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Reflex Human Papilloma Virus Infection Testing Detects the Same Proportion of Cervical Intraepithelial Neoplasia Grade 2–3 in Young Versus Elderly Women Isam A. Eltoum, M.D., M.B.A.1 David C. Chhieng, M.D., M.B.A.1 Janie Roberson, C.T.1 Drew McMillon, B.S.E.E.1 Edward E. Partridge, M.D.2
BACKGROUND. The American Society for Colposcopy and Cervical Pathology recently recommended triage of women with atypical squamous cells of undetermined significance (ASCUS) through reflex human papilloma virus (HPV) infection testing. Because age is an important determinant of the incidence of both ASCUS and HPV infection, the current study was conducted to determine the impact of age on reflex HPV infection test performance.
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METHODS. The authors reviewed all liquid-based Papanicolaou tests submitted for reflex HPV infection testing to the University of Alabama Medical Center during the period from January 2002 to January 2004. To assess the impact of age on reflex HPV infection testing, women were divided into 6 age groups (⬍ 25, 25–34, 35– 44, 45–54, 55– 64, ⬎64 years). Trend of different diagnostic categories with age was assessed using the chi-square test for linear trend. As a measure of test performance, the proportions of cervical intraepithelial neoplasia (CIN) lesions confirmed by biopsy through reflex testing (of the total CIN lesions detected in the study population) were then compared among different age groups. RESULTS. During the study period, 25,164 ThinPrep tests were performed during the study period, with 2672 (11%) cases of ASCUS and 1371 (5%) cases of squamous intraepithelial lesions (SILs). A total of 2309 (86%) ASCUS cases were tested for HPV infection, of which 789 (34%) were positive. Biopsy specimens were obtained from 831 patients with SILs and from 316 patients with HPV⫹ASCUS. Of 246 patients with CIN Grade 2–3 (CIN2–3) detected on biopsy, 36 (15%) were triaged through reflex HPV infection testing and the rest were triaged through cytologic diagnosis of SILs. Despite the trend in the prevalence of HPV⫹ASCUS and SILs with increasing age (P ⬍ 0.05), there was no significant difference in the proportion (range, 9 –23%) of CIN2–3 detected through reflex HPV infection testing in different age groups. CONCLUSIONS. Reflex HPV infection testing performed similarly for women in different age groups, including perimenopausal and postmenopausal women. Cancer (Cancer Cytopathol) 2005;105:194 – 8. © 2005 American Cancer Society.
Department of Pathology, the University of Alabama at Birmingham, Birmingham, Alabama.
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Department of Obstetrics and Gynecology, the University of Alabama at Birmingham, Birmingham, Alabama.
KEYWORDS: atypical squamous cells of undetermined significance, human papilloma virus infection, old age, cervical intraepithelial neoplasia. Address for reprints: Isam A. Eltoum, M.D., Department of Pathology, the University of Alabama at Birmingham, 19th 7 Avenue South-Kracke Building 609, Birmingham, AL 35233; Fax: (205) 934-7094; E-mail:
[email protected] Received December 21, 2004; revision received February 8, 2005; accepted February 14, 2005.
ased on the findings of several clinical trials,1–3 the American Society of Colposcopy and Cervical Pathology (ASCCP) recently recommended the use of human papilloma virus infection (HPV)DNA reflex testing for patients with atypical squamous cells of undetermined significance (ASCUS).4 This shift in the management of cervical carcinoma precursors is subsequent to the development of
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© 2005 American Cancer Society DOI 10.1002/cncr.21061 Published online 17 May 2005 in Wiley InterScience (www.interscience.wiley.com).
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new generations of HPV infection tests and to an increase in our understanding of the natural history of HPV infection and its associated lesions. As with many other infections, age is an important determinant of HPV infection and its associated cervical squamous intraepithelial lesions (SILs). Irrespective of the frequency of exposure, the highest prevalence of infection occurs in the age group 18 –30 years old and declines thereafter.5–7 Recently, a “second peak” of HPV infection prevalence has occurred among women ⱖ 50 years in several populations.8,9 In addition to a tendency of the ASCUS-to-SIL ratio to increase with age,10 this second peak of infection necessitates the evaluation of the performance of reflex HPV infection testing among older women. The objectives of the current study are to assess the age trends of SILs, ASCUS, and HPV⫹ASCUS and to determine the impact of these trends on the proportions of cervical intraepithelial neoplasia (CIN) lesions detected through reflex HPV-DNA testing among different age groups.
