Combining Human Papillomavirus Testing Or Cervicography with Cytology to Detect Cervical Neoplasia

Combining Human Papillomavirus Testing or Cervicography With Cytology to Detect Cervical Neoplasia

Michelle Howard, MSc; John W. Sellors, MD, MSc; Alice Lytwyn, MD, MSc; Paula Roth, MD; James B. Mahony, PhD

● Context.—Cervicography and oncogenic human papil- sensitivity or specificity if the second test performed ran- lomavirus (HPV) testing have been proposed for improving domly was estimated. the accuracy of cervical cancer screening. Results.—Cervical intraepithelial neoplasia 2 or 3 was Objective.—To examine whether cervicography and found in 11.8% (36/304) of the women and invasive squa- HPV testing can improve beyond chance the detection of mous cell carcinoma in 0.3% (1/304). The sensitivity of cervical intraepithelial neoplasia (CIN) 2 or 3 in women cytology for detecting CIN 2 or 3 was 73.0% and increased with atypical cells of undetermined significance or low- by 21.6% to 94.6% with the addition of a cervigram show- grade squamous intraepithelial lesions on cytology. ing a low-grade lesion or higher or a positive HPV test result. These gains were reduced to 8.1% and 10.8% above Design.—Cross-sectional analysis. Oncogenic HPV test- the sensitivities expected if the additional tests performed ing by Hybrid Capture II assay or cervicography combined randomly. The corresponding specificities decreased from with cytology was compared with the reference standard 49.1% to 32.2% and 33.0%. There was insufficient power of colposcopy with directed biopsy. to determine whether observed sensitivities were statisti- Setting.—Community family practices. cally significantly higher than the expected sensitivities. Participants.—Three hundred four women with low- Conclusion.—Adjunctive HPV testing or cervicography grade cytologic abnormality. may provide similar gains in sensitivity, but they can ap- Main Outcome Measures.—The gain in accuracy for de- pear misleadingly large if chance increases are not taken tecting histologic CIN 2 or 3 or carcinoma. Because an into account. adjunct test may improve sensitivity by chance alone, the (Arch Pathol Lab Med. 2004;128:1257–1262)

ytologic screening programs to detect cervical intra- cytology, the correlation with histologic findings has C epithelial neoplasia (CIN) have led to reductions in caused concern for conservative management, because ap- morbidity and mortality from cervical cancer in several proximately 10% to 20% of these women will have CIN 2 countries.1 These successes notwithstanding, rigorously or 3 on histologic examination.3–7 Cervicography and hu- conducted meta-analyses have shown that the sensitivity man papillomavirus (HPV) testing are adjunctive meth- of a cytology smear is low (30%–87%), in contrast to its ods that may allow effective triage of women with low- specificity of 86% to 100% for high-grade (CIN 2 or 3) grade lesions on screening cytology. cervical lesions.2 Among women with a low-grade squa- Cervicography is a procedure in which a projected pho- mous intraepithelial lesion (LSIL) or atypical squamous tographic slide of the cervix simulates colposcopic mag- cells of undetermined significance (ASCUS) on screening nification of 16 times.8 Studies6,9–13 that have evaluated cervicography in women with low-grade lesions on cytology, without verification bias, have found that the sensitivity Accepted for publication June 11, 2004. and specificity for the detection of CIN 2 or 3 range from From the Departments of Family Medicine (Ms Howard and Dr Sel- lors) and Pathology and Molecular Medicine (Dr Mahony) and Col- 54.5% to 90.0% and from 30.0% to 90.2%, respectively. poscopy Clinic (Dr Roth), McMaster University, Hamilton, Ontario; the Testing for oncogenic types of HPV in cervical speci- Program for Appropriate Technology in Health, Seattle, Wash (Dr Sel- mens is another possible method to triage women with lors); and the Department of Pathology, Sunnybrook and Women’s Col- low-grade abnormalities on cytologic screening and has lege of Health Sciences Centre, Toronto, Ontario (Dr Lytwyn). Dr Ly- been found to have sensitivity and specificity ranging twyn is now affiliated with the Department of Laboratory Medicine, McMaster University. from 65% to 98% and from 52% to 74%, respectively, for The Hybrid Capture II test assays used for detection of HPV and the detecting high-grade lesions.4,14–17 use of a luminometer were provided free of charge by Digene Cor- The use of a second test in parallel with the first test poration. (ie, cytology) must result in the same or increased sensi- The authors have no relevant financial interest in the products or tivity and the same or decreased specificity. These increas- companies described in this article. Reprints: Michelle Howard, MSc, Department of Family Medicine, es in sensitivity may be misleading because improvements McMaster University, 1200 Main St W, Room HSC-2V10, Hamilton, in sensitivity would be expected by adding a second test, Ontario, Canada L8N 3Z5 (e-mail: [email protected]). even if the second test performed randomly with respect Arch Pathol Lab Med—Vol 128, November 2004 Cervicography and HPV Testing—Howard et al 1257 Flow diagram of the studies from which women were analyzed. ASCUS indicates atypical squamous cells of undetermined significance; LSIL, low-grade squamous intraepithelial lesion; HPV, human papillomavirus; R, ran- domization; asterisk, colposcopy, cervicography, and cytology (at colposcopy clinic); and dagger, referral to colposcopy as soon as HPV detected.

