Liquid-based cytology versus conventional cytology for detection of uterine cervical lesions: a prospective observational study

Liquid-based cytology versus conventional cytology for detection of uterine cervical lesions: a... Abstract Objective Liquid-based cytology (LBC) and conventional cytology (CS) are routine diagnostic techniques in cervical cytology, but few studies have compared their diagnostic performances with each other and with histologic diagnosis. This study aimed to compare the diagnostic performances of these techniques in subjects with abnormal cervical cytology of atypical cells of undetermined significance (ASC-US) or worse. Methods A total of 312 patients diagnosed with ASC-US or worse were enrolled in this prospective study in Japan from 2013 to 2014. LBC and CS samples were prepared by a split-sampling technique and evaluated blindly. The results were classified using the Bethesda System 2001. Colposcopy and biopsy were conducted simultaneously or within 4 weeks of cytology-specimen collection in all cases. Diagnostic performance was calculated in terms of sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for detection of CIN2 or worse, with a cut-off ASC-US or worse. Results There was one unsatisfactory CS sample and the remaining 311 cases were evaluated. The sensitivities of LBC and CS were 100.0% and 98.8%, specificities were 17.2% and 23.8%, PPVs were 56.1% and 57.9% and NPVs were 100.0% and 94.7%, respectively. LBC had slightly higher sensitivity and NPV for detection of CIN2, but there was no significant difference between the two methods. Conclusions There was no significant difference in the diagnostic performances of LBC and CS in patients with ASC-US or worse. LBC may therefore be an alternative approach to CS for cervical cancer screening. liquid-based cytology, conventional cytology, cervical cancer screening Introduction Cervical cancer is one of the most common gynecologic malignancies in Western countries (1). Invasive cervical cancer develops from dysplasia with human papillomavirus (HPV) infection over a period of 10 years or more (2), during which time screening allows the detection and treatment of early dysplasia, thus preventing its development into cancer (3). Widespread application of screening programs in recent decades has achieved significant reductions in cervical cancer incidence and mortality (4,5). Cervical cancer screening involves the examination of cytological preparations, including conventional cytology sampling (CS), in which cells are smeared directly onto a glass slide, or liquid-based cytology (LBC), in which samples are stirred in a cell-fixation solvent in a suspended state using a sampling device (6–8). CS is a well-established and useful method, but has several problems, including the need to prepare samples over a short period of time, variable sample quality and occasionally unusable samples caused by drying artifacts or overlapping debris. LBC overcomes these problems, and has the advantage of collecting a large number of cells through the use of a specialized sample-preparation device (9), thus preventing variation among samples and reducing the number of unsatisfactory samples (10–12). In addition, following cytology, LBC samples can also be used directly for HPV testing (13–17), which is considered to be an effective method of screening. However, despite these benefits of LBC, CS is still used in about 90% of cases, and LBC remains to be fully integrated into the screening system in Japan. One of the reasons is that we are not quite confident that LBC has equal or even better diagnostic performance comparing with CS. Although several studies have compared the diagnostic performances of CS and LBC globally (18–28), few have been reported in Japan. Hirai (29) reported sensitivities of 71.3% and 77.4% and specificities of 99.0% and 98.9% for CS and LBC, respectively, for the detection of CIN2 or worse in Japanese patients with ASC-H (indicating high-grade squamous intraepithelial lesion) or worse. However, although LBC showed higher sensitivity, their study was based on limited biopsies performed only in patients with abnormal cytological or colposcopic abnormalities. The establishment of LBC in cervical cancer screening in Japan requires a prospective comparison of the diagnostic performances of LBC and CS based on histology in a broader population, including patients with negative cytology (NILM). Here we first conducted a prospective study in Japan to evaluate the diagnostic performances of CS and LBC for detecting cervical lesions compared with histology in Japanese patients with abnormal cervical cytology. Materials and methods Study design The design of this multicenter, observational study is summarized in Fig. 1. Eligible patients were aged 30–64 years, with previously reported abnormal cytology of ASC-US or greater detected within 24 weeks before provision of informed consent (Table 1). Institutional review board approval was obtained from the ethics committee at each participating institution before patient registration (UMIN ID: 00001146). The investigators explained the study to patients and obtained written informed consent. Cytology and histology by colposcopy and biopsy were performed within a 2-week interval. The interval between the previous biopsy and the biopsy taken for this study was at least 4 weeks, to reduce the patient burden. Subject enrollment was carried out using an online central enrollment system. Figure 1. View largeDownload slide Study design. Split sampling was performed with initial smearing for CS with a broom brush and then preparation of LBC samples using ThinPrep® was carried out. *Biopsy was performed simultaneously with cytology or within 2 weeks, and at least 4 weeks after the last previous biopsy. In cases of apparent invasive cancer, biopsy was allowed within 4 weeks following the previous biopsy. **Slides were evaluated blindly at PCL Japan, Inc. (BML, Inc. Group, Tokyo, Japan). Figure 1. View largeDownload slide Study design. Split sampling was performed with initial smearing for CS with a broom brush and then preparation of LBC samples using ThinPrep® was carried out. *Biopsy was performed simultaneously with cytology or within 2 weeks, and at least 4 weeks after the last previous biopsy. In cases of apparent invasive cancer, biopsy was allowed within 4 weeks following the previous biopsy. **Slides were evaluated blindly at PCL Japan, Inc. (BML, Inc. Group, Tokyo, Japan). Table 1. Eligibility criteria Inclusion criteria  Patients aged 30–64 years old at the time of informed consent  Abnormal cytology of ASC-US or worse suggested within 24 weeks before informed consent Exclusion criteria  Patients with previous history of invasive cervical cancer  Patients who underwent cervical conization  Patients who underwent hysterectomy  Patients who were pregnant  Patients who received tissue biopsy within 4 weeks Inclusion criteria  Patients aged 30–64 years old at the time of informed consent  Abnormal cytology of ASC-US or worse suggested within 24 weeks before informed consent Exclusion criteria  Patients with previous history of invasive cervical cancer  Patients who underwent cervical conization  Patients who underwent hysterectomy  Patients who were pregnant  Patients who received tissue biopsy within 4 weeks View Large Table 1. Eligibility criteria Inclusion criteria  Patients aged 30–64 years old at the time of informed consent  Abnormal cytology of ASC-US or worse suggested within 24 weeks before informed consent Exclusion criteria  Patients with previous history of invasive cervical cancer  Patients who underwent cervical conization  Patients who underwent hysterectomy  Patients who were pregnant  Patients who received tissue biopsy within 4 weeks Inclusion criteria  Patients aged 30–64 years old at the time of informed consent  Abnormal cytology of ASC-US or worse suggested within 24 weeks before informed consent Exclusion criteria  Patients with previous history of invasive cervical cancer  Patients who underwent cervical conization  Patients who underwent hysterectomy  Patients who were pregnant  Patients who received tissue biopsy within 4 weeks View Large The primary endpoint was difference in diagnostic performance between CS and LBC for detecting cervical lesions of CIN2 or worse, with a cut-off of ASC-US or worse. Diagnostic performance was evaluated based on sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). The methods used to calculate the parameters are shown in Table 2. Following cytology and histology diagnosis, we categorized each sample as A–D, and evaluated each parameter as shown (Table 2, upper row). The secondary endpoint was the difference in diagnostic performance between CS and LBC for detecting cervical lesions of CIN1 or worse, with a cut-off of ASC-US or worse (Table 2, lower row). Table 