The significance of high-risk human papillomavirus detection in women aged ≥ 50 years with atypical squamous cells of undetermined significance cytologic preparations


  • This article is a US Government work and, as such, is in the public domain in the United States of America.



The use of high-risk human papillomavirus (HRHPV) testing in the triage of women with a cytologic diagnosis of atypical squamous cells of undetermined significance (ASC-US) has gained widespread acceptance. To date, very little has been reported on the significance of the detection of HRHPV in elderly women.


Results of HRHPV testing performed on women aged ≥50 years were examined for a 20-month period. Reflex testing for HRHPV was performed on residual liquid-based cytology specimens from women who were diagnosed with ASC-US by using the Digene Hybrid Capture method. Follow-up information on women who had HRHPV detected was obtained from subsequent pathology reports (cytology and surgical).


HRHPV testing was performed on 762 specimens from women aged ≥50 years; virus was detected in 105 specimens (13.8%), and follow-up was available in 63 of those women (60%). Follow-up results included negative tests in 32 women (50.8% of those with follow-up available), low-grade squamous intraepithelial lesion in 26 women (41.3%), and high-grade squamous intraepithelial lesion (HSIL) in 5 women (7.9%). No glandular neoplasia or invasive carcinoma was identified. Relative light units/cutoff (RLU/CO) values ranged from 1 RLU/CO to 1705 RLU/CO; no significant associations were identified between RLU/CO values and follow-up results.


Although HRHPV was detected in a minority of samples, HSIL was present in 7.9% of those with HRHPV. This confirms that the presence of HRHPV in women aged ≥50 years with ASC-US specimens needs clinical follow-up. There were no significant differences in RLU/CO values between women with positive versus negative follow-up to allow for further discrimination for follow-up. Cancer (Cancer Cytopathol) 2007. Published 2007 by the American Cancer Society.

It has been demonstrated that the use of reflex testing for the presence of high-risk human papillomavirus (HRHPV) DNA for women who have a cervical smear result of atypical squamous cells of undetermined significance (ASC-US) is sensitive for the detection of significant underlying squamous abnormalities.1, 2 This approach has gained acceptance as a method of evaluation for women with ASC-US smears, and it is the preferred approach, when available, according to the 2001 guidelines of the American Society for Colposcopy and Cervical Pathology.3

In general, the prevalence of both cervical intraepithelial neoplasia and detectable HRHPV decreases as women age. However, there are scant data regarding the significance of the presence of HRHPV in older women when it is detected at the time of a concurrent ASC-US cervical smear. The objective of the current study was to examine the clinical significance of the detection of HRHPV in women aged ≥50 years.


This study was performed at Wilford Hall Medical Center, a tertiary care facility in San Antonio, Texas, and was granted exempt status by the Institutional Review Board. A search of the laboratory information system was performed for results of HRHPV testing from June 2002 through January 2004. HRHPV DNA testing was performed exclusively using Hybrid Capture II (Digene, Gaithersburg, Md) almost entirely as reflex testing on residual ThinPrep Pap Tests (Cytyc, Marlborough, Mass) that had a cytologic interpretation of ASC-US. For those patients who had HRHPV results, preceding ThinPrep Pap tests were sought, and those with a cytologic diagnosis of ASC-US were used in the study.

Results from women aged ≥50 years were included in this study. For women who had HRHPV detected, follow-up cytologic or surgical pathology results were sought through electronic pathology results and ranged from 6 months to 26 months from the initial result. The most significant follow-up finding was recorded and was categorized as normal, as low-grade squamous intraepithelial lesion (LSIL), as high-grade squamous intraepithelial lesion (HSIL), or as a more significant finding (carcinoma).

Descriptive statistics of follow-up are reported for the women who had ASC-US smears and positive HRHPV testing. Comparison of follow-up results for the overall group was performed using the Fisher exact test. In addition, the relative light units/cutoff (RLU/CO) values from the initial Hybrid Capture II testing were recorded for women who had follow-up results. The RLU/CO values were compared between groups of women with normal and abnormal follow-up using the Kruskal-Wallis test (comparing all 4 groups of follow-up results, as defined above), or the Mann-Whitney U test for condensing follow-up results into dichotomous variables (normal vs any squamous intraepithelial lesion [SIL]; LSIL or less vs HSIL and greater). Statistical analysis was performed using SPSS version 12 for Windows (SPSS, Chicago, Ill).


During the study period, HRHPV testing was performed on 7334 specimens. Of these, 762 specimens (10.4%) were from women aged ≥50 years, and HRHPV was detected in 105 (13.8%) of these specimens. The patients who had a cytologic interpretation of ASC-US and positive HRHPV testing ranged in age from 50 years to 91 years (median age, 56 years).