to disease identification. For this reason, the method de- the cervical os and the transformation zone with a Dacron swab scribed by Franco18 was used to determine if any gain in at the last follow-up visit before colposcopy referral. The swab test performance from the addition of the second test was was placed into specimen transport medium for Hybrid Capture greater than that expected if the second test offered no II (Digene Corporation, Gaithersburg, Md) testing. At colposcopy, diagnostic information. the smear for cervical cytology was obtained by a colposcopy This study assessed whether there was a gain in diag- clinic nurse, using an extended-tip Ayre spatula and, as required, an endocervical brush. Cervicography was then performed by 1 nostic accuracy over cervical cytology, above what would of 2 trained nurses, after washing the cervix with 5% acetic acid. be expected by chance, by adding cervicography or HPV Finally, a colposcopic examination with directed biopsy or en- testing in a group of women with low-grade cytologic ab- docervical curettage (if deemed necessary) was performed. The normalities. A cross-sectional analysis was performed us- physicians performing colposcopy were blinded to the cervico- ing the data from 2 randomized controlled trials of cytol- graphic findings and the HPV results. Colposcopy was done at ogy and HPV testing.3,17 the McMaster University Colposcopy Clinic. The cervigrams were read by National Testing Laboratories MATERIALS AND METHODS (Fenton, Mo). Cervigram results were reported by the laborato- Women were eligible if they were aged 16 to 50 years and ries as (1) negative if (a) components of the transformation zone received a result of LSIL or ASCUS on routine cytologic screen- were visible, with no abnormal lesion or (b) components of the ing. Women were recruited consecutively from 118 community transformation zone were not visible; (2) atypical, recommending family practices and 1 university student health clinic in Ontario repeat cervigram and Papanicolaou test in 6 to 12 months, if (a) between May 1995 and October 1998. Written informed consent a lesion of doubtful significance was inside the zone or (b)ale- was obtained at the physicians’ offices. The studies were ap- sion of doubtful significance was outside the zone; (3) positive, proved by the Hamilton Health Sciences Research Ethics Board. with colposcopy recommended, if the cervigram indicated (a)a The objective of the study was to determine the efficacy of HPV probable normal variant and colposcopy was warranted to ex- testing compared with repeat cytology for detecting CIN 2 or 3 clude significant disease, (b) results compatible with a low-grade or cancer, by randomizing women to repeat cytology alone (HPV lesion, (c) results compatible with a high-grade lesion, or (d) re- testing was done in these groups but not used in the randomized sults compatible with cancer; or (4) technically defective if (a)the trials) or to repeat cytology with HPV testing to direct referral to colposcopy (Figure). The maximum follow-up was 24 months, view of the cervix was obscured by mucus, blood, or an incorrect or sooner if a high-grade squamous epithelial lesion was found position, (b) there was insufficient acetic acid reaction, or (c)the on a repeat cytologic examination for any woman, or if the HPV cervigram was out of focus, overexposed, or underexposed. test result was positive in a woman randomized to the HPV test- For the purpose of analysis, cervigram interpretations that ing strategy. The cytology and cervicography results presented were normal, revealed lesions of doubtful significance, or were in this article are those obtained at the time of colposcopy (ie, judged as a probable normal variant were considered negative. study exit). The 2 positive cutoffs for the cervigram interpretation were (1) At enrollment, a written questionnaire on demographics and results compatible with a low-grade or high-grade lesion or car- sexual health history was self-completed by the women, as pre- cinoma (categories 3b, c, or d in the previous paragraph) and (2) viously described.3,17 The HPV test findings used in the present results compatible with a high-grade lesion or carcinoma (cate- analysis were obtained by the family physician from the area of gories 3c or d in the previous paragraph). Cervigrams in which 1258 Arch Pathol Lab Med—Vol 128, November 2004 Cervicography and HPV Testing—Howard et al Table 1. Demographic Characteristics and History of the Women in the 2 Randomized Controlled Trials* (154 ؍ Six-Month Study (n (150 ؍ Characteristic Two-Year Study (n Age, mean (SD), y 34.3 (9.2) 32.0 (8.8) Age at first intercourse, mean (SD), y 17.9 (3.2) 17.3 (2.7) No. of lifetime sexual partners, median or interquartile range 4 (6) 4 (5) Married or common law 57.4 (81/141) 49.6 (69/139) Previous pregnancy 65.5 (93/142) 55.8 (77/138) Ever used oral contraceptive 91.9 (125/136) 87.0 (120/138) Current smoker 36.6 (52/142) 35.3 (49/139) * Data are given as percentage (numerator divided by denominator) unless otherwise indicated.

