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Interobserver variability and accuracy of p16/Ki-67 dual immunocytochemical staining on conventional cervical smears.

ABSTRACT: BACKGROUND:p16/Ki-67 dual immunocytochemical staining (DS) has been proven as a sensitive and specific test for triage of HPV positive women with good reproducibility and accuracy. However, implementation of the test into an organized screening program (OSP) is not easy. The aims of this study were to compare the performance and agreement of DS results among three Slovenian cytopathological laboratories involved in the national OSP, and to define cases where staining results can be difficult to interpret. METHODS:Cervical smears were obtained for DS from 129 women referred to colposcopy. Smears were evaluated blindly in three laboratories by a cytotechnologist and a cytopathologist after initial training. Results were positive, suspicious, negative or inadequate. Five characteristics of DS staining were recorded. After primary evaluation, an extensive expert-led additional training was undertaken, including a discussion of difficult cases and a practical exam. Smears were re-evaluated and results compared to primary evaluation. RESULTS:After the additional training, the overall percentage of agreement among the three laboratories increased from 77.5 to 89.9% and kappa increased from 0.70 to 0.86. Sensitivity for CIN2+ increased in two laboratories, to 90.5 and 85.7%, without the loss of specificity (75.8%). In one laboratory, the sensitivity slightly decreased from 90.5 to 88.9%, but the specificity increased from 63.6 to 68.2%. Difficult cases had significantly less DS cells, weak intensity of p16 staining, suboptimal cell morphology and background staining compared to positive cases. CONCLUSION:Additional expert-led training and discussion of difficult cases are necessary for accurate interpretation of DS in laboratories involved in OSP. The most difficult cases were those with single stained cells and weak p16 staining. Training protocol for safe implementation of p16/Ki-67 DS in OSP is proposed.

SUBMITTER: Kloboves Prevodnik V

PROVIDER: S-EPMC6533697 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Interobserver variability and accuracy of p16/Ki-67 dual immunocytochemical staining on conventional cervical smears.

Kloboves Prevodnik Veronika V Jerman Tine T Nolde Nataša N Repše Fokter Alenka A Jezeršek Sandra S Pohar Marinšek Živa Ž Klopčič Ulrika U Hutter Čelik Simona S Gornik Kramberger Kristina K Primic Žakelj Maja M Ivanuš Urška U

Diagnostic pathology 20190524 1

<h4>Background</h4>p16/Ki-67 dual immunocytochemical staining (DS) has been proven as a sensitive and specific test for triage of HPV positive women with good reproducibility and accuracy. However, implementation of the test into an organized screening program (OSP) is not easy. The aims of this study were to compare the performance and agreement of DS results among three Slovenian cytopathological laboratories involved in the national OSP, and to define cases where staining results can be diffi ...[more]

PMID: 31122253

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Project description:Background p16/Ki-67 dual immunocytochemical staining (DS) is considered easy to interpret if evaluators are properly trained, however, there is no consensus on what constitutes proper training. In the present study we evaluated a protocol for teaching DS evaluation on students inexperienced in cervical cytology. Methods Initial training on 40 DS conventional smears was provided by a senior cytotechnologist experienced in such evaluation. Afterwards, two students evaluated 118 cases. Additional training consisted mainly of discussing discrepant cases from the first evaluation and was followed by evaluation of new 383 cases. Agreement and accuracy of students' results were compared among the participants and to the results of the reference after both evaluations. We also noted time needed for evaluation of one slide as well as intra-observer variability of the teacher's results. Results At the end of the study, agreement between students and reference was higher compared to those after initial training (overall percent agreement [OPA] 81.4% for each student, kappa 0.512 and 0.527 vs. OPA 78.3% and 87.2%, kappa 0.556 and 0.713, respectively). However, accuracy results differed between the two students. After initial training sensitivity was 4.3% points and 2.9% points higher, respectively compared to the reference, while specificity was 30.6% points and 24.4% points lower, respectively, compared to the reference. At the end of the study, the sensitivity reached by one student was the same as that of the reference, while it was 2.6% points lower for the other student. There was a statistically significant difference in specificity between one student and the reference and also between students (16.7 and 15.1% points). Towards the end of the study, one student needed 5.2 min for evaluating one slide while the other needed 8.2 min. The intra-observer variability of the senior cytotechnologist was in the range of "very good" in both arms of the study. Conclusions In teaching DS evaluation, the students' progress has to be monitored using several criteria like agreement, accuracy and time needed for evaluating one slide. The monitoring process has to continue for a while after students reach satisfactory results in order to assure a continuous good performance. Monitoring of teacher's performance is also advisable.

