance. Diagn CytopathoI1989;5:221-7. 15. Bonfiglio TA. Quality assurance in cytopathology recommendations and ongoing quality assurance activities of the American society of clinical pathologists. Acta Cytol 1989;33:431-4. 16. Penner DW. An overview of the college of American pathologists' programs in surgical pathology and cytopathology data summary of diagnostic performance in cervical cytopathology. Acta Cytol 1989;33:439-42. 17. Horn PL, Lowell DM, LiVolsi VA, Boyle CA. Reproducibility of the cytologic diagnosis of human papillomavirus infection. Acta Cytol 1985;29:692-4. 18. de Villiers E-M, Wagner D, Schneider A, et al. Human papillomavirus infections in women with and without abnormal cervical cytology. Lancet 1987;2:703-6. 19. Kiviat NB, Koutsky LA, Paavonen .J A, et al. Prevalence of genital papillomavirus infection among women attending a college student health clinic or a sexually transmitted disease clinic.] Infect Dis 1989; 159:293-302.
Key words: Human papillomavirus, cytology, rescreening The strong association between cervical neoplasia and human papillomavirus (HPV) infection" 2 has stimulated interest in HPV diagnostic tests. Currently, the Papanicolaou smear is used as the primary screening test for cervical disease, including condyloma and intraepithelial neoplasia." However, the Papanicolaou smear appears to be relatively insensitive, detecting only 50% to 80% of the abnormalities found by histologic examination of cervical biopsy specimens obtained by colposcopy. Hi Routine colposcopy remains a time-consuming and cost-ineffective means to identify From the Department oj Pediatrics," the Departments oj Laboratory Medicine and Stomatology," and the Department of Anatomic Patholog),,' Ulliversit~ of California, San Francisco, and the Department oj Im1Jlunology and Medical Microbiology, Stanford University," Supported in part b~ funds provided by the State of California and allocated on tlte recommendations oj tlte University-wide Task Force on Acquired IlIlmunodeficienc~ Syndrome, a Howard Hughes lHediml Institute fellowship award, Hampton Medical School fund to tlte University of California, San Francisro (No. MSC 48), Bureau of Maternal and Child Health and Resource Development Training Grant No. MC/000978, an Acquired Immunodeji(ienc~ S)ndrome Clinical Research Center grant to the University ofCalifiJntia, San Francisco (National Institute of Acquired itnmullodejiciency Syndrome grant No. P01AI21912), and University of California Cancer Research Coordinating Committee. Receivedfor publication August 9, 1990; revised January 11, 1991 ; accepted/anuary 18,1991. Reprint requests: Anna-Barbara Moscichi, MD, 400 Parnassus Ave., Room AC-Ol, Box 0374, Division of Adolescent Medicine, Univenity of CalifiJrllia-San Francisco, San Francisco, CA 94143. 611128066
cervical disease. Therefore tests that identify HPV deoxyribonucleic acid (DNA) have been developed in the hope that these techniques may augment our ability to precisely identify women who are infected with HPV and who may be at risk for cervical intraepithelial neoplasia.' The indications for and clinical utility of these DNA detection tests have not yet been established. However, the correlation between HPV DNA status and cytologic abnormalities is not absolute"; i.e., some women with abnormal Papanicolaou smears will not have detectable HPV DNA and, conversely, many patients with detectable HPV DNA appear to have normal Papanicolaou smears. Therefore, whereas HPV detection tests could augment cytologic testing, they are not equivalent replacements. It has been suggested that HPV DNA testing may be used to identify women at high risk for cervical intraepithelial neoplasia. Previous studies have reported that when cytologic smears from women who are HPV positive and who have "normal" cytologic results are reviewed additional cytologic abnormalities (including intraepithelial neoplasia) can be detected."" These findings seem to indicate that the combined use of HPV DNA testing and the Papanicolaou smear might improve the identification of women with cervical disease. Unfortunately, the results of these studies are difficult to interpret because the smears were reviewed by pathologists who knew the HPV DNA status of the spec-
67
68
Moscicki et al.