MATERIALS AND METHODS Setting This is a retrospective cytohistologic correlational study that has been approved by the University of Alabama at Birmingham (UAB) institutional review board. The UAB Medical Center (UABMC) is Alabama’s major tertiary care center serving approximately 35,000 patients annually. The UAMBC laboratory receives approximately 23,000 Papanicolaou (Pap) tests a year with an average ASCUS rate of 10%, a SIL rate of 5%, and an ASCUS-to-SIL ratio of 2:1. The mean false-negative fraction is 5%, calculated as (estimated false negative)/(true positive ⫹ estimated false negative) using low-grade SIL (LGSIL) as a cutoff value for positive cases. Since August 2001, ThinPrep Pap tests (Cytyc, Boxborough, MA) have increased gradually to reach a current rate of 90% of our total volume of Pap tests. The remaining Pap tests are conventional smears received mostly from the colposcopy clinic at UABMC. Slides were screened by 7 cytotechnologists (range of experience, 5–25 years) and reviewed by 5 cytopathologists (range of experienece, 3–13 years). Patients were triaged according to the new ASCCP guidelines.4 Most of the colposcopies were performed at UAB and nearby clinics. Three surgical pathologists read the cervical biopsy results (ranged of experience, 4 –15 years). Clinical and laboratory data were archived in a laboratory information system (Cerner Classic, Kansas City, MO).
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Study Population All ThinPrep Pap tests received from January 2002 to January 2004 were identified. Conventional cervical smears and all vaginal (conventional and ThinPrep) specimens were excluded from the study. Only biopsy results obtained after the ThinPrep Pap test were reviewed and correlated with cytologic and virologic findings. If patients had multiple biopsies or Pap tests, the worst diagnosis was considered in the analysis. An HPV infection test for high-risk viral DNA (HPV type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 68) was performed using the Digene HC2 method (Digene Corporation, Gaithersburg, MD). Patients were considered to be positive for HPV infection at the 1 pg/mL level.
Statistical Analysis For statistical analysis, extracted data were reentered in the SPSS statistical program (SPSS Inc., Chicago, IL). Women were grouped into 6 age groups: ⬍ 25, 25–34, 35– 44, 45–54, 55– 64, and ⬎64 years old. Positive rates for SIL and ASCUS (prevalence) and their trends with age were assessed using the chi-square test for linear trend. Rates of HPV⫹ASCUS (triage rate) and HPV⫹ASCUS that were positive for CIN on biopsy (true positive ratio) were calculated and its trend with age was assessed using the chi-square test for linear trend. In addition to the ASCUS-to-SIL ratio, two other ratios were calculated and their trend with age was assessed using the chi-square test for linear trend, i.e., the HPV⫹ASCUS-to-SIL ratio (ASCUS cases that were HPV positive to the SIL ratio) and the CINrx-to-CIN ratio (i.e., the ratio of CIN detected through reflex HPV infection testing to the ratio of CIN detected through SIL triage [LGSIL and above ⫺ atypical squamous cells of undetermined significance, cannot rule out a high grade lesion (ASC-H) ⫺ included in the high-grade lesion]). Lastly, the proportions of CIN Grade 2–3 (CIN2–3) detected in biopsy specimens through reflex HPV infection testing, LGSIL, or high-grade SIL (HSIL)⫹ among different age groups were compared using the chi-square test. Because of the few patients with CIN2–3 in the ⬎44-year-old age group, these patients were collapsed into one.
RESULTS Figure 1 is a flow chart of ThinPrep tests received during the study period. A total of 1147 biopsy specimens were received, representing 60% (831 of 1371) of women triaged through the SIL/ASC-H arm and 40% (316 of 789) of women triaged through the reflex HPV test arm. Information about follow-up was not avail-
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FIGURE 1. Flow chart of ThinPrep tests.
FIGURE 3. Prevalence of human papilloma virus (HPV)⫹ atypical squamous cells of undetermined significance (ASCUS) cases and cervical intraepithelial neoplasia (CIN) and CIN2–3 within HPV⫹ ASCUS cases. Straight line: HPV⫹ ASCUS cases; dotted line: CIN within HPV⫹ ASCUS; dashed line: CIN2–3 within HPV⫹ ASCUS cases.