2. Sensitivity, specificity, PPV and NPVs of CS and LBC for detecting CIN2 or worse (primary endpoint) and CIN1 or worse (secondary endpoint) with a cut-off level of ASC-US or worse Histology results Cytology results Normal/CIN1 CIN2/CIN3/SCC CIN2 or worse NILM A B ASC-US, LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy C D Histology results Cytology results Normal/CIN1 CIN2/CIN3/SCC CIN2 or worse NILM A B ASC-US, LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy C D Normal CIN1/CIN2/CIN3/SCC CIN1 or worse NLIM E F ASC-US,LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy G H Normal CIN1/CIN2/CIN3/SCC CIN1 or worse NLIM E F ASC-US,LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy G H Sensitivity: D/(B + D) and (H/F + H); specificity: A/(A + C) and E/(F + G); PPV: D/(C + D) and H/(G + H); NPV: A/(A + B) and E/(E + F). CIN, cervical intraepithelial neoplasia; NILM, negative for intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells that cannot exclude; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; AGC, atypical glandular cells; AIS, adenocarcinoma in situ. Table 2. Sensitivity, specificity, PPV and NPVs of CS and LBC for detecting CIN2 or worse (primary endpoint) and CIN1 or worse (secondary endpoint) with a cut-off level of ASC-US or worse Histology results Cytology results Normal/CIN1 CIN2/CIN3/SCC CIN2 or worse NILM A B ASC-US, LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy C D Histology results Cytology results Normal/CIN1 CIN2/CIN3/SCC CIN2 or worse NILM A B ASC-US, LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy C D Normal CIN1/CIN2/CIN3/SCC CIN1 or worse NLIM E F ASC-US,LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy G H Normal CIN1/CIN2/CIN3/SCC CIN1 or worse NLIM E F ASC-US,LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy G H Sensitivity: D/(B + D) and (H/F + H); specificity: A/(A + C) and E/(F + G); PPV: D/(C + D) and H/(G + H); NPV: A/(A + B) and E/(E + F). CIN, cervical intraepithelial neoplasia; NILM, negative for intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells that cannot exclude; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; AGC, atypical glandular cells; AIS, adenocarcinoma in situ. Cytology and interpretation Cells were collected using a Rovers® Cervix-Brush® (Oss, The Netherlands). The cells were divided into CS samples smeared on glass slides and LBC samples prepared using PreservCyt Solution and a ThinPrep® Processor. CS samples on glass slides were immersed in 95% ethanol and fixed and stained by Papanicolaou staining, and then evaluated blindly at PCL Japan, Inc. (BML, Inc. Group, Tokyo, Japan; hereinafter referred to as PCL). LBC samples were collected in ThinPrep® PreservCyt Solution Vials and then sent to PCL. No clinical information was provided to PCL, to ensure equal and independent blinded evaluation of the two methods. The obtained cytology results were compared with the histology results assessed at each institution. Diagnostic criteria Cytology results were reported in accordance with the Bethesda System 2001. Reasons for unsatisfactory samples were recorded, and re-collection was not allowed for samples determined to be unsatisfactory using CS or LBC. Colposcopic findings were classified based on the New Colposcopic Classification of The Japan Society of Gynecologic Oncology, 2005. Colposcopic data were not collected in this study. Histological findings were classified using the General Rules for Clinical and Pathological Management of Uterine Cervical Cancer (3rd edition) at each study site, as follows: normal; CIN1 or worse; CIN2 or worse; CIN3 or worse; cancer; and not evaluable. Colposcopy, biopsy and histological diagnosis Colposcopy and colposcopy-guided biopsy were carried out within 2 weeks after cell collection. Colposcopy was performed according to the General Rules for Clinical and Pathological Management of Uterine Cervical Cancer. Biopsies were performed under colposcopic guidance and the number of biopsy samples was determined by the investigator. Histological diagnosis was performed using the normal procedure at each study site and the findings were classified. If the histology results varied among multiple biopsies, the highest diagnosis was recorded. Statistical design and analysis The target number of subjects was estimated to be 300, based on the following information. A total of 1059 patients with suspected abnormal histology of ASC-US or worse were referred to Keio University Hospital and underwent CS and histology from January to December 2011 (unpublished data). Using a cut-off of ASC-US or worse, cytology results were negative in 393 patients and positive in 666. No CIN2 or worse lesion was detected by histology in 379 of the 393 cytology-negative patients, and CIN2 or worse lesions were detected by histology in 415 of the 666 cytology-positive patients. A cytological diagnosis of NILM and a histological diagnosis of CIN1 or better, or a cytological diagnosis of ASC-US or worse and a histological diagnosis of CIN2 or worse was considered to be accurate. The diagnostic accuracy was therefore 75.0%, calculated as the proportion of the accurately diagnosed population among all patients. The reported sensitivity and specificity of ThinPrep® LBC were slightly higher or comparable to CS, and given an accuracy of LBC of 82.0%, a sample size of 300 subjects would be adequate to demonstrate significant superiority of LBC compared with CS. Demographic data and other data at enrollment were summarized as descriptive statistics, based on the types and distribution of data. Average differences in diagnostic performances between LBC and CS with 95% confidence intervals (CI) were calculated. Cross tables of histology results with CS and LBC results were prepared. Sensitivity, specificity, PPV and NPV with 95% CIs were estimated and analyzed by McNemar tests. Results Patient population A total of 326 cases were enrolled at three institutions between April 2013 and December 2014. Fourteen cases were excluded for withdrawal of consent (n = 1), cytology or histology not performed (n = 7) and interval between cytology and histology >2 weeks (n = 6). Of the remaining 312 cases, one was excluded from the analysis because of an unsatisfactory CS sample. A total of 311 cases were therefore finally evaluated (Fig. 2). The median age of the 311patients was 38.0 (30.0–64.0) years. Figure 2. View largeDownload slide Patient enrollment and analysis. Figure 2. View largeDownload slide Patient enrollment and analysis. Cytology and histology The results of CS and LBC are summarized in Table 3. High-grade squamous intraepithelial lesion was the most frequent diagnosis (46.9% for CS and 44.9% for LBC). Although all the patients were referred to the study institutions with previously reported ASC-US or worse cytology, the overall rates of NILM were 12.2% in CS and 8.3% in LBC, compared with 87.8% in CS and 91.7% in LBC among patients with ASC-US or worse. For cases with a cut-off of low-grade squamous intraepithelial lesion or higher, the rates were 73.3% for CS and 80.1% for LBC. Table 3. Results of CS and LBC Items Category CS LBC n = 312 n = 312 Specimen Unsatisfactory 1 (0.3) 0 (0.0) Satisfactory 311 (99.7) 312 (100) Items Category CS LBC n = 312 n = 312 Specimen Unsatisfactory 1 (0.3) 0 (0.0) Satisfactory 311 (99.7) 312 (100) Analysis of accepted samples Items Category Patients in total Patients in total n = 311 n = 312 Results NILM 38 (12.2) 26 (8.3) ASC-US 45 (14.5) 36 (11.5) LSIL 29 (9.3) 41 (13.1) ASC-H 25 (8.0) 37 (11.9) HSIL 146 (46.9) 140 (44.9) SCC 20 (6.4) 21 (6.7) AGC 4 (1.3) 7 (2.2) AIS 1 (0.3) 2 (0.6) Adenocarcinoma 3 (1.0) 2 (0.6) Other malignancy 0 (0.0) 0 (0.0) Results with the cutoff of ASC-US or worse ASC-US or higher 273 (87.8) 286 (91.7) NILM 38 (12.2) 26 (8.3) Results with the cutoff of LSIL or worse LSIL or higher 228 (73.3) 250 (80.1) NILM, ASC-US 83 (26.7) 62 (19.9) Analysis of accepted samples Items Category Patients in total Patients in total n = 311 n = 312 Results NILM 38 (12.2) 26 (8.3) ASC-US 45 (14.5) 36 (11.5) LSIL 29 (9.3) 41 (13.1) ASC-H 25 (8.0) 37 (11.9) HSIL 146 (46.9) 140 (44.9) SCC 20 (6.4) 21 (6.7) AGC 4 (1.3) 7 (2.2) AIS 1 (0.3) 2 (0.6) Adenocarcinoma 3 (1.0) 2 (0.6) Other malignancy 0 (0.0) 0 (0.0) Results with the cutoff of ASC-US or worse ASC-US or higher 273 (87.8) 286 (91.7) NILM 38 (12.2) 26 (8.3) Results with the cutoff of LSIL or worse LSIL or higher 228 (73.3) 250 (80.1) NILM, ASC-US 83 (26.7) 62 (19.9) CIN, cervical intraepithelial neoplasia; NILM, negative for intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells that cannot exclude; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; AGC, atypical glandular cells; AIS, adenocarcinoma in situ. Table 3. Results of CS and LBC Items Category CS LBC n = 312 n = 312 Specimen Unsatisfactory 1 (0.3) 0 (0.0) Satisfactory 311 (99.7) 312 (100) Items