July 1991 Am J Obstet Gynecol
Table I. Population characteristics Age (yr, mean ± SE) Age at initial sexual intercourse (mean ± SE) No. of lifetime sexual partners 0-3 4-10 2: 11
No. of sexual partners in past 2 mo (mean ± SE) Race-ethnicity (%) White Black Hispanic Other Reason for visit (%) Family planning Sexually transmitted disease related
17.1 ± 1.5 15.5 ± 1.5
65% 25%
10%
1.2 ± 0.7
71% 5%
10%
14% 90% 10%
imens at the time the reviews were conducted, possibly biasing the outcome. We describe a study that examines the association between HPV DNA status and diagnostic interpretations on review of cytologic results by two independent pathologists who were without knowledge of the HPV DNA results. Methods
This study was part of a large epidemiologic study examining the prevalence of HPV DNA in sexually active adolescents. 10 All female adolescents (aged 13 to 19 years) who were attending a private, suburban family planning clinic and having a pelvic examination for any reason were asked to enter this study according to the guidelines set by the Committee for Human Research to screen for the presence of cervical HPV DNA. The refusal rate was < 10%. Populations attending the clinic were predominantly middle-class and the racialethnic distribution of the clinic was 60% white, 20% black, 20% Hispanic, and 10% other. All female adolescents who entered the study were asked to complete a self-administered questionnaire before examination; this included demographic and sexual behavior information. Pelvic examination included a Papanicolaou smear obtained with a Dacron swab and spatula followed by an endocervical and exocervical sample for HPV DNA with a Dacron swab. Slides for cytologic testing were immediately fixed in alcohol and were sent to a contracted private cytology laboratory for initial review per routine protocol. Slides from 134 consecutive subjects during the second month through the fourth month of the study were identified and released for review by two independent pathologists. Although the two pathologists were without knowledge of the HPV DNA status of each specimen and were not apprised of the specific purpose of the review, they were asked to carefully review the slides for the epidemiology study.
Cytology. Papanicolaou smears were prepared by standard methods at the private cytology laboratory and were then screened according to the following routine protocol. All smears were first screened by a cytotechnician. Those smears found to have abnormal findings including inflammation, atypia, and changes suggestive of condyloma and neoplasia, as well as 10% of the benign smears, were then reviewed by a supervising pathologist. Cytologic diagnoses from the private laboratory were categorized into one of the following: benign, atypia, or cervical intraepithelial neoplasia with or without koilocytosis. The term cervical intraepithelial neoplasia is used for easier reference in this article although it is understood that cytologic testing cannot determine the site of origin of neoplastic cells. Pathologist A then reviewed each slide independently without knowledge of either the HPV DNA status or the private laboratory's cytologic diagnosis at X 10 magnification with higher magnification used for the examination of questionable or abnormal areas. For comparison, the second pathologist (pathologist B) used a method more common to cytology laboratories; that is, a cytotechnician screened the smears first and then pathologist B verified the diagnosis of all the smears. The diagnoses reported by the reviewing pathologists were categorized as described here. Standard criteria were used to diagnose neoplasia and koilocytosis. II Atypia was diagnosed when rare abnormal cells (neoplastic or koilocytes) were found or if some but not all of the features of cervical intraepithelial neoplasia or koilocytosis were found on Papanicolaou smears. Since cytologic correlates for condyloma are controversial, cells suggestive of condyloma (koilocytes) but without the nuclear features of neoplasia also were categorized as atypia. Slides that appeared to have been air-dried or inadequately stained were excluded from the analysis. The presence or absence of endocervical cells was noted by the reviewing pathologists. HPV DNA hybridization. HPV DNA was identified with a commercially available ribonucleic acid-DNA dot blot hybridization technique (ViraPap).12 Cervical swabs for HPV were immediately placed into 1 ml of the sample transport medium and refrigerated at 4° C for no longer than 2 weeks before processing. Samples were processed with the ViraPap method according to the manufacturer's recommendation. 12 This method allows for identification of HPV types 6, 11, 16, 18, 31, 33, and 35. Unused portions of the samples that were positive for the HPV probe mixture were then studied under similar conditions to determine the HPV type more precisely. Samples were filtered onto three separate membranes, and each membrane was then hybridized with one of three separate radioactively labeled probe mixtures: type 6111, type 16/18, or type 31133/35.1l
