DOI:10.1111/cyt.12176

Interobserver variability of cervical cytology in HIV-infected women I. Heard*,†, V. Potard‡,§,¶, C. Bergeron**, I. Cartier†† and D. Costagliola‡,§ *Centre National de Reference des papillomavirus humains, Institut Pasteur, †Groupe Hospitalier Piti e-Salp^ etri ere, ‡

Sorbonne Universites, UPMC Universite Paris 06, UMR_S 1136, §INSERM, UMR_S 1136, Paris, France, ¶INSERM

TRANSFERT, **Laboratoire Cerba, Cergy Pontoise,

††

Laboratoire Cartier, Paris, France

Accepted for publication 21 June 2014

I. Heard, V. Potard, C. Bergeron, I. Cartier and D. Costagliola Interobserver variability of cervical cytology in HIV-infected women Objectives: Our objectives were to determine the reproducibility of cytological specimen interpretation between two pathologists in human immunodeficiency virus (HIV)-infected women (from the VIHGY, ANRS CO17 study of human papillomavirus genital pathology among HIV-positive women) and to analyse the improvement, if any, between conventional and liquid-based cytology (LBC) interpretations. Materials and methods: A sample of all abnormal and 40% of randomly selected normal Papanicolaou (Pap) tests was randomly ordered and read blindly by a second pathologist using the revised Bethesda terminology 2001. For both conventional and liquid-based preparations, unweighted and Cicchetti–Allisonweighted kappa and their 95% confidence intervals (CIs) were calculated. Kappa values were then compared using the Altman rule to classify the reproducibility of cytological specimen interpretation. Results: Two hundred and seventy-seven conventional Pap tests were reviewed, including 79 abnormal and 10 unsatisfactory results. Overall agreement between the two observers was 78%, with an estimated Cicchetti–Allison-weighted kappa of 0.69 (95%CI, 0.61–0.77). The corresponding values for the 268 LBCs, including 123 abnormal and two unsatisfactory results, were 84% and 0.82 (95%CI, 0.76–0.87), respectively. The reproducibility of LBC interpretations was significantly higher than that of conventional preparations (P = 0.009) and, for both laboratories, the percentages of unsatisfactory results were significantly lower for LBC. Conclusion: In HIV-infected women in the combination antiretroviral therapy era, the strength of agreement was better for LBCs than for conventional preparations, with a lower percentage of unsatisfactory results. When available, LBC should be preferred because of its higher reproducibility. Keywords: cervical cancer screening, conventional cytology, liquid-based cytology, reproducibility, human immunodeficiency virus, interobserver variation

Introduction It is now widely accepted that human immunodeficiency virus (HIV)-positive women have an increased risk of human papillomavirus (HPV)-associated lower genital tract neoplasia, particularly cervical

Correspondence: I. Heard, Centre National de Reference des papillomavirus humains, Institut Pasteur, 25–28 rue du Dr Roux, 75015 Paris, France Tel.: +33 1 4061 3139; Fax: +33 1 4438 9484; E-mail: [email protected]

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intraepithelial neoplasia (CIN), and a higher incidence and prevalence of CIN have been reported in HIV-1-positive women.1 HIV therapies have led to a decrease in mortality related to HIV and in life prolongation.2 Nevertheless, these therapies do not prevent or clear HPV infection, and effective screening and treatment of precancerous lesions are needed to prevent the progression to invasive cervical cancer. Low incidence rates of invasive cervical cancer have been found among adequately screened HIV-positive women in the USA and France.3,4 For over the last 50 years, screening for cervical cancer using conventional Papanicolaou (Pap) smears © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 362–367