Category CS LBC n = 312 n = 312 Specimen Unsatisfactory 1 (0.3) 0 (0.0) Satisfactory 311 (99.7) 312 (100) Analysis of accepted samples Items Category Patients in total Patients in total n = 311 n = 312 Results NILM 38 (12.2) 26 (8.3) ASC-US 45 (14.5) 36 (11.5) LSIL 29 (9.3) 41 (13.1) ASC-H 25 (8.0) 37 (11.9) HSIL 146 (46.9) 140 (44.9) SCC 20 (6.4) 21 (6.7) AGC 4 (1.3) 7 (2.2) AIS 1 (0.3) 2 (0.6) Adenocarcinoma 3 (1.0) 2 (0.6) Other malignancy 0 (0.0) 0 (0.0) Results with the cutoff of ASC-US or worse ASC-US or higher 273 (87.8) 286 (91.7) NILM 38 (12.2) 26 (8.3) Results with the cutoff of LSIL or worse LSIL or higher 228 (73.3) 250 (80.1) NILM, ASC-US 83 (26.7) 62 (19.9) Analysis of accepted samples Items Category Patients in total Patients in total n = 311 n = 312 Results NILM 38 (12.2) 26 (8.3) ASC-US 45 (14.5) 36 (11.5) LSIL 29 (9.3) 41 (13.1) ASC-H 25 (8.0) 37 (11.9) HSIL 146 (46.9) 140 (44.9) SCC 20 (6.4) 21 (6.7) AGC 4 (1.3) 7 (2.2) AIS 1 (0.3) 2 (0.6) Adenocarcinoma 3 (1.0) 2 (0.6) Other malignancy 0 (0.0) 0 (0.0) Results with the cutoff of ASC-US or worse ASC-US or higher 273 (87.8) 286 (91.7) NILM 38 (12.2) 26 (8.3) Results with the cutoff of LSIL or worse LSIL or higher 228 (73.3) 250 (80.1) NILM, ASC-US 83 (26.7) 62 (19.9) CIN, cervical intraepithelial neoplasia; NILM, negative for intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells that cannot exclude; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; AGC, atypical glandular cells; AIS, adenocarcinoma in situ. The concordance between the CS and LBC results is shown in Table 4. The concordance rate between the two methods was 64.0% (95% CI, 58.4–69.3%). Table 4. Concordance between CS and LBC Category LBC results n NILM ASC-US LSIL ASC-H HSIL SCC AGC AIS Adeno Other CS results NILM 20 10 2 4 1 0 1 0 0 0 38 ASC-US 3 17 12 10 2 0 1 0 0 0 45 LSIL 0 2 22 0 5 0 0 0 0 0 29 ASC-H 2 1 1 9 10 0 1 0 1 0 25 HSIL 0 4 3 13 117 7 1 1 0 0 146 SCC 0 0 0 0 5 14 1 0 0 0 20 AGC 1 1 1 1 0 0 0 0 0 0 4 AIS 0 0 0 0 0 0 0 1 0 0 1 Adeno 0 0 0 0 0 0 2 0 1 0 3 Other 0 0 0 0 0 0 0 0 0 0 0 Total 26 35 41 37 140 21 7 2 2 0 311 Category LBC results n NILM ASC-US LSIL ASC-H HSIL SCC AGC AIS Adeno Other CS results NILM 20 10 2 4 1 0 1 0 0 0 38 ASC-US 3 17 12 10 2 0 1 0 0 0 45 LSIL 0 2 22 0 5 0 0 0 0 0 29 ASC-H 2 1 1 9 10 0 1 0 1 0 25 HSIL 0 4 3 13 117 7 1 1 0 0 146 SCC 0 0 0 0 5 14 1 0 0 0 20 AGC 1 1 1 1 0 0 0 0 0 0 4 AIS 0 0 0 0 0 0 0 1 0 0 1 Adeno 0 0 0 0 0 0 2 0 1 0 3 Other 0 0 0 0 0 0 0 0 0 0 0 Total 26 35 41 37 140 21 7 2 2 0 311 Shadowed cases represent concordant cases. NILM, negative for intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells that cannot exclude; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; AGC, atypical glandular cells; AIS, adenocarcinoma in situ; Adeno: adenocarcinoma; Other, other malignancy. Table 4. Concordance between CS and LBC Category LBC results n NILM ASC-US LSIL ASC-H HSIL SCC AGC AIS Adeno Other CS results NILM 20 10 2 4 1 0 1 0 0 0 38 ASC-US 3 17 12 10 2 0 1 0 0 0 45 LSIL 0 2 22 0 5 0 0 0 0 0 29 ASC-H 2 1 1 9 10 0 1 0 1 0 25 HSIL 0 4 3 13 117 7 1 1 0 0 146 SCC 0 0 0 0 5 14 1 0 0 0 20 AGC 1 1 1 1 0 0 0 0 0 0 4 AIS 0 0 0 0 0 0 0 1 0 0 1 Adeno 0 0 0 0 0 0 2 0 1 0 3 Other 0 0 0 0 0 0 0 0 0 0 0 Total 26 35 41 37 140 21 7 2 2 0 311 Category LBC results n NILM ASC-US LSIL ASC-H HSIL SCC AGC AIS Adeno Other CS results NILM 20 10 2 4 1 0 1 0 0 0 38 ASC-US 3 17 12 10 2 0 1 0 0 0 45 LSIL 0 2 22 0 5 0 0 0 0 0 29 ASC-H 2 1 1 9 10 0 1 0 1 0 25 HSIL 0 4 3 13 117 7 1 1 0 0 146 SCC 0 0 0 0 5 14 1 0 0 0 20 AGC 1 1 1 1 0 0 0 0 0 0 4 AIS 0 0 0 0 0 0 0 1 0 0 1 Adeno 0 0 0 0 0 0 2 0 1 0 3 Other 0 0 0 0 0 0 0 0 0 0 0 Total 26 35 41 37 140 21 7 2 2 0 311 Shadowed cases represent concordant cases. NILM, negative for intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells that cannot exclude; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; AGC, atypical glandular cells; AIS, adenocarcinoma in situ; Adeno: adenocarcinoma; Other, other malignancy. All patients underwent punch biopsies (median, 2; range, 1–5). The biopsy results are summarized in Table 5. CIN3 was the most-frequently diagnosed condition in biopsies, followed by CIN1. Table 5. Biopsy results Histology diagnosis Category Total patients n = 311 Normal No pathological abnormality 2 (0.6) Reactive or inflammatory lesion confirmed 77 (24.7) Squamous metaplasia 4 (1.3) Others 1 (0.3) CIN1 (Mild dysplasia) 67 (21.5) CIN2 CIN2 (Moderate dysplasia) 62 (19.9) CIN3 CIN3 (Severe dysplasia or squamous cell carcinoma in situ) 81 (26.0) Adenocarcinoma in situ 1 (0.3) Cancerous Microinvasive squamous cell carcinoma, squamous cell carcinoma 11 (3.5) Microinvasive adenocarcinoma, adenocarcinoma, adenosquamous cell carcinoma 6 (1.9) Histology diagnosis Category Total patients n = 311 Normal No pathological abnormality 2 (0.6) Reactive or inflammatory lesion confirmed 77 (24.7) Squamous metaplasia 4 (1.3) Others 1 (0.3) CIN1 (Mild dysplasia) 67 (21.5) CIN2 CIN2 (Moderate dysplasia) 62 (19.9) CIN3 CIN3 (Severe dysplasia or squamous cell carcinoma in situ) 81 (26.0) Adenocarcinoma in situ 1 (0.3) Cancerous Microinvasive squamous cell carcinoma, squamous cell carcinoma 11 (3.5) Microinvasive adenocarcinoma, adenocarcinoma, adenosquamous cell carcinoma 6 (1.9) CIN, cervical intraepithelial neoplasia. Table 5. Biopsy results Histology diagnosis Category Total patients n = 311 Normal No pathological abnormality 2 (0.6) Reactive or inflammatory lesion confirmed 77 (24.7) Squamous metaplasia 4 (1.3) Others 1 (0.3) CIN1 (Mild dysplasia) 67 (21.5) CIN2 CIN2 (Moderate dysplasia) 62 (19.9) CIN3 CIN3 (Severe dysplasia or squamous cell carcinoma in situ) 81 (26.0) Adenocarcinoma in situ 1 (0.3) Cancerous Microinvasive squamous cell carcinoma, squamous cell carcinoma 11 (3.5) Microinvasive adenocarcinoma, adenocarcinoma, adenosquamous cell carcinoma 6 (1.9) Histology diagnosis Category Total patients n = 311 Normal No pathological abnormality 2 (0.6) Reactive or inflammatory lesion confirmed 77 (24.7) Squamous metaplasia 4 (1.3) Others 1 (0.3) CIN1 (Mild dysplasia) 67 (21.5) CIN2 CIN2 (Moderate dysplasia) 62 (19.9) CIN3 CIN3 (Severe dysplasia or squamous cell carcinoma in situ) 81 (26.0) Adenocarcinoma in situ 1 (0.3) Cancerous Microinvasive squamous cell carcinoma, squamous cell carcinoma 11 (3.5) Microinvasive adenocarcinoma, adenocarcinoma, adenosquamous cell carcinoma 6 (1.9) CIN, cervical intraepithelial neoplasia. Diagnostic performances of CS and LBC for detecting CIN2 or worse The primary endpoint was the difference in diagnostic performances between CS and LBC for detecting cervical lesions of CIN2 or worse, with a cut-off of ASC-US or worse (Table 6). The sensitivities of LBC and CS were 98.8% and 100.0%, specificities were 23.8% and 17.2%, PPVs were 57.9% and 56.1% and NPVs were 94.7% and 100.0%, respectively. LBC had higher sensitivity for detecting CIN2, but there was no significant difference between LBC and CS (%difference between LBC and CS, 1.3; 95% CI, −0.5–3.0) (Fig. 3A). Table 6. Diagnostic performances of CS and LBC for detection of CIN2 or worse Statics Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 98.8 95.6–99.8  Specificity (%) 23.8 17.3–31.4  PPV (%) 57.9 51.8–63.8  NPV (%) 94.7 82.3–99.4 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 100.0 97.7–100.0  Specificity (%) 17.2 11.6–24.2  PPV (%) 56.1 50.2–62.0  NPV (%) 100.0 86.8–100.0 Statics Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 98.8 95.6–99.8  Specificity (%) 23.8 17.3–31.4  PPV (%) 57.9 51.8–63.8  NPV (%) 94.7 82.3–99.4 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 100.0 97.7–100.0  Specificity (%) 17.2 11.6–24.2  PPV (%) 56.1 50.2–62.0  NPV (%) 100.0 86.8–100.0 Table 6. Diagnostic performances of CS and LBC for detection of CIN2 or worse Statics Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 98.8 95.6–99.8  Specificity (%) 23.8 17.3–31.4  PPV (%) 57.9 51.8–63.8  NPV (%) 94.7 82.3–99.4 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 100.0 97.7–100.0  Specificity (%) 17.2 11.6–24.2  PPV (%) 56.1 50.2–62.0  NPV (%) 100.0 86.8–100.0 Statics Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 98.8 95.6–99.8  Specificity (%) 23.8 17.3–31.4  PPV (%) 57.9 51.8–63.8  NPV (%) 94.7 82.3–99.4 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 100.0 97.7–100.0  Specificity (%) 17.2 11.6–24.2  PPV (%) 56.1 50.2–62.0  NPV (%) 100.0 86.8–100.0 Figure 3. View largeDownload slide (A) Diagnostic performances of CS and LBC for detection of CIN2 or worse: comparison of CS and LBC (%difference). (B) Diagnostic performances of CS and LBC for detection of CIN1 or worse: comparison of conventional cytology and LBC (%difference). PPV, positive predictive value; NPV, negative predictive value. Figure 3. View largeDownload slide (A) Diagnostic performances of CS and LBC for detection of CIN2 or worse: comparison of CS and LBC (%difference). (B) Diagnostic