Cytologic rescreening in HPV-positive women
Volume HiS :-lumber I
69
Table II. Comparison of cytologic diagnoses among pathologists A and B and the private cytologic laboratory for HPV-positive and HPV-negative subjects Primtate cytologic laboratory: HPV /)()sitivf Benign Pathologist A Benign Atypia Cervical intraepithelial neoplasia Pathologist B Benign Atypia Cervical intraepithelial neoplasia
9 :)
I
Atypical
I
Cervical intraepitltelia/ /teoll/asia
0 I
4
"
13 3 2
0 :3 :3
Data analysis. For each of the reviewers, X" tests were performed to examine the relationship between HPV DNA status and diagnosis. Interrater reliability is reported for agreement between the two reviewing pathologists. The effect of rescreening was assessed by calculating z scores. This test examined whether rescreening of HPV-positive subjects and HPV-negative subjects resulted in a significant difference in the proportion of subjects with a diagnosis of cervical intraepithelial neoplasia by the private cytology laboratory (first screen), when compared with diagnoses assigned by pathologist A or B (rescreens). Because the private cytology laboratory did not diagnose any cases of cervical intraepithelial neoplasia, a second variable for cytologic diagnosis was created for comparison. This variable compared only two categories: benign and any abnormality (atypia or cervical intraepithelial neoplasia). The z test was also performed to examine the difference in the proportion of subjects with any abnormality. Results
Population characteristics. Table I describes the demographic characteristics of the subjects including sexual behaviors and reasons for visit. Twenty-four subjects (19%) had positive HPV DNA results: 5 were positive for group 6111, 11 for group 16118,4 for group 31/33/35, and 1 for both group 16118 and group 31/33/35. Three subjects had undetermined types with the described dot blot method. Interrater reliability between the pathologists. The overall interrater reliability between the two pathologists with three cytologic categories (benign, atypia, and cervical intraepithelial neoplasia with or without koilocytosis) was 79% (K = 0.28, P = 0.001). When categories were subdivided into benign and any cytologic abnormality, the interrater reliability was 86% (K = 0.51, P = 0.001). For the latter analysis, the interrater reliability was 75% (K = 0.51, P < 0.002) in the HPVpositive group and 89% (K = 0.208, P < 0.03) in the HPV-negative group.
Privatate cytologic laboratory: HPV negative Benign
I
Atypical
0 0 0
92 6
I 0
0
2
0 0 0
93 4 I
I
Cervical intraepithelia/ neoplasia 0 0 0 0 0 0
Comparison of cytologic diagnosis and HPV DNA status. There was a significant association between abnormal cytologic findings and positive HPV DNA status for the private cytology laboratory, pathologist A, and pathologist B. The private cytology laboratory found that none of the patients had cervical intraepithelial neoplasia; however, 6 of 24 HPV-positive suqjects had atypia, compared with 3 of 101 HPV-negative subjects (X 2 = 10.98, P < 0.0009). Pathologist A diagnosed abnormal cytologic findings in 15 of 24 women in the HPV -positive group (9 with cervical intraepithelial neoplasia and 6 with atypia) compared with 8 of 101 women in the HPV -negative group (2 with cervical intraepithelial neoplasia and 6 with atypia) (X" = 42.2, P < 0.0001). A similar association was found for pathologist B; 11 of 24 HPV-positive women had abnormal cytologic findings (5 with cervical intraepithelial neoplasia and 6 with atypia) and 7 of 101 HPV -negative women had abnormal cytologic findings (2 with cervical intraepithelial neoplasia and 5 with atypia) (X 2 = 24.59, P < 0.0001). Comparison of cytologic findings among the private laboratory and pathologists A and B within HPVpositive and HPV-negative groups. Table II compares the cytologic diagnoses of the private cytology laboratory with those of pathologist A and pathologist B by HPV DNA ,[dIllS. Of the cervical intraepithelial neoplasia cases di,H~ll()sed, only 2 had grade 2; the rest had grade I. h< ,I h (~l'(" of grade 2 were assigned the same diagnosis l)v IJd~h pathologist A and pathologist B; both of these \\'Onllll were HPV positive. Pathologist A diagnosed 38% of the HPV-positive patients as having cervical intraepithelial neoplasia, compared with none diagnosed by the private cytology laboratory (z = 3.09, P < 0.005); pathologist A diagnosed any cytologic abnormality in 63% of the HPV-positive patients, compared with 25% by the private cytology laboratory (z = 2.39, P < 0.03). In contrast, there was no significant difference in the proportion of abnormal cytologic smears diagnosed in the HPV-negative group. Specifically, 2% of HPV-negative subjects were diagnosed to
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Moscicki et al.