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has resulted in a dramatic decrease in cervical cancer cases in industrialized countries. Liquid-based cytology (LBC) is now often used despite being neither more sensitive nor more specific than conventional Pap smears.5–7 A substantial reduction in the number of unsatisfactory samples has been found with this technique.5 At present, there is no clear consensus as how best to prevent and manage cervical disease in HIV-positive women. This is in part a result of the lack of reliable information regarding the clinical effectiveness of cytology testing in HIV-infected women. Discrepancies in the sensitivity of the Pap test for CIN in HIV-infected women were found in early studies.8 Little is known about the sensitivity, specificity and reproducibility of reporting of cytological diagnoses in the setting of HIV. The question of Pap test accuracy is important, particularly as primary HPV testing in cervical screening may not be appropriate in these women with a high rate of genital HPV infection. The VIHGY study was a prospective, multicentre cohort study of HIV-infected women conducted in France between 2008 and 2012. The main objective was to assess and characterize genital HPV infection and related disease among HIV-infected women. At the cohort initiation, only conventional cytology was used. For several reasons, including a high rate of unsatisfactory samples and of recalls of patients, we moved to LBC at the third trimester of Year 2 of the cohort study. The purpose of this study was to assess the interobserver variability in the classification of cervical lesions with conventional and liquidbased methods. Given its design, the cohort did not allow the evaluation of the performances of conventional cytology and LBC in terms of sensitivity and specificity, but we did evaluate whether moving from the first to the second technique was associated with a change in the percentage of unsatisfactory results or reproducibility. Materials and methods Specimens were obtained during gynaecological examination of HIV-infected women enrolled in a prospective cohort of the French National Agency for Research on Aids and Viral Hepatitis study: ANRS CO17, VIHGY. Participants were recruited from hospital-based gynaecology consultations. Questionnaires were administered at each visit and collected data on demographic characteristics as well as gynaecological and sexual history. All women underwent a Pap test. © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 362–367

Women with abnormal cytology (atypical squamous cells of undetermined significance or worse; ASCUS+) were referred for colposcopy and biopsy, and treatment when necessary. The specimens for conventional cytology and LBC were collected using the Roversâ Cervex-Brushâ. Conventional smears were made by smearing cellular material obtained with the collection device onto the slide, followed by immediate fixation with a spray fixative. The LBC samples were prepared by transferring the sampled cells from the brush to the transport medium by firmly rotating and pushing the brush against the vial wall several times. The liquid-based system used was PreservCytâ. LBC samples were processed at the Laboratoire Cerba (Cergy pontoise, France) with the Thin-Prep 3000 processor (Hologic Inc.). Each slide was categorized as satisfactory or unsatisfactory and, if satisfactory, into the following five categories based on the criteria proposed by the 2001 Bethesda System terminology: within normal limit (WNL), ASC-US, low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL) and suggestive of invasive cervical carcinoma. In the present study, experienced cytotechnologists from Laboratoire Cerba (laboratory A) reviewed the cytology and showed the slides to an expert pathologist (C.B.) in cases of abnormal findings (ASC-US+). For LBC, the slides were read by the cytotechnologists according to the 22 fields selected by the pre-reading ThinPrepâ imager system (Hologic Inc.). A cytotechnologist from Laboratoire Cartier (laboratory B) read blindly the selected cervical smears and used the same categorization. Abnormal smears were reviewed by an expert pathologist (I.C.). As specified in the protocol of the study, the reproducibility of the cytological diagnoses was first evaluated at the end of Year 1 of the VIHGY cohort. The specimens reviewed were all unsatisfactory results, abnormal results (ASC-US+) and normal results associated with a positive Hybrid Capture 2 (HC2) test, and a random sample of normal cytology results associated with a negative HC2 test. As proposed by the scientific committee, and after approval of the Ethics Committee on September 2009, it was decided to move to LBC. A second reproducibility study of cytological diagnoses was conducted on liquid-based samples collected during 2010. A similar selection of slides was used and reviewed by Laboratory B.

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For the agreement between the two cytology raters, we calculated the total agreement with a binomial 95% confidence interval (95% CI). To evaluate agreement, and because kappa treats equally any disagreement and does not account for the increasing severity of cervical lesions, we calculated the Cicchetti–Allison linear-weighted kappa with 95% CI for the ordered cytology categories. Kappa values of less than 0.20 were interpreted as poor, values between 0.21 and 0.40 were interpreted as fair, values between 0.41 and 0.60 were interpreted as moderate, values between 0.61 and 0.80 were interpreted as good and values greater than 0.80 were interpreted as very good, using thresholds by Altman.9 Finally, kappa values were compared using the chi-

squared test. As only women with ASC-US+ were referred for colposcopy, we were unable to estimate the sensitivity and specificity for CIN2+ in LBC compared with conventional cytology. Results Two hundred and seventy-seven conventional Pap tests performed in 216 women and 268 LBCs performed in 210 women were reviewed. They were selected according to the Laboratory A diagnoses. They consisted of all unsatisfactory results (10 conventional and two LBC), all abnormal results (79 conventional and 123 LBC), and 188 and 143 normal results for conventional cytology and LBC, respectively (Figure 1),

