Objective
The primary cervical cancer screening strategy for women over age 30 is high-risk human papillomavirus (HPV) testing combined with Papanicolaou (Pap) testing (cotesting) every 5 years. This combination strategy is a preventive service that is required by the Affordable Care Act to be covered with no cost-sharing by most health insurance plans. The cotesting recommendation was made based entirely on prospective data from an insured population that may have a lower proportion of women with HPV positive and Pap negative results (ie, discordant results). The discordant group represents a very difficult group to manage. If the frequency of discordant results among underserved women is higher, health care providers may perceive the cotesting strategy to be a less favorable screening strategy than traditional Pap testing every 3 years.
Study Design
The Centers for Disease Control and Prevention’s Cervical Cancer Study was conducted at 15 clinics in 6 federally qualified health centers across Illinois. Providers at these clinics were given the option of cotesting for routine cervical cancer screening. Type-specific HPV detection was performed on residual extracts using linear array.
Results
Pap test results were abnormal in 6.0% and HPV was positive in 7.2% of the underserved women screened in this study (mean age, 45.1 years). HPV prevalence decreased with age, from 10.3% among 30- to 39-year-olds to 4.5% among 50- to 60-year-olds. About 5% of the women had a combination of a positive HPV test and normal Pap test results; HPV 16/18 was identified in 14% of discordant women.
Conclusion
The rate of discordant results among underserved women was similar to those reported throughout the US in a variety of populations. Typing for HPV 16/18 appears to assist in the management in a small proportion of women with discordant results.
Since 2003, in the United States, cervical cancer screening with human papillomavirus (HPV) and Papanicolaou (Pap) tests (“cotesting”) has been recommended by the American College of Obstetricians and Gynecologists and the American Cancer Society. In 2012, the United States Preventive Services Task Force endorsed cotesting every 5 years as an alternative screening strategy to Pap-testing alone every 3 years among women 30 to 65 years of age. The American Cancer Society and American College of Obstetricians and Gynecologists made similar recommendations, although they deemed cotesting as the preferred strategy. However, provider and patient surveys have indicated that cotesting and the increase in screening intervals have not been widely adopted; in fact, lengthening screening intervals beyond annual screening is uncommon.
In addition to potentially improving acceptability and compliance with appropriate lengthened screening intervals, an important need has been to define the optimal follow-up of women who have HPV-positive and Pap-negative results (from here on, this will be called discordant results). Women with discordant results have a low prevalence of cervical intraepithelial neoplasia of grade 2 or worse (CIN 2+). In 2006, the American Society for Colposcopy and Cervical Pathology (ASCCP) published guidelines recommending that these women either be retested with both HPV and Pap tests in a year, or tested for HPV 16 and 18 (using a test approved by the US Food and Drug Administration) and directed to colposcopy if results are positive. According to data from a large managed care organization that includes over half a million women, followed prospectively, approximately 4% of women 30 and older can be expected to have discordant results. However, there is concern that data from a managed care population may not be generalizable to low-income, underinsured women. Moreover, if the frequency of discordant results among underserved women is higher, health care providers may perceive the cotesting strategy to be less favorable than traditional Pap testing every 3 years.
Our Centers for Disease Control and Prevention’s (CDC) Cervical Cancer Study (referred to as the Cx3 Study) offered a unique opportunity to examine the performance of cotesting in a cohort of underserved women presenting to federally qualified health centers (FQHCs) for routine cervical cancer screening. We also sought to compare the results of HPV type-specific testing in this population of women with that of other populations, to estimate the effect of type-specific HPV testing on referral to colposcopy. This population of women is of increased concern when determining optimal management strategies to ensure proper follow-up for those at highest risk of cervical cancer.
Materials and Methods
Participants
The data for this study were obtained from women, recruited between Sept. 2009 and May 2011 as part of CDC’s Cx3 Study. The study was conducted in 15 clinics associated with 6 FQHCs serving low-income women in Illinois. FQHCs provide comprehensive primary health care services to medically underserved communities and vulnerable populations in high-need areas across the United States. Women between the ages of 30 and 60 who were being seen in one of the FQHCs for a routine screening Pap test were identified through medical chart review by clinic staff. This group had no abnormal Pap test results in the preceding year survey, no history of cervical cancer, no record of being HIV positive, and no hysterectomy. They were invited to participate in our study when they arrived at the clinic for their routine visit. Further details on study design can be found in Benard et al. This study was approved by CDC’s Institutional Review Board. Informed consent was obtained from all study participants.