Project description:BACKGROUND:Human Papilloma Virus (HPV) DNA tests are highly sensitive and can triage women with mild lesions, improving the prognosis and diagnosis of cervical lesions. However, additional efficient strategies should be developed to improve the specificity of these tests. METHODS:This study aimed to evaluate the clinical value of HPV DNA load in improving the diagnosis and prognosis of cervical lesions by p16/Ki-67 testing. Histological samples were collected from 350 women with HR-HPV genotyping and analyzed by qRT-PCR. Immunohistochemical staining was used to assess p16 and Ki-67 expression and clinical performance characteristics were calculated. RESULTS:Of the cases, 271 had detectable HR-HPV infection, in which HPV-16 was most prevalent (52.0%), followed by HPV-58 (22.5%). P16/Ki-67-positivity increased with histological severity but not for HR-HPV infection. Amongst the 13 HR-HPV genotypes, only HPV-16 (P = 0.016) and HPV-58 (P = 0.004) viral loads significantly correlated with lesion severity. The P16/Ki-67/HPV DNA load co-test indicated an increased sensitivity for the detection of cervical intraepithelial neoplasia (CIN) lesions compared to p16/Ki-67 staining in HPV-16 and/or 58 positive cases. Viral load did not improve the sensitivity of p16/Ki-67 co-test in non-HPV-16 or 58 positive cases. The clinical performance of the p16/Ki-67/HPV DNA load co-test was limited for the prediction of the outcome of CIN1 lesions. However, amongst the 12 HPV-16 and/or 58 positive CIN2 cases in which return visit results were obtained, the behavior of the lesions could be predicted, with a sensitivity, specificity, positive prediction rate (PPV), and negative prediction rate (NPV) of 0.667, 1, 1 and 0.5, respectively. CONCLUSION:Combination of the assessment of HPV DNA load with the intensity of p16 and Ki-67 staining could increase the sensitivity of CIN lesion diagnosis and predict the outcome of CIN2 in patients with a HPV-16 and/or 58 infection.

Project description:ImportanceAs cervical cancer screening transitions to primary human papillomavirus (HPV) testing, effective triage and management of HPV-positive women is critical to avoid unnecessary colposcopy referral and associated harms while maintaining high sensitivity for cervical precancer. Triage with p16/Ki-67 dual-stain (DS) testing has shown high sensitivity and specificity for detection of cervical precancers; however, longitudinal studies are needed to determine the long-term risk of precancer following a negative DS result.ObjectiveTo evaluate the longitudinal performance of p16/Ki-67 DS triage for detection of cervical precancer in HPV-positive women over 5 years of follow-up in the context of clinical management thresholds.Design, setting, and participantsProspective cohort study of HPV-positive women 30 years or older undergoing routine cervical cancer screening in 2012 with HPV and Papanicolaou (hereinafter "cytology") co-testing within the Kaiser Permanente Northern California health care system. Follow-up of medical records was conducted through 2017.ExposuresAll p16/Ki-67 DS testing was performed on residual SurePath material, and slides were evaluated for p16/Ki-67 positivity.Main outcomes and measuresHistological end points were ascertained from the clinical database through 2017. We estimated 5-year cumulative risks of cervical intraepithelial neoplasia grades of 2 or worse (≥CIN2) or grades 3 or worse (≥CIN3) by baseline DS and cytology at yearly intervals using Logistic Weibull models. Risks were compared with clinical management thresholds for colposcopy referral and a 1-year return interval.ResultsAmong the 1549 HPV-positive women in this study, the mean age at enrollment was 42.2 years, and the median follow-up time was 3.7 years (range, 0.2-5.4 years). Positive DS results were associated with significantly higher cumulative 5-year risks of ≥CIN2 compared with abnormal cytology (31.0%; 95% CI, 27.2%-35.3% vs 25.0%; 95% CI, 21.7%-28.7%; P = .03). Women with DS-negative findings had significantly lower 5-year risks of ≥CIN2 compared with women with normal cytology (8.5%; 95% CI, 6.5%-11.1% vs 12.3%; 95% CI, 9.8%-15.4%; P = .04). In DS-negative women, the risks of both ≥CIN2 and ≥CIN3 remained below the colposcopy referral threshold for all 5 years, crossing the 1-year return threshold at 3 years.Conclusions and relevanceTriage with p16/Ki-67 DS provides better long-term risk stratification than cytology over 5 years. The low risk of cervical precancer in p16/Ki-67 DS-negative women permits safe extension of follow-up intervals for 3 years.