FIGURE 2. Prevalence of squamous intraepithelial lesions (SIL) as detected by cytology among different age groups. Straight line: SIL; dotted line: atypical squamous cells of undetermined significance; dashed line: high-grade squamous intraepithelial lesions positive.
able for the rest of the patients. A total of 246 CIN2–3 and 2 cases of squamous cell carcinomas (SCC) were identified. Thirty-six (15%) of CIN2–3⫹ and 154 (26%) of CIN-1, but none of the SCC, were detected in the HPV⫹ASCUS group. There was a significant trend with age for both ASCUS (2 ⫽ 5.0, P ⬍ 0.0001) and SIL (2 ⫽ 5.0, P ⬍ 0.0001). The highest prevalence of ASCUS (19.8%) and SIL (11.4%) was encountered in the age group ⬍ 25 years old. The prevalence then tapered gradually for the ASCUS group to reach 8.6% in women ⱖ 65 years (a 0.9% decrease for each decade of life) compared with the prevalence of the SIL group, which reached 1.6% in women ⱖ 65 years (a 2% decrease for each decade of life), indicating a slight increase over the age 55– 65 age group (Fig. 2). Within the ASCUS category, there was a significant trend with age for the HPV⫹ASCUS rate (2 test
for trend ⫽ 203, P ⬍ 0.01) with a decrease of 7% for each decade of life. Unlike the ASCUS distribution, the distribution of ASCUS-HPV⫹ closely resembles that of SIL lesions apart from the appearance of the spur, the so-called second peak of HPV infection, in the ⱖ 65 year-old group. Regardless of these trends, the rate of CIN detected within HPV⫹ASCUS was not significantly different among the age groups (Fig. 3). When comparing the ASCUS rate in different settings or populations, the ASCUS-to-SIL ratio is commonly used to control the variation of ASCUS rates with disease prevalence. For evaluation of reflex HPV infection test performance, we proposed two similar ratios—the HPV⫹ASCUS-to-SIL ratio and the CINrxto-CIN ratio. The latter is the ratio of CIN detected through HPV-DNA tests (CINrx) to the ratio of CIN detected through cytology-only triage (LGSIL, HGSIL, and ASC-H). The CINrx-to-CIN ratio was approximately 0.3:1 in all age groups and there was no trend with age compared with the ASCUS-to-SIL ratio, which ranged from 1.2:1 to ⬎ 3:1 (2 ⫽ 203, P ⬍ 0.01, with a 7% increase for each decade of life) and the HPV⫹ASCUS-to-SIL ratio, which ranged from 0.7:1 to 1.1:1 with a significant trend (2 ⫽ 6.7, P ⬍ 0.01, with a 4% increase for each decade of life) (Fig. 4). There was no significant association between age and the number of patients with CIN2–3 detected through the reflex HPV infection test (2 ⫽ 3.985, P ⬍ 0.26) (Table 1). For patients ⬎44 years, the proportion of CIN2–3 detected through the reflex HPV infection test (7 of 31) was not significantly different from the proportion
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DISCUSSION
FIGURE 4.
Ratio of atypical squamous cells of undetermined significance (ASCUS) to squamous intraepithelial lesions (SIL), ratio of human papilloma virus⫹ to SIL, and the ratio of cervical intraepithelial lesions detected through reflex human papilloma virus (HPV) infection testing (CINrx) to CIN among different age groups. Straight line: ASCUS-to-SIL ratio; dotted line: HPV⫹ ASCUS⫹-SIL ratio; dashed line: CINrx-to-CIN ratio.
TABLE 1 Frequency (Percent of Raw) of Final Histologic Diagnosis as Triaged by Reflex HPV Infection or Cytology (LGSIL/HGSILⴙASC-H) Age (yrs)
CIN2-3 by HPVⴙASCUS
CIN2-3 by LGSIL
CIN2-3 by HGSIL
Total CIN2-3
⬍ 25 25–34 35–44 ⬎ 44
17 (19) 8 (9) 4 (10) 7 (23)
38 (43) 28 (32) 16 (39) 8 (26)
34 (38) 51 (59) 21 (51) 16 (51)
89 87 41 31
HPV: human papilloma virus; ASCUS: atypical squamous cells of undetermined significance; LGSIL: low-grade squamous intraepithelial lesion; HGSIL: high-grade squamous intraepithelial lesion; ASC-H:; CIN2-3: cervical intraepithelial neoplasia Grade 2–3.
detected through triage of LGSIL (8 of 31) (2 ⫽ 0.097, P ⫽ 0.755) (Table 1). There were a total of 2339 (9.3%) women in the ⱖ 65 year-old group. Sixty-one (2.6%) patients had a cytologic diagnosis of SIL, 22 had a diagnosis of HGSIL (1%), and 1 patient had SCC. There were 201 (8.5%) women with ASCUS, of whom 161 (80%) were tested for HPV infection. Fifty-six (35%) of these were HPV positive and 18 (32%) underwent biopsies, resulting in 7 women with CIN1 and 1 woman with CIN2. The latter patient was a 69-year-old woman who continued to have normal cytology from 1995 to 1999. She had an atypical glandular cells (AGC)/ASCUS diagnosis in 2001 and 2002. The HPV infection test was positive in 2002 and she underwent colposcopy and biopsy, which showed CIN2.