Two samples of exfoliated cervical cells were collected during the pelvic examination after visualization of the cervix. The first sample was collected per clinic protocol for routine Pap tests (either liquid or conventional) using the Bethesda system for reporting. The second sample was collected with the Digene Cervical Sampler (Qiagen Inc., Valencia, CA) and placed in specimen transport media (STM; Qiagen) for high-risk HPV testing (Hybrid Capture 2, HC2; Digene, Gaithersburg, MD). The STM specimens were stored and shipped to CDC at ambient temperature within 1 week of collection.
Laboratory
Specimens received at CDC were stored at 4°C and processed within 1 week of receipt. High-risk HC2 testing was performed on 250 μL aliquot, according to the manufacturer’s specification. A positive result indicates the presence of 1 or more of the 13 high-risk types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68. A second 200 μL aliquot was treated with Proteinase K at 65°C to lyse cells and the DNA from lysate was purified using the automated Chemagic Magnetic Separation Module 1 (PerkinElmer chemagen Technologie GmbH, Baesweiler, Germany) with the ViralNA/gDNA kit (Chemagen). The resulting extract (100 μL total volume) was tested immediately or stored at −20°C. Water blanks were processed through all laboratory steps as contamination control.
All DNA extracts were tested with the Research Use Only Linear Array genotyping assay (Roche Diagnostics, Indianapolis, IN). This assay uses HPV L1 consensus polymerase chain reaction (PCR) with PGMY09/11 primers and consensus PCR with β-globin primers as an internal control for amplification and cellular DNA. The typing strips include probes for 37 different HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, XR(52), 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, 89, IS39). There are 14 specific high-risk types examined in this assay: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68. The manufacturer’s protocol was modified to use 10 μL extract in the 100 μL PCR reaction and automated hybridization and wash steps with Bee Blot instrument (Bee Robotics, Caernafon, UK). Because HPV52 is detected with XR probe that cross-hybridizes with HPV 33, 35, and 58, all XR- high-risk positive samples with 1 or more cross-hybridizing types present were tested using a type-specific HPV 52 quantitative PCR. We had 15 samples that were considered negative for high-risk HC2, but these were positive for high risk by linear array. We excluded these in the analysis that was based on the assumption that HC2 would be the first test used. Samples negative for all HPV types and β-globin were considered inadequate and were omitted from further analysis.
Analysis
We analyzed baseline data from the 2246 women enrolled in the study who had both HPV and Pap testing. Pap test results defined as positive included atypical squamous cells of undetermined significance. Atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion, low-grade squamous intraepithelial lesion, high-grade squamous intraepithelial lesion, squamous cell carcinoma, and atypical glandular cells (atypical glandular cells of undetermined significance, adenocarcinoma). Results for cotesting included negative (HPV negative and Pap normal), discordant (HPV positive and Pap normal) or positive (HPV positive and Pap positive; HPV negative and Pap positive). Binomial exact 95% confidence intervals (CIs) were calculated for Pap and HPV results prevalence. Typing results are reported only for those samples that were HC2 HPV-positive. Other factors that were collected included the woman’s age and clinic location, including Chicago area and nonChicago area (ie, southern Illinois and midIllinois). Prevalence ratios (HPV/Pap) with 95% CIs were calculated to show the relative contribution of HPV to Pap for screening positive by cotesting. McNemar’s χ 2 test was used to test for statistical differences ( P < .05) for testing HPV-positive vs testing Pap-positive for all women and within 10-year age groups. Logistic regression was used to test the statistical differences of overall HPV positivity (HPV positive vs HPV negative) and discordant result (HPV negative and Pap negative vs all other categories combined) by 10-year age groups. The 50- to 60-year-old age group was included as the reference category. We used Stata version 12.1 (StataCorp, College Station, TX) for statistical analyses.