Figure 1. Specimen flow of enrolment. © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 362–367

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Table 1. Adequacy of conventional and liquid-based preparations Laboratory A

Conventional preparation LBC P value

Laboratory B

Satisfactory

Unsatisfactory

Total

Satisfactory

Unsatisfactory

Total

267 266

10 (3.6%) 2 (0.7%) 0.02

277 268

263 265

14 (5.1%) 3 (1.1 %) 0.008

277 268

LBC, liquid-based cytology.

for a roughly similar number of specimens in both rounds. Population study Two hundred and seventy-seven conventional Pap tests performed in 216 women aged 20-4 to 69.6 years (median 41.4 years) were reviewed. Eighty-eight per cent were on combination antiretroviral therapy. Almost half (46.3%) of these had a CD4+ cell count above 500 and the HIV load was lower than 50 copies/ ml in 68.5% at the time when the cytology was taken. Two hundred and sixty-eight LBC samples performed in 210 women aged 27.1 to 69.8 years (median 43.5 years) were reviewed. Ninety-one per cent were on combination antiretroviral therapy. More than half (55.2%) of these had a CD4+ cell count above 500 when the cytology was taken and 75.4% had an HIV load lower than 50 copies/ml. Finally, 354 women had more than one sample (72 conventional and one LBC).

rates with conventional cytology were 3.6% and 5.1% for Laboratory A and B respectively. Using LBC, the rates were 0.7% and 1.1%, respectively. Kappa increased from 0.39 (95% CI, 0.14–0.65) to 0.80 (95% CI, 0.41–1.00) according to the Pap test method (P = 0.08). Conventional cytology Table 2 shows a comparison of the cytological interpretation by the two raters among 256 satisfactory cases classified by both Laboratory A and B. Laboratory A called 70.3% of the samples as WNL (180/ 256), 10.2% as ASC-US, 9.8% as LSIL, 9.8% as HSIL and none suggestive of carcinoma. Laboratory B called 68.4% of the samples as WNL (175/256), 8.6% as ASC-US, 12.1% as LSIL, 9.4% as HSIL and 1.6% as suggestive of carcinoma. The crude agreement was 78.1% (95% CI, 73.1–83.2%) and the weighted kappa 0.69 (95% CI, 0.61–0.77). Liquid-based cytology

Rate of satisfactory Pap tests The proportion of women with an unsatisfactory cytology result was significantly reduced with LBC for both Laboratory A and B (Table 1). The unsatisfactory

A similar inter-rater agreement study was performed on 268 LBC samples. Table 3 shows a comparison of the cytological interpretation by the two laboratories among 265 cases classified as satisfactory by both

Table 2. Inter-rater agreement for cytological interpretation of conventional preparations with a satisfactory result Laboratory B

Laboratory A

Diagnosis

Normal n

ASC-US n

LSIL n

HSIL n

ICC n

Total n (% Laboratory A)

Normal ASC-US LSIL HSIL ICC Total: n (% Laboratory B)

160 12 2 1 0 175 (68.4)

13 6 3 0 0 22 (8.6)

2 4 19 6 0 31 (12.1)

4 4 1 15 0 24 (9.4)

1 0 0 3 0 4 (1.6)

180 26 25 25 0 256

(70.3) (10.2) (9.8) (9.8)

ASC-US, atypical squamous cells of undetermined significance; HSIL, high-grade squamous intraepithelial lesion; ICC, invasive cervical cancer; LSIL, low-grade squamous intraepithelial lesion. © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 362–367

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Table 3. Inter-rater agreement for cytological interpretation of liquid-based preparations with a satisfactory result Laboratory B