Table 2. The Performance of Cervical Cytology, Cervicography, and the Cervical Swab for Hybrid Capture II Human Papillomavirus (HPV) Testing for the Detection of 37 Women With a Biopsy-Proven Cervical Intraepithelial Neoplasia (CIN) 2 or 3 or Invasive Squamous Cell Carcinoma* Positive Predictive Negative Predictive Triage Test Triage Positive Threshold Sensitivity Specificity Value Value Cytology alone ՆAtypical squamous cells of 73.0 (55.9–86.2) 49.1 (43.1–55.1) 16.6 (10.9–22.3) 92.9 (87.3–96.6) undetermined significance Cervigram alone Low grade or higher 83.8 (68.0–93.8) 65.5 (59.8–71.2) 25.4 (17.7–33.1) 96.2 (92.2–98.4) High grade or carcinoma 24.3 (11.8–41.2) 95.9 (92.7–97.9) 45.0 (23.1–68.5) 90.1 (85.9–93.0) HPV DNA test alone Positive for oncogenic type 86.5 (75.5–97.5) 59.2 (53.0–65.4) 22.7 (15.8–29.6) 96.9 (93.0–99.0) * Data are given as percentage (95% confidence interval). the view of the cervix was obscured and those that were tech- Differences were considered statistically significant at ␣Ͻ0.05 nically defective were classified as negative. (2-sided). The Hybrid Capture II assay detects 13 of the most common oncogenic HPV types, and testing was carried out as previously RESULTS 14 described. Specimens with relative light unit readings equal to Of the 469 women enrolled, complete data on cervicog- or greater than the mean relative light unit reading of triplicate positive controls containing 1 pg/mL of HPV DNA were consid- raphy, HPV testing, cervical cytology, and the end point ered positive. Testing was performed at the McMaster University assessment of colposcopy were available for 304 (64.8%). Regional Virology and Chlamydiology Laboratory. The mean Ϯ SD number of days between the HPV test Tissue specimens were processed in the Department of Pa- and colposcopy was 116.0 Ϯ 88.7 days. The demographic thology, Hamilton Health Sciences. Results were reported using characteristics of the women are shown separately for the CIN terminology. Human papillomavirus effects were catego- 2 trials in Table 1. rized as CIN 1 if no greater degree of dysplasia was present. Cervical cytology performed at colposcopy showed Two expert gynecologic pathologists independently reviewed all 46.4% (141/304) of women had normal cytology, 31.9% biopsy and curettage material (␬ϭ0.88 for high-grade dysplasia (97/304) had ASCUS, 16.1% (49/304) had LSIL, 5.3% (16/ and ␬ϭ0.83 for any grade of dysplasia, P Ͻ .001 for both) and 304) had a high-grade squamous epithelial lesion, and achieved consensus on any discordant specimens. For cases with more than 1 histologic specimen available, the most severe di- 0.3% (1/304) had carcinoma. There was no statistically sig- agnosis was recorded. A colposcopic diagnosis of normal was nificant association between the time since study entry recorded if the examination revealed no lesion and no biopsy was and the cytology result (negative vs others) (data not taken, or if the tissue obtained showed no CIN or cancer. Cervical shown). Cervical biopsies were performed on 55.9% (170/ cytology specimens taken at the colposcopy clinic were read in 304) of the women at colposcopy. On colposcopy, CIN 2 the Department of Pathology using Bethesda system terminol- or 3 was found in 36 (11.8%) of the 304 women, CIN 1 ogy. was found in 29 (9.5%), carcinoma was found in 1 (0.3%), The results of colposcopic examination and biopsy were used and the cervix was normal in 238 (78.3%). Cervigram re- as the reference standard for the other tests. Sensitivity, specific- sults were consistent with a low-grade lesion (category 3b) ity, and positive and negative predictive values were calculated c for cervical cytology (taken at the time of colposcopy), cervicog- in 33.6% (102/304), a high-grade lesion (category 3 )in raphy, and HPV testing. To estimate the magnitude of the gain 5.9% (18/304), and carcinoma (category 3d) in 0.7% (2/ in sensitivity with the addition of cervicography or HPV testing, 304) of the women. Findings were negative or atypical but the proportion of additional true positives identified by the sec- not requiring colposcopy in the remaining 59.9% (182/ ond test (cervicography or HPV testing) that were missed by 304) of the women, including 0.7% (2/304) and 0.3% (1/ cytology alone was calculated. To estimate the loss of specificity 304) that were obscured views and technically defective, when adding a second test, the proportion of additional false respectively. The prevalence of HPV infection