Project description:ImportanceAs cervical cancer screening transitions from Papanicolaou cytologic screening to primary human papillomavirus (HPV) testing worldwide, effective triage tests are needed to decide who among the HPV-positive women should receive further diagnostic evaluation to avoid unnecessary colposcopies and biopsies.ObjectiveTo evaluate the performance of the p16/Ki-67 dual stain (DS) and HPV16/18 genotyping for the triage of HPV-positive women.Design, setting, and participantsA prospective observational study was conducted within the cervical cancer screening program at Kaiser Permanente Northern California of 3225 HPV-positive women undergoing HPV and Papanicolaou cytologic testing with a valid DS result from September 16 to October 31, 2015, with follow-up through December 31, 2018.ExposuresHuman papillomavirus screening with partial genotyping and cytologic triage compared with DS triage.Main outcomes and measuresCervical intraepithelial neoplasia grade 3 or more severe (CIN3+) and grade 2 or more severe (CIN2+), diagnosed within 3 years after sample collection.ResultsA total of 3225 women (mean [SD] age, 37.9 [11.3] years) participated in the study. For triage of HPV-positive women with partial genotyping, DS showed better risk stratification for CIN3+ than did Papanicolaou cytologic testing, with women with positive DS results having a higher risk than women with positive Papanicolaou test results for CIN3+ (218 of 1818 [12.0%; 95% CI, 10.5%-13.5%] vs 219 of 2128 [10.3%; 95% CI, 9.0%-11.6%]; P = .005). Similarly, DS showed better risk stratification for CIN3+ compared with Papanicolaou cytologic testing in HPV-positive women, irrespective of genotyping. The greatest reassurance against CIN3+ was observed in HPV16/18-negative women with negative DS results, with a risk low enough to extend retesting intervals. Dual stain triage strategies required substantially fewer colposcopies per detection of CIN3+ compared with Papanicolaou cytologic testing, with a 32.1% (859 of 2677) reduction of colposcopies compared with the currently recommended triage strategy of HPV screening with Papanicolaou cytologic testing. Results for CIN2+ were very similar.Conclusions and relevanceTriage of HPV-positive women with DS was superior to Papanicolaou cytologic testing in this study, demonstrating equal immediate detection of precancerous lesions and substantially reduced referral to colposcopy. These findings suggest that DS can safely replace Papanicolaou cytologic testing as a triage strategy for primary HPV screening, and that retesting intervals in HPV16/18-negative women with negative DS results can be safely extended to 3 years.

Dataset Information

Interobserver variability and accuracy of p16/Ki-67 dual immunocytochemical staining on conventional cervical smears.

Publications

Interobserver variability and accuracy of p16/Ki-67 dual immunocytochemical staining on conventional cervical smears.

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