Laboratory A

Diagnosis

Normal n

ASC-US n

LSIL n

HSIL n

Total n (% Laboratory A)

Normal ASC-US LSIL HSIL Total: n (% Laboratory B)

130 4 2 0 136 (51.3)

10 18 2 0 30 (11.3)

1 11 60 3 75 (28.3)

2 4 3 15 24 (9.1)

143 37 67 18 265

(54.0) (14.0) (25.3) (6.8)

ASC-US, atypical squamous cells of undetermined significance; HSIL, high-grade squamous intraepithelial lesion; ICC, invasive cervical cancer; LSIL, low-grade squamous intraepithelial lesion.

Laboratory A and B. Laboratory A called 54.0% of the samples as WNL, 14.0% as ASC-US, 25.3% as LSIL and 6.8% as HSIL. Laboratory B called 51.3% of the samples as WNL, 11.3% as ASC-US, 28.3% as LSIL and 9.1% as HSIL. The crude agreement was 84.2% (95% CI, 79.8–88.5%) and the weighted kappa 0.82 (95% CI, 0.76–0.87). Kappa’s comparison, performed to compare agreement for diagnostic categories between conventional cytology and LBC, showed that the reproducibility of cytological interpretation was significantly higher with the liquid-based method (P = 0.009). Discussion In this analysis, we found moderate to good agreement between the two pathologists who evaluated cervical cytology from HIV-infected women using conventional methods. Good agreement was observed when using LBC, with a Cicchetti–Allisonweighted kappa value of 0.82. The reproducibility of LBC interpretations was significantly higher than the reproducibility of conventional preparations. Similar to other studies performed in the general population, the rate of unsatisfactory samples was significantly lower with LBC.6,7 The interobserver agreement for conventional smears was better than that reported in different studies in the general population.10 This might be related to the low number of ASC-US in the set of smears, as it has been shown that ASC-US smears have poor interobserver agreement.11 Interobserver variation was observed in one case of suspicion of cancer (Laboratory B) classified as normal by Laboratory A. Unfortunately, no biopsy was performed in this case as the follow-up of the patient was based on the diagnosis of Laboratory A. Normal cytology has been observed for

this woman on four consecutive Pap tests during follow-up. As others, we observed a good agreement between the two laboratories in cytology using the liquidbased method, and the reproducibility for each diagnosis was higher than in conventional cytology.10–12 As our objective was to assess interobserver variability between the conventional method and the LBC method, we did not take into account the results of histology in our analysis. Few studies have compared the interobserver variability between liquid-based and conventional methods, but a similar significant difference between these two methods has been shown in a set of liquid-based slides and their conventional smears.10 The improved cytological preservation and presentation of the liquid-based method over the conventional method are probable explanations for the observed differences in interobserver agreement. In most developed countries, guidelines recommend that HIV-infected women should have a Pap test at HIV diagnosis and 6 months later, with subsequent lifetime annual tests. These recommendations are based on the fact that the sensitivity of the Pap test is limited and that HIV-infected women are at higher risk for cervical cancer. HIV-infected women face a high load of HIV care and many do not receive an annual Pap test.13 The difficulty and intensity of their clinical management for HIVrelated diseases justify that, when a Pap test for cervical screening is performed, the risk of an inadequate sample and unnecessary recall is low. The substantial reduction in the number of inadequate samples observed in our study (reduction from 5% to 1.1%) favours the use of LBC as the preferred screening technique, at least when its extra cost can be afforded. Another benefit of LBC preparation © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 362–367