Because biopsy is not performed in HPV-negative cases under ASCCP guidelines, conventional sensitivity and specificity cannot be calculated to evaluate the performance of reflex HPV infection testing. Comparing the reflex HPV infection test with SIL (LGSIL, HGSIL, and ASC-H) triage and determining the proportion of CIN2–3 detected through HPV triage as we did in the current study offer an alternative option. Despite the variation of the ASCUS-to-SIL ratio, the HPV⫹ASCUS rate, and the HPV⫹ASCUS-to-SIL ratio with age, we found that the proportion of CIN2–3 detected through reflex HPV-DNA triage was not significantly different among the various age groups, including perimenopausal and postmenopausal women. For women ⬎ 44 years, triage through reflex HPV infection testing detected the same proportion of CIN2–3 as that of triage through LGSIL, supporting the previous data that the risk for developing high-grade CIN for patients with HPV⫹ASCUS is identical to that of LGSIL.11 The diagnosis of ASCUS is hampered by the lack of both reproducibility and specificity.2,12–15 Despite the finding that the majority of ASCUS cases show insignificant changes on histologic examination, the sheer volume of this category allows it to contribute ⱕ 39% of the total CIN2–3 detected within a routinely screened population.16 That is perhaps the strongest argument for pursuing a better triage of patients with ASCUS. Like other studies, reviewed recently by Arbyn et al.,3 the current study demonstrated that HPV infection testing remarkably reduced the number of patients triaged for colposcopy and, depending on age, 9 –23% of the CIN2–3 cases was detected through reflex HPV infection triage. Although age is an important determinant of both the incidence of ASCUS and HPV infection, few studies have stratified the performance of the reflex HPV infection test result by age to include perimenopausal and postmenopausal women.3 Lin et al.17 studied Taiwanese women ⬎ 50 years and found an HPV-positive rate of 52.7% (39 of 96) among women with ASCUS/LGSIL with a high sensitivity and specificity for detecting high-grade CIN. In the ALTS trial,18 women ⬎ 40 years comprised 14.5% of the ASCUS population and HPV infection testing at 1.0 the pg/mL level referred 19.5% of patients for colposcopy with 6 of 7 with CIN3 or higher detected through HPV triage. There is a continuous debate regarding the balance of risks, benefits, and cost of screening for cervical carcinoma in older women.19,20 Mandelblatt et al.20 recently reported that a high percentage (25%) of new cases and increased mortality rates (41%) of cervical carcinoma occur among women ⱖ 65 years.
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Conversely, Sawaya19 argue that cytology lacks specificity, leading to a large number of tests and procedures that result in few true-positive cases. Sawaya et al.21 followed 2500 women with normal Pap smears for 2 years and identified 110 women with cytologic abnormalities and only 1 woman with mild to moderate dysplasia. Because numerous procedures were performed in this cohort to detect mild to moderate lesions, Sawaya et al. concluded that screening in this age group should be personalized and informed. Both Sawaya19 and Mandelblatt et al.20 concluded that HPV infection triage could help in improving the predictive value of screening in this age group. In the current study, women ⱖ 65 years represent 9% of the study population (approximately 13% of the Alabama population is ⬎ 65 years), 8% of the ASCUS cases, 5% of the total SIL cases, 3% of the HGSIL cases on cytology, and 3% of the CIN3⫹ cases on histology with 1 case of SCC. Like studies from other countries, we observed a spur in HPV⫹ASCUS prevalence in this age group.8,9 Several reasons to explain this spur include cohort effect, immune senescence, and new exposure.9,20 This spur may also reflect the tendency of cytologists to down-call true lesions, leading to an increase in the pool of HPV-positive cases in both the normal and ASCUS categories. Although the absolute number of cases was small compared with cases in the younger population (a reflection of the number of women ⬎ 65 years in the general population), HPV infection testing is as useful in the triage of this age group as in younger women. In conclusion, the reflex HPV infection test performs equally for elderly and young women and in women ⬎ 44, its detection of CIN2–3 is equal to that detected through LGSIL triage.
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