performances of CS and LBC for detection of CIN1 or worse: comparison of conventional cytology and LBC (%difference). PPV, positive predictive value; NPV, negative predictive value. Diagnostic performances of CS and LBC for detecting CIN1 or worse The secondary endpoint of this study was the difference in performances of CS and LBC for detecting cervical lesions of CIN1 or worse, with a cut-off of ASC-US or worse (Table 7). The sensitivities of CS and LBC were 95.2% and 97.8%, specificities were 32.1% and 25.0%, PPVs were 79.1% and 77.9% and NPVs were 77.1% and 80.8%, respectively. LBC also had higher sensitivity for detecting CIN1, but again there was no significant difference between LBC and CS (%difference between LBC and CS, 1.3; 95% CI, −0.5 to 3.0) (Fig. 3B). Overall, there were no significant differences in diagnostic performances between CS and LBC. Table 7. Diagnostic performance of CS and LBC for detection of CIN1 or worse Parameter Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 95.2 91.5–97.6  Specificity (%) 32.1 22.4–43.2  PPV (%) 79.1 73.8–83.8  NPV (%) 71.1 54.1–84.6 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 97.8 94.9–99.3  Specificity (%) 25.0 16.2–35.6  PPV (%) 77.9 72.6–82.6  NPV (%) 80.8 60.6–93.4 Parameter Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 95.2 91.5–97.6  Specificity (%) 32.1 22.4–43.2  PPV (%) 79.1 73.8–83.8  NPV (%) 71.1 54.1–84.6 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 97.8 94.9–99.3  Specificity (%) 25.0 16.2–35.6  PPV (%) 77.9 72.6–82.6  NPV (%) 80.8 60.6–93.4 Table 7. Diagnostic performance of CS and LBC for detection of CIN1 or worse Parameter Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 95.2 91.5–97.6  Specificity (%) 32.1 22.4–43.2  PPV (%) 79.1 73.8–83.8  NPV (%) 71.1 54.1–84.6 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 97.8 94.9–99.3  Specificity (%) 25.0 16.2–35.6  PPV (%) 77.9 72.6–82.6  NPV (%) 80.8 60.6–93.4 Parameter Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 95.2 91.5–97.6  Specificity (%) 32.1 22.4–43.2  PPV (%) 79.1 73.8–83.8  NPV (%) 71.1 54.1–84.6 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 97.8 94.9–99.3  Specificity (%) 25.0 16.2–35.6  PPV (%) 77.9 72.6–82.6  NPV (%) 80.8 60.6–93.4 Discussion The primary endpoint of this study concerned the abilities of CS and LBC to detect histological lesions of CIN2 or worse. Although LBC demonstrated slightly higher sensitivity compared with CS, there was no significant difference in diagnostic performances between the two methods. Evaluation of the diagnostic performances of LBC and CS requires validation of the cytological results with those of histology or colposcopy (30). Although several studies have evaluated the diagnostic performances of these two methods by comparing test-positivity rates (18–21), few have included information on the histologic diagnosis of every case in a prospective setting (24,30). It is also necessary to evaluate cytological methods in different countries, given that screening programs differ among countries, and cytology diagnosis involves subjective elements that may be influenced by the educational background of the specific country. We therefore conducted this first prospective study in Japan, designed to evaluate the primary endpoint of detection of CIN2 or worse. This endpoint provided an appropriate basis to assess performance in this study, because spontaneous disappearance and progression to cancer rates of 60% and 1% for CIN1, and 40% and 5% for CIN2 have been reported (31). Furthermore, the 2012 American Society for Colposcopy and Cervical Pathology consensus guidelines and 2014 Guidelines for Gynecology in Japan recommended that CIN2 should be managed by excision or ablation, rather than by follow-up without treatment (32). This study found no statistical superiority of LBC compared with CS in terms of sensitivity. However, previous studies have drawn different conclusions regarding the relative diagnostic performances of these two methods. For example, Ferreccio et al. (17) found sensitivities of 85.71% and 62.96% and specificities of 87.80%and 93.73% for LBC and CS (ThinPrep®; Hologic, Inc.), respectively, for detecting CIN3 or worse in patients with ASC-US or worse. In contrast, a meta-analysis by Arbyn et al. (33) found that LBC was neither more sensitive nor more specific for the detection of high-grade CIN, consistent with our results. However, LBC is associated with additional benefits, notably the ability to use the same samples for other tests, such as HPV (13–16), which is considered to increase the detection rate of CIN or worse compared with Pap smears (34,35). Moreover, the uniform thin epithelial layer in LBC could facilitate cytological interpretation, and could be used with automated screening devices (33). These additional advantages suggest that LBC could be an alternative to CS for cervical cancer screening. We have initiated population based prospective randomized trial (CITRUS study) comparing LBC with or without HPV test intervention for cervical cancer screening (36). In this study, all patients with suspected abnormal cytology of ASC-US or greater were referred to the investigators’ institutions where trained gynecologists and cytopathologists were available. Cytology and colposcopy/biopsy were therefore performed in every patient in a normal clinical setting, with no ethical issues. Negative-cytology cases were also included in the results, because some patients initially suspected of having abnormal cytology at the primary institutions were subsequently diagnosed with NILM at the second cytology examination. This study therefore provides useful information for comparing CS and LBC in cases ranging from NILM to squamous cell carcinoma, and the results should thus help to improve cervical cancer screening among populations with large numbers of NILM cases. There were several limitations to this study. First, we used split sampling, which could lead to non-comparable cellular representation which could affect diagnostic accuracy. As we found in the previous studies, split-sample studies found higher proportion of unsatisfactory slides because of the lack of endocervial components compared with the CS slides (24). Second, smears and biopsies were only taken by highly experienced gynecologists, and it may therefore not be possible to generalize the cytology and histology results to all institutions in Japan. The conventional staining protocols differed slightly among centers, and no central pathological review of biopsy samples was included in the study protocol. Thirdly, cytology diagnosis was performed thoroughly by well-trained cytopathologists, and the results may thus not reflect the general level of cytology screening. Finally, cost-effectiveness was not compared between the two methods. LBC method requires additional cost which varies according to the collecting devices, laboratory and additional tests. However, previous studies showed that conflicting results with regard to cost-effectiveness. (37–39). de Bekker-Grob et al. (37) pointed out that this discrepancy could result from the sensitivity of LBC method, which differs among institutions or countries. Namely, if LBC has higher sensitivity compared with CS, LBC may have higher cost-effectiveness. Because the sensitivity was high and only one unsatisfactory sample was obtained in the CS arm, additional studies are needed to verify the cost-effectiveness of the LBC method. To the best of our knowledge, this was the first study in Japan to compare prospectively the diagnostic performances of LBC and CS with management of overlap between the two methods. LBC had slightly higher sensitivity and higher NPV for the detection of CIN2 or greater compared with CS, though the difference was not significant. However, the additional benefits of LBC, especially the availability of HPV testing using the same sample, suggest that LBC may be an attractive alternative to CS for cervical cancer screening. Future studies in screening populations are needed to confirm these preliminary results. Acknowledgements We are grateful to all the patients and co-investigators for their cooperation in this study. We also thank the following investigators for their contributions to the study: Mr. Hideki Kono, data manager, Ms. Takako Ogawa, project manager, and Prof. Masanori Fukushima, director of the Translational Research Informatics Center. We also thank Ms. Yumi Funato for reviewing manuscripts and Ms. Keiko Abe for assistance with writing the manuscript. Funding This study was supported by the Japanese Society of Clinical Cytology. Conflict of interest statement Following authors indicated financial or other interests that is relevant to this study. Honoria: Daisuke Aoki from Roche Pharmaceutical and Hologic, Inc. Takashi Iwata from Roche Pharmaceutical. Expert testimony fee: Daisuke Aoki from Sysmex Corp. and Sekisui Medical. Research Funding: Takashi Iwata from Sekisui Medical. References 1 Wallis L . Cervical cancer incidence may be higher than previously thought . Am J Nurs 2014 ; 114 : 17 . 2 Gravitt PE . The known unknowns of HPV natural history . 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For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Japanese Journal of Clinical Oncology Oxford University Press