have cervical intraepithelial neoplasia by pathologist A and none were diagnosed by the private cytology laboratory (z = 0.714, difference not significant). Similarly, no difference was found for diagnosing "any cytologic abnormality" in the HPV-negative group (z = 0.15; difference not significant). In addition, 5 of the 6 subjects who were HPV positive and were diagnosed as having atypia by the private cytology laboratory had cervical intraepithelial neoplasia. Two of the subjects with atypia and who were HPV negative had cervical intraepithelial neoplasia. Pathologist B also found significantly more subjects with cervical intraepithelial neoplasia on rescreening the HPV-positive group than were diagnosed by the private cytology laboratory, 21 % by pathologist B versus 0% by the private cytology laboratory (z = 1.98, P < 0.(5). However, there was no difference between the private cytology laboratory and pathologist B in the proportion of subjects with a diagnosis of cervical intraepithelial neoplasia among the HPV-negative group (z = 0.714, difference not significant). Pathologist B diagnosed any cytologic abnormality in 46% of HPVpositive subjects, compared with 25% diagnosed by the private cytology laboratory (z = 1.2; difference not significant). There was also no significant difference found between the proportion of those subjects with a diagnosis of any cytologic abnormality between pathologist B and the private cytology laboratory among the HPVnegative group (z = 0.15, difference not significant). When readings between the two groups were compared by each HPV category, the significant differences found between the two readings remained similar for each HPV group (data not shown). In addition, the presence or absence of endocervical cells did not influence the differences in diagnosis on reviews. Comment
This study demonstrates that a significant proportion of HPV-positive women in our study were diagnosed as having cervical intraepithelial neoplasia when their Papanicolaou smears were rescreened. In contrast, cytologic rescreening from HPV-negative women did not result in a significant proportion of additional abnormal diagnoses. This association was less clear for diagnosing the category any cytologic abnormality because pathologist A was more likely than the private cytology laboratory to identify atypia on smears from HPV DNA-positive women, whereas no such association was found for pathologist B. However, the clinical implications for atypia are controversial and this cytologic category is considered less predictive of a significant lesion. Our findings suggest that the reported insensitivity of the Papanicolaou smear is due in part to screening error. Currently, the Papanicolaou smear is the primary
July 1991 Am J Obstet Gynecol
screening tool to identify women with cervical disease, specifically intraepithelialneoplasia. Although this kind of cytologic screening remains the most cost-effective method, numerous studies have identified the limitations of the Papanicolaou smear, including substantially lower sensitivity rates than those of colposcopy and histology. 16 These limitations have been attributed to sampling error, difficulty in cytologic interpretation, and fatigue (in both cytotechnicians and pathologists) resulting in screening error. II. I., Although most laboratories set high standards for quality assurance, previous studies have documented that reviews of cytologic smears will result in a fivefold increase in the number of significant abnormalities as compared with the results from the initial routine screening. 16 Our study both confirms and extends these findings. We showed that additional abnormalities were found when cytologic smears were reviewed. However, when the rescreening results were analyzed by HPV status, the majority of additional significant abnormalities were present in the HPV DNA-positive group even though the reviewers were blinded to the HPV DNA status. This finding suggests that it would be more efficacious to review smears from this positive population. Although our findings apppear to have important implications for the cytologic identification of cervical intraepithelial neoplasia, these results must be interpreted with caution. First, the clinical significance of the reviews is unknown because we did not perform colposcopic examinations and obtain histologic confirmations. It is possible that some of our reviews reflect erroneous diagnoses; however, this is unlikely since the positive predictive value of the Papanicolaou smear to identify disease is considered to be relatively high." Our pathologists used strict criteria to diagnose disease. In addition, statistical analysis of our data indicated that the association between HPV DNA status and abnormal reviews was greater than chance alone. Second, our findings may not necessarily be generalizable because the quality of screening can vary considerably among laboratories. 16 As a result, it is possible that our findings simply reflected a higher-quality screening process. However, we feel that our results were not biased by this possibility for several reasons. First, it should be stressed that, although the reviewers were without knowledge of the HPV status, they were aware that they were reviewing slides for a study that possibly reflected an increased "effort per slide" and were reviewing slides without the conventional pressures associated with rapid bulk screening. This situation may reflect a similar situation found when slides are reviewed for routine quality assurance measures. In addition, if the reviewers were significantly "better" at screening, it would have been more likely that additional abnormalities would have been found propor-
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tionately in both groups. Finally, the percent agreement between the pathologists reflected "differences in opinion" similar to those found in other studies. I7 To help remove the "type-of-reader" bias, we selected two types of review: One involved an independent pathologist and the other involved a screening cyto-technicianpathologist team. The two groups occasionally disagreed; however, both were significantly more likely to find additional abnormalities in the HPV-positive group and not in the HPV-negative group. Another factor that influences the generalizability of our findings is the population examined. Our population consisted of sexually active young women with a high prevalence of HPV and cervical intraepithelial neoplasia (14% had cervical intraepithelial neoplasia on the reviewed smears). The prevalence of these factors may affect the outcome in such studies. Last, our abnormal smears with cervical intraepitheIial neoplasia were associated with a high rate of DNA positivity (86%). This rate is higher than that reported in other studies,I8. 19 which may reflect the high prevalence of these specific HPV types in our population and the association between these types and abnormal cytologic findings. In summary, reviewing cytologic results in patients who are HPV DNA positive increased the number of diagnosed cases of cervical intraepithelial neoplasia. This suggests that in a population at high risk for HPV and cervical intraepithelial neoplasia but with normal cytologic findings, routine screening for HPV DNA would be beneficial if the cytologic findings from HPV DNA-positive women are reviewed. Longitudinal studies that include colposcopy and histology are currently under way to confirm these findings. We thank Dr. Barbara Winkler for her assistance in the reviews of the Papanicolaou smears. We also thank Roy Rodriguez for his assistance in manuscript and figure preparation. REFERENCES I. Broker TR, Botchan M. Papillomaviruses: retrospectives and prospectives cancer cells. Cold Spring Harbor Symp Quant Bioi 1986;4: 17-36. 2. Durst M, Gissman L, Ikenberg H, Zur Hausen H. A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc Nat! Acad Sci USA 1983;80:3812-5.