was 46.4% positives identified by the second test was calculated. It was ex- (141/304); the prevalence of HPV in LISL was 65.3% (64/ pected that these proportions would be significantly greater than 98) and 37.4% (77/206) in ASCUS. zero. The 95% confidence intervals (CIs) were calculated using Table 2 shows the sensitivities, specificities, and positive the normal approximation to the binomial distribution.19 Differences between the observed sensitivities and specificities and negative predictive values of cervicography and cer- and the actual sensitivities and specificities (those calculated after vical HPV testing alone. The sensitivities of cytology and adjusting for the chance increases expected if the second test per- HPV testing for detecting CIN 2 or higher were 73.0% and formed randomly using the method by Franco18) were compared 86.5% respectively. Sensitivity was low for cervicography using the normal approximation to the binomial distribution.20 when using the high-grade lesion or carcinoma cutoff Arch Pathol Lab Med—Vol 128, November 2004 Cervicography and HPV Testing—Howard et al 1259 Table 3. Comparison of the Observed Sensitivities and Specificities for the Combined Tests for Detecting Histologically Confirmed Cervical Intraepithelial Neoplasia 2 or 3 or Invasive Squamous Cell Carcinoma, in the 304 Women With Low-Grade Cervical Cytology at Study Entry* Sensitivity Specificity After Reduction After Reduction Because of Because of Random Test Observed P Value Random Test Observed P Value Cervical cytology (Նatypical squamous cells of undetermined significance) or Cervigram assessment of low-grade lesion or higher 83.8 94.6 (81.8–99.3) .06 29.2 32.2 (26.6–37.8) .28 Cervigram assessment of high- grade lesion or carcinoma 75.7 75.7 (58.8–88.2) .97 45.7 47.2 (41.1–56.9) .62 Human papillomavirus DNA test positive 86.5 94.6 (81.8–99.3) .14 26.2 33.0 (27.3–38.6) .01 * Using the method of Franco and Ferenczy,25 which assumes that the second test is random with respect to disease detection. Data are given as percentage (95% confidence interval) unless otherwise indicated.

(24.3%) but improved when using the low-grade lesion or after adjusting the sensitivities of the combined tests to higher cutoff (83.8%). The cervigram assessment using the take into account increases in disease detection because of high-grade lesion or carcinoma cutoff identified 1 addi- chance, these gains were reduced to 10.8% for cervicog- tional case of CIN 2 or 3, or true positive (1.50% [95% CI, raphy and to 8.1% for HPV testing. None of the differ- 0.04%–8.20%]), increasing sensitivity from 73.0% to 75.7%. ences between the observed and actual sensitivities after The cervigram assessment of a low-grade lesion or higher adjustment were statistically significant; however, our and the HPV test identified 8 additional true positives power to detect these differences was small (20%–30%). (21.6% [95% CI, 9.8%–38.2%]), increasing sensitivity to Studies examining gains in the diagnostic use of com- 94.6%. The addition of the cervigram using the high-grade bined tests should be interpreted with caution. Six stud- lesion or carcinoma cutoff resulted in 5 additional false ies4,5,21–24 of combined cervical cytology and HPV testing positives (1.8% [95% CI, 0.7%–4.6%]), reducing specificity for the detection of histologically confirmed cervical ab- from 52.5% for cytology alone to 47.2%. The addition of normalities were summarized by Franco and Ferenczy.25 the cervigram using the low-grade lesion or higher cutoff Five of the studies4,5,21,23,24 demonstrated statistically sig- and the HPV test identified 45 (16.9% [95% CI, 12.0%– nificant gains in sensitivity for the combined method; 22.7%]) and 43 (16.1% [95% CI, 12.0%–21.2%]) additional however, after using an adjustment to take into account false positives, reducing specificity to 32.2% and 33.0% the chance increase in sensitivity from adding a second respectively. test, only 2 of the 5 studies4,23 were found to have statis- Actual sensitivities after adjusting for the increased sen- tically significantly improved sensitivity. Previous stud- sitivity that would be expected if the second test per- ies6,9,26 examining the usefulness of cervicography as an formed randomly were 83.8% (vs 94.6% without adjust- adjunct to cytology for women with low-grade cytologic ment, P ϭ .06) for the cervigram assessment and 86.5% abnormalities have reported increases in sensitivity of (vs 94.6% without