Liquid-based cytology versus conventional cytology for detection of uterine cervical lesions: a prospective observational study

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Abstract

Abstract Objective Liquid-based cytology (LBC) and conventional cytology (CS) are routine diagnostic techniques in cervical cytology, but few studies have compared their diagnostic performances with each other and with histologic diagnosis. This study aimed to compare the diagnostic performances of these techniques in subjects with abnormal cervical cytology of atypical cells of undetermined significance (ASC-US) or worse. Methods A total of 312 patients diagnosed with ASC-US or worse were enrolled in this prospective study in Japan from 2013 to 2014. LBC and CS samples were prepared by a split-sampling technique and evaluated blindly. The results were classified using the Bethesda System 2001. Colposcopy and biopsy were conducted simultaneously or within 4 weeks of cytology-specimen collection in all cases. Diagnostic performance was calculated in terms of sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for detection of CIN2 or worse, with a cut-off ASC-US or worse. Results There was one unsatisfactory CS sample and the remaining 311 cases were evaluated. The sensitivities of LBC and CS were 100.0% and 98.8%, specificities were 17.2% and 23.8%, PPVs were 56.1% and 57.9% and NPVs were 100.0% and 94.7%, respectively. LBC had slightly higher sensitivity and NPV for detection of CIN2, but there was no significant difference between the two methods. Conclusions There was no significant difference in the diagnostic performances of LBC and CS in patients with ASC-US or worse. LBC may therefore be an alternative approach to CS for cervical cancer screening. liquid-based cytology, conventional cytology, cervical cancer screening Introduction Cervical cancer is one of the most common gynecologic malignancies in Western countries (1). Invasive cervical cancer develops from dysplasia with human papillomavirus (HPV) infection over a period of 10 years or more (2), during which time screening allows the detection and treatment of early dysplasia, thus preventing its development into cancer (3). Widespread application of screening programs in recent decades has achieved significant reductions in cervical cancer incidence and mortality (4,5). Cervical cancer screening involves the examination of cytological preparations, including conventional cytology sampling (CS), in which cells are smeared directly onto a glass slide, or liquid-based cytology (LBC), in which samples are stirred in a cell-fixation solvent in a suspended state using a sampling device (6–8). CS is a well-established and useful method, but has several problems, including the need to prepare samples over a short period of time, variable sample quality and occasionally unusable samples caused by drying artifacts or overlapping debris. LBC overcomes these problems, and has the advantage of collecting a large number of cells through the use of a specialized sample-preparation device (9), thus preventing variation among samples and reducing the number of unsatisfactory samples (10–12). In addition, following cytology, LBC samples can also be used directly for HPV testing (13–17), which is considered to be an effective method of screening. However, despite these benefits of LBC, CS is still used in about 90% of cases, and LBC remains to be fully integrated into the screening system in Japan. One of the reasons is that we are not quite confident that LBC has equal or even better diagnostic performance comparing with CS. Although several studies have compared the diagnostic performances of CS and LBC globally (18–28), few have been reported in Japan. Hirai (29) reported sensitivities of 71.3% and 77.4% and specificities of 99.0% and 98.9% for CS and LBC, respectively, for the detection of CIN2 or worse in Japanese patients with ASC-H (indicating high-grade squamous intraepithelial lesion) or worse. However, although LBC showed higher sensitivity, their study was based on limited biopsies performed only in patients with abnormal cytological or colposcopic abnormalities. The establishment of LBC in cervical cancer screening in Japan requires a prospective comparison of the diagnostic performances of LBC and CS based on histology in a broader population, including patients with negative cytology (NILM). Here we first conducted a prospective study in Japan to evaluate the diagnostic performances of CS and LBC for detecting cervical lesions compared with histology in Japanese patients with abnormal cervical cytology. Materials and methods Study design The design of this multicenter, observational study is summarized in Fig. 1. Eligible patients were aged 30–64 years, with previously reported abnormal cytology of ASC-US or greater detected within 24 weeks before provision of informed consent (Table 1). Institutional review board approval was obtained from the ethics committee at each participating institution before patient registration (UMIN ID: 00001146). The investigators explained the study to patients and obtained written informed consent. Cytology and histology by colposcopy and biopsy were performed within a 2-week interval. The interval between the previous biopsy and the biopsy taken for this study was at least 4 weeks, to reduce the patient burden. Subject enrollment was carried out using an online central enrollment system. Figure 1. View largeDownload slide Study design. Split sampling was performed with initial smearing for CS with a broom brush and then preparation of LBC samples using ThinPrep® was carried out. *Biopsy was performed simultaneously with cytology or within 2 weeks, and at least 4 weeks after the last previous biopsy. In cases of apparent invasive cancer, biopsy was allowed within 4 weeks following the previous biopsy. **Slides were evaluated blindly at PCL Japan, Inc. (BML, Inc. Group, Tokyo, Japan). Figure 1. View largeDownload slide Study design. Split sampling was performed with initial smearing for CS with a broom brush and then preparation of LBC samples using ThinPrep® was carried out. *Biopsy was performed simultaneously with cytology or within 2 weeks, and at least 4 weeks after the last previous biopsy. In cases of apparent invasive cancer, biopsy was allowed within 4 weeks following the previous biopsy. **Slides were evaluated blindly at PCL Japan, Inc. (BML, Inc. Group, Tokyo, Japan). Table 1. Eligibility criteria Inclusion criteria  Patients aged 30–64 years old at the time of informed consent  Abnormal cytology of ASC-US or worse suggested within 24 weeks before informed consent Exclusion criteria  Patients with previous history of invasive cervical cancer  Patients who underwent cervical conization  Patients who underwent hysterectomy  Patients who were pregnant  Patients who received tissue biopsy within 4 weeks Inclusion criteria  Patients aged 30–64 years old at the time of informed consent  Abnormal cytology of ASC-US or worse suggested within 24 weeks before informed consent Exclusion criteria  Patients with previous history of invasive cervical cancer  Patients who underwent cervical conization  Patients who underwent hysterectomy  Patients who were pregnant  Patients who received tissue biopsy within 4 weeks View Large Table 1. Eligibility criteria Inclusion criteria  Patients aged 30–64 years old at the time of informed consent  Abnormal cytology of ASC-US or worse suggested within 24 weeks before informed consent Exclusion criteria  Patients with previous history of invasive cervical cancer  Patients who underwent cervical conization  Patients who underwent hysterectomy  Patients who were pregnant  Patients who received tissue biopsy within 4 weeks Inclusion criteria  Patients aged 30–64 years old at the time of informed consent  Abnormal cytology of ASC-US or worse suggested within 24 weeks before informed consent Exclusion criteria  Patients with previous history of invasive cervical cancer  Patients who underwent cervical conization  Patients who underwent hysterectomy  Patients who were pregnant  Patients who received tissue biopsy within 4 weeks View Large The primary endpoint was difference in diagnostic performance between CS and LBC for detecting cervical lesions of CIN2 or worse, with a cut-off of ASC-US or worse. Diagnostic performance was evaluated based on sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). The