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3. Meisels A, Fortin R, Roy M. Condylomatous lesions of the cervix: cytologic, colposcopic and histopathologic study. Acta Cytol 1977;21:379-90. 4. Shulman]], Ley ton M, Hamilton R. The papanicolaou smear: an insensitive case-finding procedure. AM] OBSTET GV:\ECOL 1974;120:446-51. 5. Waeckerlin RW, Potter N], Cheatham GR]r. Correlation of cytologic, colposcopic, and histologic studies with immunohistochemical studies of human papillomavirus structural antigens in an unselected patient population. AM] OBSTET GVI\:ECOL 1988; 158: 1394-402. 6. Ritter DB, Kadish AS, Vermund SH, Romney SL, Villari D, Burk RD. Detection of human papillomavirus deoxyribonucleic acid in exfoliated cervicovaginal cells as a predictor of cervical neoplasia in a high-risk population. AM ] OBSTET GV:'>IEcoLl988;159:1517-25. 7. Roman A, Fife KH. Human papillomaviruses: are we ready to type? Clin Microbiol Rev 1989;2:166-90. 8. Martinez], Smith R, Farmer M, et al. High prevalence of genital tract papillomavirus infection in female adolescents. Pediatrics 1988;82:604-8. 9. Schneider A, Meinhardt G, De-Villiers EM, Gissmann L. Sensitivity of the cytologic diagnosis of cervical condyloma in comparison with HPV-DNA hybridization studies. Diagn Cytopathol 1987;3:250-5. 10. Moscicki AB, Palefsky], Gonzales], Schoolnik GK. Human papillomavirus infection in sexually active adolescent females: Prevalence and risk factors. Pediatr Res 1990; 28:507-13. II. Ferenzy A, Winkler B. Cervical intra-epithelial neoplasia and condyloma. In: Kurman RJ, ed. Blaustein's pathology of the female genital tract. 3rd ed. New York: SpringerVerlag, 1987:177-217. 12. Product information. ViraPap human papillomavirus (HPV) DNA detection kit. Gaithersburg, Maryland: Life Technologies, Bethesda Research Laboratories, 1989. 13. ViraType HPV DNA typing kit manuals. Gaithersburg, Maryland: Life Technologies, Bethesda Research Laboratories, 1989. 14. Genack L, Schumann GB. Chain of events in papanicolaou smear testing: impact on quality assurance. Diagn CytopathoI1989;5:221-7. 15. Bonfiglio TA. Quality assurance in cytopathology recommendations and ongoing quality assurance activities of the American society of clinical pathologists. Acta Cytol 1989;33:431-4. 16. Penner DW. An overview of the college of American pathologists' programs in surgical pathology and cytopathology data summary of diagnostic performance in cervical cytopathology. Acta Cytol 1989;33:439-42. 17. Horn PL, Lowell DM, LiVolsi VA, Boyle CA. Reproducibility of the cytologic diagnosis of human papillomavirus infection. Acta Cytol 1985;29:692-4. 18. de Villiers E-M, Wagner D, Schneider A, et al. Human papillomavirus infections in women with and without abnormal cervical cytology. Lancet 1987;2:703-6. 19. Kiviat NB, Koutsky LA, Paavonen .J A, et al. Prevalence of genital papillomavirus infection among women attending a college student health clinic or a sexually transmitted disease clinic.] Infect Dis 1989; 159:293-302.