Follow-up pathology results were available for 63 of the 105 women (60%) who had HRHPV detected. This included repeat cytology only for 15 women and some form of surgical specimen for the remaining women. Normal follow-up was noted in 32 women (50.8%). Of the 31 women (49.2%) who had some type of abnormal follow-up, the majority (26 women; 41.3%) had LSIL identified; 5 women (7.9%) had HSIL identified, and each HSIL diagnosis was confirmed on a surgical specimen. Carcinoma and glandular neoplasia were not identified on follow-up of any in these women. HRHPV testing was performed on both cervical and vaginal ThinPrep Pap tests, and the results described above included all specimens (Table 1). Of the 63 patients who had follow-up, 8 patients (12.7%) had vaginal smears only. Of those 8 women, abnormalities were identified on follow-up in 4 women (50%) and included 3 women with LSIL and 1 woman with HSIL. The distribution of abnormalities between cervical and vaginal smears did not differ significantly (Fisher exact test; P = 1).

In women who had follow-up available, RLU/CO values ranged from 1 RLU/CO to 1705 RLU/CO. These values, categorized according to follow-up diagnoses, are depicted in Figure 1. For all potential groups, according to follow-up, no difference was observed when RLU/CO values were compared (normal, LSIL and HSIL; P = .46). A comparison of RLU/CO values for women who had normal results versus any SIL and for women who had LSIL or less versus HSIL and greater also revealed no significant associations (P = .383 and P = .169, respectively).

Figure 1.

Relative light unit/cutoff values (RLU/CO) for high-risk human papillomavirus (HRHPV) testing plotted against follow-up (F/U) results for women who had specimens that were classified as atypical squamous cells of undetermined significance and had positive HRHPV test results. There was no significant correlation between RLU/CO values and follow-up results. LSIL indicates low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion.

Table 1. Overall Follow-up Results Evaluated by Initial Specimen Source*
Follow-upNo. of specimens (%)
OverallCervical specimensVaginal specimens
  • LSIL indicates low-grade squamous intraepithelial lesion (includes human papillomavirus-related changes and mild dysplasia); HSIL, high-grade squamous intraepithelial lesion (encompasses moderate and severe dysplasia).

  • *

    The differences were not statistically significant by source (P = 1; Fisher exact test).

Normal32 (50.8)28 (50.9)4 (50)
LSIL26 (41.3)23 (41.2)3 (37.5)
HSIL5 (7.9)4 (7.3)1 (12.5)


SIL was identified on follow-up in nearly 50% of women aged ≥50 years who had ASC-US cytologic specimens and positive reflex HRHPV testing. A high-grade lesion was identified in 7.9% of these women.

In comparing our results with those from the ASC-US-Low-Grade Squamous Intraepithelial Lesion Triage Study (ALTS) trial, a similar rate (approximately 50%) of squamous intraepithelial lesions in women with ASC-US smears and positive HRHPV testing was identified.1 However, in the ALTS study, 27.5% of women had a follow-up diagnosis of HSIL or worse (HSIL+), compared with 7.9% in this our study. In the ALTS study, the mean age was 29 years; results in older women were not reported specifically. The higher rate of HSIL+ in the ALTS study probably was because of the inclusion of a greater number of young women, in whom a higher prevalence of high-grade lesions would be expected.

Studies of squamous atypia on cervical and vaginal smears from older women have revealed varying rates of dysplasia on follow-up; and significant squamous abnormalities, in general, are identified less frequently among older women compared with younger women. Flynn and Rimm analyzed follow-up information from 150 women aged >50 years and compared their results with those from a control cohort of women aged <50 yearse.4 Those authors reported SIL in 12.8% of the older group compared with 29.5% in the control group and HSIL+ rates of 4.8% and 7.2%, respectively. Kaminsky et al. evaluated follow-up information on 115 patients aged >50 years who had squamous atypia on a Pap smear.5 In that study, SIL was identified in only 5 women (4.3%), and HSIL was identified in only 2 women (1.7%). The current results differ from prior studies, because we used HRHPV triage after a cytology result of ASC-US. We observed much higher rates of LSIL on follow-up compared with these prior investigations, and we observed slightly higher rates of HSIL.

Viral load, as determined by RLU/CO values at the time of reflex testing, is not used currently in the treatment algorithm for women with ASC-US smears because of the lack of association between viral load and clinical outcome.6–8 Our findings demonstrated a broad range of RLU/CO values without any noticeable correlation between viral load and the presence or absence of SIL on follow-up, reinforcing the lack of a significant clinical correlation between viral load and abnormal follow-up in this population of older women.

The overall results from this study were primarily from women with cervices, although 8 of the specimens (12.7%) were from vaginal smears. Currently, reflex testing is approved by the U.S. Food and Drug Administration for use with cervical specimens only; however, our experience is that providers often also request testing on vaginal specimens. The overall rate of abnormality did not differ significantly for either type of specimen for women who had HRHPV detected. The use of HRHPV testing as triage for women with ASC-US vaginal specimens is an area that warrants further investigation.

In summary, squamous abnormalities were detected in nearly 50% of women aged ≥50 years when HRHPV was detected on reflex testing for ASC-US specimens, confirming that an ASC-US specimens with HRHPV detected for women in this age range is clinically significant. Assessment of viral load through RLU/CO values did not predict which patients were more likely to have a significant lesion on follow-up.