methods used to calculate the parameters are shown in Table 2. Following cytology and histology diagnosis, we categorized each sample as A–D, and evaluated each parameter as shown (Table 2, upper row). The secondary endpoint was the difference in diagnostic performance between CS and LBC for detecting cervical lesions of CIN1 or worse, with a cut-off of ASC-US or worse (Table 2, lower row). Table 2. Sensitivity, specificity, PPV and NPVs of CS and LBC for detecting CIN2 or worse (primary endpoint) and CIN1 or worse (secondary endpoint) with a cut-off level of ASC-US or worse Histology results Cytology results Normal/CIN1 CIN2/CIN3/SCC CIN2 or worse NILM A B ASC-US, LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy C D Histology results Cytology results Normal/CIN1 CIN2/CIN3/SCC CIN2 or worse NILM A B ASC-US, LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy C D Normal CIN1/CIN2/CIN3/SCC CIN1 or worse NLIM E F ASC-US,LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy G H Normal CIN1/CIN2/CIN3/SCC CIN1 or worse NLIM E F ASC-US,LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy G H Sensitivity: D/(B + D) and (H/F + H); specificity: A/(A + C) and E/(F + G); PPV: D/(C + D) and H/(G + H); NPV: A/(A + B) and E/(E + F). CIN, cervical intraepithelial neoplasia; NILM, negative for intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells that cannot exclude; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; AGC, atypical glandular cells; AIS, adenocarcinoma in situ. Table 2. Sensitivity, specificity, PPV and NPVs of CS and LBC for detecting CIN2 or worse (primary endpoint) and CIN1 or worse (secondary endpoint) with a cut-off level of ASC-US or worse Histology results Cytology results Normal/CIN1 CIN2/CIN3/SCC CIN2 or worse NILM A B ASC-US, LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy C D Histology results Cytology results Normal/CIN1 CIN2/CIN3/SCC CIN2 or worse NILM A B ASC-US, LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy C D Normal CIN1/CIN2/CIN3/SCC CIN1 or worse NLIM E F ASC-US,LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy G H Normal CIN1/CIN2/CIN3/SCC CIN1 or worse NLIM E F ASC-US,LSIL, ASC-H, HSIL, SCC, AGC, AIS, adenocarcinoma, other malignancy G H Sensitivity: D/(B + D) and (H/F + H); specificity: A/(A + C) and E/(F + G); PPV: D/(C + D) and H/(G + H); NPV: A/(A + B) and E/(E + F). CIN, cervical intraepithelial neoplasia; NILM, negative for intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells that cannot exclude; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; AGC, atypical glandular cells; AIS, adenocarcinoma in situ. Cytology and interpretation Cells were collected using a Rovers® Cervix-Brush® (Oss, The Netherlands). The cells were divided into CS samples smeared on glass slides and LBC samples prepared using PreservCyt Solution and a ThinPrep® Processor. CS samples on glass slides were immersed in 95% ethanol and fixed and stained by Papanicolaou staining, and then evaluated blindly at PCL Japan, Inc. (BML, Inc. Group, Tokyo, Japan; hereinafter referred to as PCL). LBC samples were collected in ThinPrep® PreservCyt Solution Vials and then sent to PCL. No clinical information was provided to PCL, to ensure equal and independent blinded evaluation of the two methods. The obtained cytology results were compared with the histology results assessed at each institution. Diagnostic criteria Cytology results were reported in accordance with the Bethesda System 2001. Reasons for unsatisfactory samples were recorded, and re-collection was not allowed for samples determined to be unsatisfactory using CS or LBC. Colposcopic findings were classified based on the New Colposcopic Classification of The Japan Society of Gynecologic Oncology, 2005. Colposcopic data were not collected in this study. Histological findings were classified using the General Rules for Clinical and Pathological Management of Uterine Cervical Cancer (3rd edition) at each study site, as follows: normal; CIN1 or worse; CIN2 or worse; CIN3 or worse; cancer; and not evaluable. Colposcopy, biopsy and histological diagnosis Colposcopy and colposcopy-guided biopsy were carried out within 2 weeks after cell collection. Colposcopy was performed according to the General Rules for Clinical and Pathological Management of Uterine Cervical Cancer. Biopsies were performed under colposcopic guidance and the number of biopsy samples was determined by the investigator. Histological diagnosis was performed using the normal procedure at each study site and the findings were classified. If the histology results varied among multiple biopsies, the highest diagnosis was recorded. Statistical design and analysis The target number of subjects was estimated to be 300, based on the following information. A total of 1059 patients with suspected abnormal histology of ASC-US or worse were referred to Keio University Hospital and underwent CS and histology from January to December 2011 (unpublished data). Using a cut-off of ASC-US or worse, cytology results were negative in 393 patients and positive in 666. No CIN2 or worse lesion was detected by histology in 379 of the 393 cytology-negative patients, and CIN2 or worse lesions were detected by histology in 415 of the 666 cytology-positive patients. A cytological diagnosis of NILM and a histological diagnosis of CIN1 or better, or a cytological diagnosis of ASC-US or worse and a histological diagnosis of CIN2 or worse was considered to be accurate. The diagnostic accuracy was therefore 75.0%, calculated as the proportion of the accurately diagnosed population among all patients. The reported sensitivity and specificity of ThinPrep® LBC were slightly higher or comparable to CS, and given an accuracy of LBC of 82.0%, a sample size of 300 subjects would be adequate to demonstrate significant superiority of LBC compared with CS. Demographic data and other data at enrollment were summarized as descriptive statistics, based on the types and distribution of data. Average differences in diagnostic performances between LBC and CS with 95% confidence intervals (CI) were calculated. Cross tables of histology results with CS and LBC results were prepared. Sensitivity, specificity, PPV and NPV with 95% CIs were estimated and analyzed by McNemar tests. Results Patient population A total of 326 cases were enrolled at three institutions between April 2013 and December 2014. Fourteen cases were excluded for withdrawal of consent (n = 1), cytology or histology not performed (n = 7) and interval between cytology and histology >2 weeks (n = 6). Of the remaining 312 cases, one was excluded from the analysis because of an unsatisfactory CS sample. A total of 311 cases were therefore finally evaluated (Fig. 2). The median age of the 311patients was 38.0 (30.0–64.0) years. Figure 2. View largeDownload slide Patient enrollment and analysis. Figure 2. View largeDownload slide Patient enrollment and analysis. Cytology and histology The results of CS and LBC are summarized in Table 3. High-grade squamous intraepithelial lesion was the most frequent diagnosis (46.9% for CS and 44.9% for LBC). Although all the patients were referred to the study institutions with previously reported ASC-US or worse cytology, the overall rates of NILM were 12.2% in CS and 8.3% in LBC, compared with 87.8% in CS and 91.7% in LBC among patients with ASC-US or worse. For cases with a cut-off of low-grade squamous intraepithelial lesion or higher, the rates were 73.3% for CS and 80.1% for LBC. Table 3. Results of CS and LBC Items Category CS LBC n = 312 n = 312 Specimen Unsatisfactory 1 (0.3) 0 (0.0) Satisfactory 311 (99.7) 312 (100) Items Category CS LBC n = 312 n = 312 Specimen Unsatisfactory 1 (0.3) 0 (0.0) Satisfactory 311 (99.7) 312 (100) Analysis of accepted samples Items Category Patients in total Patients in total n = 311 n = 312 Results NILM 38 (12.2) 26 (8.3) ASC-US 45 (14.5) 36 (11.5) LSIL 29 (9.3) 41 (13.1) ASC-H 25 (8.0) 37 (11.9) HSIL 146 (46.9) 140 (44.9) SCC 20 (6.4) 21 (6.7) AGC 4 (1.3) 7 (2.2) AIS 1 (0.3) 2 (0.6) Adenocarcinoma 3 (1.0) 2 (0.6) Other malignancy 0 (0.0) 0 (0.0) Results with the cutoff of ASC-US or worse ASC-US or higher 273 (87.8) 286 (91.7) NILM 38 (12.2) 26 (8.3) Results with the cutoff of LSIL or worse LSIL or higher 228 (73.3) 250 (80.1) NILM, ASC-US 83 (26.7) 62 (19.9) Analysis of accepted samples Items Category Patients in total Patients in total n = 311 n = 312 Results NILM 38 (12.2) 26 (8.3) ASC-US 45 (14.5) 36 (11.5) LSIL 29 (9.3) 41 (13.1) ASC-H 25 (8.0) 37 (11.9) HSIL 146 (46.9) 140 (44.9) SCC 20 (6.4) 21 (6.7) AGC 4 (1.3) 7 (2.2) AIS 1 (0.3) 2 (0.6) Adenocarcinoma 3 (1.0) 2 (0.6) Other malignancy 0 (0.0) 0 (0.0) Results with the cutoff of ASC-US or worse ASC-US or higher 273 (87.8) 286 (91.7) NILM 38 (12.2) 26 (8.3) Results with the cutoff of LSIL or worse LSIL or higher 228 (73.3) 250 (80.1) NILM, ASC-US 