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might be a significant decrease in the proportion of smears diagnosed with ASC-US in HIV-infected women.14 HPV DNA testing is now being introduced into some countries as the primary screening test because of consistent evidence that HPV DNA detection has greater one-time sensitivity than cytology-based methods for precancer lesion detection.15 An HPV test may not be ideal in the setting of HIV, because of its low specificity, resulting from a high HPV prevalence in women with normal cytology.16 Therefore, the only tool that may be proposed for cervical cancer screening is the Pap test. Women enrolled in the VIHGY cohort, aimed at evaluating the burden and natural history of HPVrelated cervical lesions, did not undergo colposcopy in cases with normal Pap tests, even when oncogenic HPVs were detected. Therefore, we were unable to evaluate the sensitivity and negative predictive value of conventional cytology and LBC. Nevertheless, the strong interobserver agreement observed confirms the accuracy of the Pap test for the detection of abnormal cells and cervical cancer screening in the setting of HIV. We have demonstrated that LBC is superior to the conventional method in terms of reproducibility and the small number of unsatisfactory samples, and might thus be preferred when available. Funding This work was supported by the Agence Nationale de Recherches sur le SIDA et les hepatites virales (ANRS), Institut National de la Sante et de la Recherche Medicale (INSERM) . References 1. Denny LA, Franceschi S, de Sanjose S et al. Human papillomavirus, human immunodeficiency virus and immunosuppression. Vaccine 2012;30(Suppl 5):F168– 74. 2. Kaulich-Bartz J, Dam W, May MT et al. Insurability of HIV-positive people treated with antiretroviral therapy in Europe: collaborative analysis of HIV cohort studies. AIDS 2013;27:1641–55. 3. Massad LS, Seaberg EC, Watts DH et al. Low incidence of invasive cervical cancer among HIV-infected US women in a prevention program. AIDS 2004;18: 109–13.

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4. Hleyhel M, Belot A, Bouvier AM et al. Risk of AIDSdefining cancers among HIV-1-infected patients in france between 1992 and 2009: results from the FHDH-ANRS CO4 cohort. Clin Infect Dis 2013;57: 1638–47. 5. Arbyn M, Bergeron C, Klinkhamer P et al. Liquid compared with conventional cervical cytology: a systematic review and meta-analysis. Obstet Gynecol 2008;111: 167–77. 6. Ronco G, Cuzick J, Pierotti P et al. Accuracy of liquid based versus conventional cytology: overall results of new technologies for cervical cancer screening: randomised controlled trial. BMJ 2007;335:28. 7. Siebers AG, Klinkhamer PJ, Grefte JM et al. Comparison of liquid-based cytology with conventional cytology for detection of cervical cancer precursors: a randomized controlled trial. JAMA 2009;302:1757–64. 8. Fruchter RG, Maiman M, Sillman FH et al. Characteristics of cervical intraepithelial neoplasia in women infected with the human immunodeficiency virus. Am J Obstet Gynecol 1994;171:531–7. 9. Altman DG. Practical statistics for medical research. London: Chapman and Hall; 1991. 10. Chhieng DC, Talley LI, Roberson J et al. Interobserver variability: comparison between liquid-based and conventional preparations in gynecologic cytology. Cancer 2002;96:67–73. 11. Crum CP, Genest DR, Krane JF et al. Subclassifying atypical squamous cells in Thin-Prep cervical cytology correlates with detection of high-risk human papillomavirus DNA. Am J Clin Pathol 1999;112:384–90. 12. Confortini M, Bergeron C, Desai M et al. Accuracy of liquid-based cytology: comparison of the results obtained within a randomized controlled trial (the New Technologies for Cervical Cancer Screening Study) and an external group of experts. Cancer Cytopathol 2010;118:203–8. 13. Oster AM, Sullivan PS, Blair JM. Prevalence of cervical cancer screening of HIV-infected women in the United States. J Acquir Immune Defic Syndr 2009;51:430–6. 14. Swierczynski SL, Lewis-Chambers S, Anderson JR et al. Impact of liquid-based gynecologic cytology on an HIV-positive population. Acta Cytol 2004;48: 165–72. 15. Castle PE, de Sanjose S, Qiao YL et al. Introduction of human papillomavirus DNA screening in the world: 15 years of experience. Vaccine 2012;30(Suppl 5): F117–22. 16. Womack SD, Chirenje ZM, Gaffikin L et al. HPVbased cervical cancer screening in a population at high risk for HIV infection. Int J Cancer 2000;85: 206–10.

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Interobserver variability of cervical cytology in HIV-infected women.

Our objectives were to determine the reproducibility of cytological specimen interpretation between two pathologists in human immunodeficiency virus (...
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