Results
For the 2246 women enrolled in the study, the mean age was 45.1 years ( Table 1 ). Two-thirds of the women were from Chicago. Two-thirds of the samples used liquid-based cytology. Overall, the HPV test result was positive in 7.2% (95% CI, 6.2–8.4%; n = 162) of the women; while 6.0% (95% CI, 5.0–7.0%; n = 134) had a positive Pap test result. Most (89.1%, 95% CI, 87.8–90.4%; n = 2002) were cotest negative and 4.9% (95% CI, 4.0–5.9%; n = 110) had discordant results (HPV positive and Pap negative results).
| Demographic | Category | n | % |
|---|---|---|---|
| Age at enrollment, by 5-y age category | 30-34 | 149 | 6.6 |
| 35-39 | 471 | 21.0 | |
| 40-44 | 473 | 21.1 | |
| 45-49 | 468 | 20.8 | |
| 50-54 | 385 | 17.1 | |
| 55-60 | 300 | 13.4 | |
| Total | 2246 | 100.0 | |
| Age at enrollment, mean (SD) | 45.1 (7.6) | ||
| Clinic is located in Chicago | No | 772 | 34.4 |
| Yes | 1474 | 65.6 | |
| Total | 2246 | 100.0 | |
| Laboratory tests | |||
| Pap test type | Liquid | 1466 | 65.3 |
| Conventional | 768 | 34.2 | |
| Not reported | 12 | 0.5 | |
| Total | 2246 | 100 | |
| Pap results | Negative | 2112 | 94.0 |
| ASC-US | 95 | 4.2 | |
| LSIL | 25 | 1.1 | |
| ASC-H | 4 | 0.2 | |
| HSIL | 5 | 0.2 | |
| AGUS | 3 | 0.1 | |
| AGC | 1 | 0.04 | |
| Adenocarcinoma | 1 | 0.04 | |
| Total | 2246 | 100.0 | |
| Pap results a | Negative | 2112 | 94.0 |
| Positive | 134 | 6.0 | |
| Total | 2246 | 100 | |
| HPV results b | Negative | 2084 | 92.8 |
| Positive | 162 | 7.2 | |
| Total | 2246 | 100 | |
| Pap and HPV combinations | Pap− / HPV− | 2002 | 89.1 |
| Pap+ / HPV − | 82 | 3.7 | |
| Pap− / HPV+ | 110 | 4.9 | |
| Pap+ / HPV+ | 52 | 2.3 | |
| 2246 | 100.0 | ||
a Negative includes within normal limits and negative for intraepithelial lesion or malignancy; Positive includes ASC-US, LSIL, HSIL, AGUS, AGC, adenocarcinoma
b Negative includes HPV negative; positive includes HPV positive.
The various combinations of HPV and Pap test results are presented in Table 2 . In general, the percentage of Pap-test results considered abnormal remained relatively constant across age categories. However, the percentage of HPV-positive tests decreased from 10.3% among women aged 30 to 39 years to 4.5% among those aged 50 to 60 years (odds ratio, 2.43; P < .001). The prevalence of discordant cotest results decreased by age group with a higher rate for women aged 30 to 39 (6.5%) than among women aged 50 to 60 years (2.9%) (odds ratio, 2.2; P = .003).
| Age group | Total | Pap + | HPV+ | Pap−/ HPV− | Pap+/ HPV− | Pap−/ HPV+ | Pap+/ HPV+ | Prevalence ratio (HPV/Pap) (95% CI) | P value a | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| n | % | n | % | n | % | n | % | n | % | n | % | n | % | |||
| 30-39 | 620 | 27.6% | 38 | 6.1% | 64 | 10.3% | 542 | 87.4% | 14 | 2.3% | 40 | 6.5% | 24 | 3.9% | 1.68 (1.26–2.26) | < .001 b |
| 40-49 | 941 | 41.9% | 57 | 6.1% | 67 | 7.1% | 834 | 88.6% | 40 | 4.3% | 50 | 5.3% | 17 | 1.8% | 1.18 (0.87–1.59) | .292 |
| 50-60 | 685 | 30.5% | 39 | 5.7% | 31 | 4.5% | 626 | 91.4% | 28 | 4.1% | 20 | 2.9% | 11 | 1.6% | 0.79 (0.54–1.17) | .248 |
| Total | 2246 | 100% | 134 | 6.0% | 162 | 7.2% | 2002 | 89.1% | 82 | 3.7% | 110 | 4.9% | 52 | 2.32% | 1.21 (1.01–1.45) | .043 a , b |
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