83 (26.7) 62 (19.9) CIN, cervical intraepithelial neoplasia; NILM, negative for intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells that cannot exclude; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; AGC, atypical glandular cells; AIS, adenocarcinoma in situ. Table 3. Results of CS and LBC Items Category CS LBC n = 312 n = 312 Specimen Unsatisfactory 1 (0.3) 0 (0.0) Satisfactory 311 (99.7) 312 (100) Items Category CS LBC n = 312 n = 312 Specimen Unsatisfactory 1 (0.3) 0 (0.0) Satisfactory 311 (99.7) 312 (100) Analysis of accepted samples Items Category Patients in total Patients in total n = 311 n = 312 Results NILM 38 (12.2) 26 (8.3) ASC-US 45 (14.5) 36 (11.5) LSIL 29 (9.3) 41 (13.1) ASC-H 25 (8.0) 37 (11.9) HSIL 146 (46.9) 140 (44.9) SCC 20 (6.4) 21 (6.7) AGC 4 (1.3) 7 (2.2) AIS 1 (0.3) 2 (0.6) Adenocarcinoma 3 (1.0) 2 (0.6) Other malignancy 0 (0.0) 0 (0.0) Results with the cutoff of ASC-US or worse ASC-US or higher 273 (87.8) 286 (91.7) NILM 38 (12.2) 26 (8.3) Results with the cutoff of LSIL or worse LSIL or higher 228 (73.3) 250 (80.1) NILM, ASC-US 83 (26.7) 62 (19.9) Analysis of accepted samples Items Category Patients in total Patients in total n = 311 n = 312 Results NILM 38 (12.2) 26 (8.3) ASC-US 45 (14.5) 36 (11.5) LSIL 29 (9.3) 41 (13.1) ASC-H 25 (8.0) 37 (11.9) HSIL 146 (46.9) 140 (44.9) SCC 20 (6.4) 21 (6.7) AGC 4 (1.3) 7 (2.2) AIS 1 (0.3) 2 (0.6) Adenocarcinoma 3 (1.0) 2 (0.6) Other malignancy 0 (0.0) 0 (0.0) Results with the cutoff of ASC-US or worse ASC-US or higher 273 (87.8) 286 (91.7) NILM 38 (12.2) 26 (8.3) Results with the cutoff of LSIL or worse LSIL or higher 228 (73.3) 250 (80.1) NILM, ASC-US 83 (26.7) 62 (19.9) CIN, cervical intraepithelial neoplasia; NILM, negative for intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells that cannot exclude; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; AGC, atypical glandular cells; AIS, adenocarcinoma in situ. The concordance between the CS and LBC results is shown in Table 4. The concordance rate between the two methods was 64.0% (95% CI, 58.4–69.3%). Table 4. Concordance between CS and LBC Category LBC results n NILM ASC-US LSIL ASC-H HSIL SCC AGC AIS Adeno Other CS results NILM 20 10 2 4 1 0 1 0 0 0 38 ASC-US 3 17 12 10 2 0 1 0 0 0 45 LSIL 0 2 22 0 5 0 0 0 0 0 29 ASC-H 2 1 1 9 10 0 1 0 1 0 25 HSIL 0 4 3 13 117 7 1 1 0 0 146 SCC 0 0 0 0 5 14 1 0 0 0 20 AGC 1 1 1 1 0 0 0 0 0 0 4 AIS 0 0 0 0 0 0 0 1 0 0 1 Adeno 0 0 0 0 0 0 2 0 1 0 3 Other 0 0 0 0 0 0 0 0 0 0 0 Total 26 35 41 37 140 21 7 2 2 0 311 Category LBC results n NILM ASC-US LSIL ASC-H HSIL SCC AGC AIS Adeno Other CS results NILM 20 10 2 4 1 0 1 0 0 0 38 ASC-US 3 17 12 10 2 0 1 0 0 0 45 LSIL 0 2 22 0 5 0 0 0 0 0 29 ASC-H 2 1 1 9 10 0 1 0 1 0 25 HSIL 0 4 3 13 117 7 1 1 0 0 146 SCC 0 0 0 0 5 14 1 0 0 0 20 AGC 1 1 1 1 0 0 0 0 0 0 4 AIS 0 0 0 0 0 0 0 1 0 0 1 Adeno 0 0 0 0 0 0 2 0 1 0 3 Other 0 0 0 0 0 0 0 0 0 0 0 Total 26 35 41 37 140 21 7 2 2 0 311 Shadowed cases represent concordant cases. NILM, negative for intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells that cannot exclude; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; AGC, atypical glandular cells; AIS, adenocarcinoma in situ; Adeno: adenocarcinoma; Other, other malignancy. Table 4. Concordance between CS and LBC Category LBC results n NILM ASC-US LSIL ASC-H HSIL SCC AGC AIS Adeno Other CS results NILM 20 10 2 4 1 0 1 0 0 0 38 ASC-US 3 17 12 10 2 0 1 0 0 0 45 LSIL 0 2 22 0 5 0 0 0 0 0 29 ASC-H 2 1 1 9 10 0 1 0 1 0 25 HSIL 0 4 3 13 117 7 1 1 0 0 146 SCC 0 0 0 0 5 14 1 0 0 0 20 AGC 1 1 1 1 0 0 0 0 0 0 4 AIS 0 0 0 0 0 0 0 1 0 0 1 Adeno 0 0 0 0 0 0 2 0 1 0 3 Other 0 0 0 0 0 0 0 0 0 0 0 Total 26 35 41 37 140 21 7 2 2 0 311 Category LBC results n NILM ASC-US LSIL ASC-H HSIL SCC AGC AIS Adeno Other CS results NILM 20 10 2 4 1 0 1 0 0 0 38 ASC-US 3 17 12 10 2 0 1 0 0 0 45 LSIL 0 2 22 0 5 0 0 0 0 0 29 ASC-H 2 1 1 9 10 0 1 0 1 0 25 HSIL 0 4 3 13 117 7 1 1 0 0 146 SCC 0 0 0 0 5 14 1 0 0 0 20 AGC 1 1 1 1 0 0 0 0 0 0 4 AIS 0 0 0 0 0 0 0 1 0 0 1 Adeno 0 0 0 0 0 0 2 0 1 0 3 Other 0 0 0 0 0 0 0 0 0 0 0 Total 26 35 41 37 140 21 7 2 2 0 311 Shadowed cases represent concordant cases. NILM, negative for intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells that cannot exclude; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; AGC, atypical glandular cells; AIS, adenocarcinoma in situ; Adeno: adenocarcinoma; Other, other malignancy. All patients underwent punch biopsies (median, 2; range, 1–5). The biopsy results are summarized in Table 5. CIN3 was the most-frequently diagnosed condition in biopsies, followed by CIN1. Table 5. Biopsy results Histology diagnosis Category Total patients n = 311 Normal No pathological abnormality 2 (0.6) Reactive or inflammatory lesion confirmed 77 (24.7) Squamous metaplasia 4 (1.3) Others 1 (0.3) CIN1 (Mild dysplasia) 67 (21.5) CIN2 CIN2 (Moderate dysplasia) 62 (19.9) CIN3 CIN3 (Severe dysplasia or squamous cell carcinoma in situ) 81 (26.0) Adenocarcinoma in situ 1 (0.3) Cancerous Microinvasive squamous cell carcinoma, squamous cell carcinoma 11 (3.5) Microinvasive adenocarcinoma, adenocarcinoma, adenosquamous cell carcinoma 6 (1.9) Histology diagnosis Category Total patients n = 311 Normal No pathological abnormality 2 (0.6) Reactive or inflammatory lesion confirmed 77 (24.7) Squamous metaplasia 4 (1.3) Others 1 (0.3) CIN1 (Mild dysplasia) 67 (21.5) CIN2 CIN2 (Moderate dysplasia) 62 (19.9) CIN3 CIN3 (Severe dysplasia or squamous cell carcinoma in situ) 81 (26.0) Adenocarcinoma in situ 1 (0.3) Cancerous Microinvasive squamous cell carcinoma, squamous cell carcinoma 11 (3.5) Microinvasive adenocarcinoma, adenocarcinoma, adenosquamous cell carcinoma 6 (1.9) CIN, cervical intraepithelial neoplasia. Table 5. Biopsy results Histology diagnosis Category Total patients n = 311 Normal No pathological abnormality 2 (0.6) Reactive or inflammatory lesion confirmed 77 (24.7) Squamous metaplasia 4 (1.3) Others 1 (0.3) CIN1 (Mild dysplasia) 67 (21.5) CIN2 CIN2 (Moderate dysplasia) 62 (19.9) CIN3 CIN3 (Severe dysplasia or squamous cell carcinoma in situ) 81 (26.0) Adenocarcinoma in situ 1 (0.3) Cancerous Microinvasive squamous cell carcinoma, squamous cell carcinoma 11 (3.5) Microinvasive adenocarcinoma, adenocarcinoma, adenosquamous cell carcinoma 6 (1.9) Histology diagnosis Category Total patients n = 311 Normal No pathological abnormality 2 (0.6) Reactive or inflammatory lesion confirmed 77 (24.7) Squamous metaplasia 4 (1.3) Others 1 (0.3) CIN1 (Mild dysplasia) 67 (21.5) CIN2 CIN2 (Moderate dysplasia) 62 (19.9) CIN3 CIN3 (Severe dysplasia or squamous cell carcinoma in situ) 81 (26.0) Adenocarcinoma in situ 1 (0.3) Cancerous Microinvasive squamous cell carcinoma, squamous cell carcinoma 11 (3.5) Microinvasive adenocarcinoma, adenocarcinoma, adenosquamous cell carcinoma 6 (1.9) CIN, cervical intraepithelial neoplasia. Diagnostic performances of CS and LBC for detecting CIN2 or worse The primary endpoint was the difference in diagnostic performances between CS and LBC for detecting cervical lesions of CIN2 or worse, with a cut-off of ASC-US or worse (Table 6). The sensitivities of LBC and CS were 98.8% and 100.0%, specificities were 23.8% and 17.2%, PPVs were 57.9% and 56.1% and NPVs were 94.7% and 100.0%, respectively. LBC had higher sensitivity for detecting CIN2, but there was no significant difference between LBC and CS (%difference between LBC and CS, 1.3; 95% CI, −0.5–3.0) (Fig. 3A). Table 6. Diagnostic performances of CS and LBC for detection of CIN2 or worse Statics Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 98.8 95.6–99.8  Specificity (%) 23.8 17.3–31.4  PPV (%) 57.9 51.8–63.8  NPV (%) 94.7 82.3–99.4 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 100.0 97.7–100.0  Specificity (%) 17.2 11.6–24.2  PPV (%) 56.1 50.2–62.0  NPV (%) 100.0 86.8–100.0 Statics Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 98.8 95.6–99.8  Specificity (%) 23.8 17.3–31.4  PPV (%) 57.9 51.8–63.8  NPV (%) 94.7 82.3–99.4 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 100.0 97.7–100.0  Specificity (%) 17.2 11.6–24.2  PPV (%) 56.1 50.2–62.0  NPV (%) 100.0 86.8–100.0 Table 6. Diagnostic performances of CS and LBC for detection of CIN2 or worse Statics Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 98.8 95.6–99.8  Specificity (%) 23.8 17.3–31.4  PPV (%) 57.9 51.8–63.8  NPV (%) 94.7 82.3–99.4 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 100.0 97.7–100.0  Specificity (%) 17.2 11.6–24.2  PPV (%) 56.1 50.2–62.0  NPV (%) 100.0 86.8–100.0 Statics Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 98.8 95.6–99.8  Specificity (%) 23.8 17.3–31.4  PPV (%) 57.9 51.8–63.8  NPV (%) 94.7 82.3–99.4 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 100.0 97.7–100.0  Specificity (%) 17.2 11.6–24.2  PPV (%) 56.1 50.2–62.0  NPV (%) 100.0 86.8–100.0 Figure 3. View largeDownload slide (A) Diagnostic performances of CS and