adjustment, P ϭ .14) for the HPV test, 25% to 45% above cytology alone. These studies did not resulting in absolute decreases of 10.8% and 8.1% respec- take into account chance increases in sensitivity and may tively, compared with the observed sensitivities (Table 3). have overestimated the benefits of combined testing. The specificities for the combined tests were lower than Although we found that cervicography using a high- for cytology alone, but were not as low as those calculated grade lesion interpretation to indicate a positive result had assuming the second test was random. For the HPV test, poor sensitivity, the use of a suspicious or atypical cervi- the observed specificities were statistically significantly gram assessment has shown moderate to good sensitivity better than the actual specificities after adjustment for (61%–92%) in women with ASCUS or LSIL on cytolo- chance increases in sensitivity. gy.6,11,12,26–29 The sensitivity of HPV testing alone for de- Classification of the 26 women with probable normal tecting CIN 2 or 3 in the present study was 86.5%, but variants or lesions of doubtful significance as negative (in- specificity was low (59.2%). These findings are similar to cluding 2 women with histologic CIN 1 and 2 women with those of previous studies7,14,15,30 using the Hybrid Capture CIN 2 or 3) resulted in overestimates of specificities by II assay, in which sensitivity ranged from 89% to 98% and approximately 10% (data not shown), but had little effect specificity from 47% to 73.9% among women referred on sensitivity. All 3 women with technically defective cer- with cytologic abnormalities. vigrams had normal histologic findings. Some previous studies describing the diagnostic accuracy of cervicography6,11,12,28,31–35 and HPV testing31,36,37 COMMENT were unable to verify disease status in women with neg- This study found that the use of a cervigram assessment ative test results, which can result in verification bias if threshold of a low-grade lesion or the use of a positive corrections are not made. In screening studies in which HPV test (Hybrid Capture II) result appeared to increase disease prevalence is low, it is often not feasible to subject the sensitivity of cytology for detecting histologically con- all women with negative test results to colposcopy. In firmed CIN 2 or 3 by an increment of 21.6% in women such cases, a randomly selected sample of women with with recent low-grade cytologic abnormalities. However, negative results can be examined by colposcopy to esti- 1260 Arch Pathol Lab Med—Vol 128, November 2004 Cervicography and HPV Testing—Howard et al mate the true performance of a screening test.18 In the ferent management strategies will be informative, and ad- present study, all women underwent colposcopy. It is rec- justments to sensitivity estimates, if used, may alter the ognized that colposcopy with directed biopsy has imper- expected improvements in cost-effectiveness. The net ben- fect sensitivity,38,39 but it is the best tool available. efits of improved sensitivity and negative predictive value An advantage of using combined tests, in addition to of combined testing must be considered in relation to the the potential for improved sensitivity, is the potential im- availability of adjunctive testing and colposcopy resources provement in negative predictive value or the ability to and to patients’ adherence with follow-up in a given set- rule out disease. This reassurance may be especially im- ting. portant in litigious societies or in settings where patient adherence with follow-up is problematic. It has been sug- This study was funded by grants MT13287 from the Medical Research Council of Canada, Ottawa, and 95-32 from the Physi- gested that an additional benefit of improved sensitivity cians’ Services Incorporated Foundation, North York, Ontario. We of combined testing is the possibility of increasing the cy- thank Stephen Walter for advice on data analysis and presenta- tologic screening interval, because the chances of missing tion. a case of high-grade disease will be reduced.40 However, improvements in sensitivity and negative predictive value References 1. Parkin DM, Pisani P, Ferlay J. Global cancer statistics. CA Cancer J Clin. when adding a second test may be undermined by the 1999;49:33–64. loss of specificity and positive predictive value, resulting 2. Nanda K, McCrory DC, Myers ER, et al. 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