LBC for detection of CIN2 or worse: comparison of CS and LBC (%difference). (B) Diagnostic performances of CS and LBC for detection of CIN1 or worse: comparison of conventional cytology and LBC (%difference). PPV, positive predictive value; NPV, negative predictive value. Figure 3. View largeDownload slide (A) Diagnostic performances of CS and LBC for detection of CIN2 or worse: comparison of CS and LBC (%difference). (B) Diagnostic performances of CS and LBC for detection of CIN1 or worse: comparison of conventional cytology and LBC (%difference). PPV, positive predictive value; NPV, negative predictive value. Diagnostic performances of CS and LBC for detecting CIN1 or worse The secondary endpoint of this study was the difference in performances of CS and LBC for detecting cervical lesions of CIN1 or worse, with a cut-off of ASC-US or worse (Table 7). The sensitivities of CS and LBC were 95.2% and 97.8%, specificities were 32.1% and 25.0%, PPVs were 79.1% and 77.9% and NPVs were 77.1% and 80.8%, respectively. LBC also had higher sensitivity for detecting CIN1, but again there was no significant difference between LBC and CS (%difference between LBC and CS, 1.3; 95% CI, −0.5 to 3.0) (Fig. 3B). Overall, there were no significant differences in diagnostic performances between CS and LBC. Table 7. Diagnostic performance of CS and LBC for detection of CIN1 or worse Parameter Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 95.2 91.5–97.6  Specificity (%) 32.1 22.4–43.2  PPV (%) 79.1 73.8–83.8  NPV (%) 71.1 54.1–84.6 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 97.8 94.9–99.3  Specificity (%) 25.0 16.2–35.6  PPV (%) 77.9 72.6–82.6  NPV (%) 80.8 60.6–93.4 Parameter Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 95.2 91.5–97.6  Specificity (%) 32.1 22.4–43.2  PPV (%) 79.1 73.8–83.8  NPV (%) 71.1 54.1–84.6 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 97.8 94.9–99.3  Specificity (%) 25.0 16.2–35.6  PPV (%) 77.9 72.6–82.6  NPV (%) 80.8 60.6–93.4 Table 7. Diagnostic performance of CS and LBC for detection of CIN1 or worse Parameter Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 95.2 91.5–97.6  Specificity (%) 32.1 22.4–43.2  PPV (%) 79.1 73.8–83.8  NPV (%) 71.1 54.1–84.6 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 97.8 94.9–99.3  Specificity (%) 25.0 16.2–35.6  PPV (%) 77.9 72.6–82.6  NPV (%) 80.8 60.6–93.4 Parameter Estimated 95% CI A. Performance of CS compared with histology diagnosis  Sensitivity (%) 95.2 91.5–97.6  Specificity (%) 32.1 22.4–43.2  PPV (%) 79.1 73.8–83.8  NPV (%) 71.1 54.1–84.6 B. Performance of LBC compared with histology diagnosis  Sensitivity (%) 97.8 94.9–99.3  Specificity (%) 25.0 16.2–35.6  PPV (%) 77.9 72.6–82.6  NPV (%) 80.8 60.6–93.4 Discussion The primary endpoint of this study concerned the abilities of CS and LBC to detect histological lesions of CIN2 or worse. Although LBC demonstrated slightly higher sensitivity compared with CS, there was no significant difference in diagnostic performances between the two methods. Evaluation of the diagnostic performances of LBC and CS requires validation of the cytological results with those of histology or colposcopy (30). Although several studies have evaluated the diagnostic performances of these two methods by comparing test-positivity rates (18–21), few have included information on the histologic diagnosis of every case in a prospective setting (24,30). It is also necessary to evaluate cytological methods in different countries, given that screening programs differ among countries, and cytology diagnosis involves subjective elements that may be influenced by the educational background of the specific country. We therefore conducted this first prospective study in Japan, designed to evaluate the primary endpoint of detection of CIN2 or worse. This endpoint provided an appropriate basis to assess performance in this study, because spontaneous disappearance and progression to cancer rates of 60% and 1% for CIN1, and 40% and 5% for CIN2 have been reported (31). Furthermore, the 2012 American Society for Colposcopy and Cervical Pathology consensus guidelines and 2014 Guidelines for Gynecology in Japan recommended that CIN2 should be managed by excision or ablation, rather than by follow-up without treatment (32). This study found no statistical superiority of LBC compared with CS in terms of sensitivity. However, previous studies have drawn different conclusions regarding the relative diagnostic performances of these two methods. For example, Ferreccio et al. (17) found sensitivities of 85.71% and 62.96% and specificities of 87.80%and 93.73% for LBC and CS (ThinPrep®; Hologic, Inc.), respectively, for detecting CIN3 or worse in patients with ASC-US or worse. In contrast, a meta-analysis by Arbyn et al. (33) found that LBC was neither more sensitive nor more specific for the detection of high-grade CIN, consistent with our results. However, LBC is associated with additional benefits, notably the ability to use the same samples for other tests, such as HPV (13–16), which is considered to increase the detection rate of CIN or worse compared with Pap smears (34,35). Moreover, the uniform thin epithelial layer in LBC could facilitate cytological interpretation, and could be used with automated screening devices (33). These additional advantages suggest that LBC could be an alternative to CS for cervical cancer screening. We have initiated population based prospective randomized trial (CITRUS study) comparing LBC with or without HPV test intervention for cervical cancer screening (36). In this study, all patients with suspected abnormal cytology of ASC-US or greater were referred to the investigators’ institutions where trained gynecologists and cytopathologists were available. Cytology and colposcopy/biopsy were therefore performed in every patient in a normal clinical setting, with no ethical issues. Negative-cytology cases were also included in the results, because some patients initially suspected of having abnormal cytology at the primary institutions were subsequently diagnosed with NILM at the second cytology examination. This study therefore provides useful information for comparing CS and LBC in cases ranging from NILM to squamous cell carcinoma, and the results should thus help to improve cervical cancer screening among populations with large numbers of NILM cases. There were several limitations to this study. First, we used split sampling, which could lead to non-comparable cellular representation which could affect diagnostic accuracy. As we found in the previous studies, split-sample studies found higher proportion of unsatisfactory slides because of the lack of endocervial components compared with the CS slides (24). Second, smears and biopsies were only taken by highly experienced gynecologists, and it may therefore not be possible to generalize the cytology and histology results to all institutions in Japan. The conventional staining protocols differed slightly among centers, and no central pathological review of biopsy samples was included in the study protocol. Thirdly, cytology diagnosis was performed thoroughly by well-trained cytopathologists, and the results may thus not reflect the general level of cytology screening. Finally, cost-effectiveness was not compared between the two methods. LBC method requires additional cost which varies according to the collecting devices, laboratory and additional tests. However, previous studies showed that conflicting results with regard to cost-effectiveness. (37–39). de Bekker-Grob et al. (37) pointed out that this discrepancy could result from the sensitivity of LBC method, which differs among institutions or countries. Namely, if LBC has higher sensitivity compared with CS, LBC may have higher cost-effectiveness. Because the sensitivity was high and only one unsatisfactory sample was obtained in the CS arm, additional studies are needed to verify the cost-effectiveness of the LBC method. To the best of our knowledge, this was the first study in Japan to compare prospectively the diagnostic performances of LBC and CS with management of overlap between the two methods. LBC had slightly higher sensitivity and higher NPV for the detection of CIN2 or greater compared with CS, though the difference was not significant. However, the additional benefits of LBC, especially the availability of HPV testing using the same sample, suggest that LBC may be an attractive alternative to CS for cervical cancer screening. Future studies in screening populations are needed to confirm these preliminary results. Acknowledgements We are grateful to all the patients and co-investigators for their cooperation in this study. 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Journal

Japanese Journal of Clinical OncologyOxford University Press

Published: Apr 13, 2018

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