LSIL, prediction of CIN2–3 in follow up biopsies and spontaneous regression of CIN2–3

Gynecologic Oncology 123 (2011) 278–283

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Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y g y n o

Comparison of different commercial methods for HPV detection in follow-up cytology after ASCUS/LSIL, prediction of CIN2–3 in follow up biopsies and spontaneous regression of CIN2–3 Irene T. Ovestad a, c, d, Undis Vennestrøm a, Liv Andersen a, Einar Gudlaugsson a, d, Ane Cecilie Munk a, b, d, Anais Malpica e, f, Weiwei Feng g, Feja Voorhorst h, Emiel A.M. Janssen a, c, Jan P.A. Baak a, d,⁎ a

Department of Pathology, Stavanger University Hospital, Stavanger, Norway Department of Gynecology, Stavanger University Hospital, Stavanger, Norway c Laboratory for Molecular Biology, Stavanger University Hospital, Stavanger, Norway d The Gade Institute, University of Bergen, Bergen, Norway e Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA f Department of Gynecologic Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA g Department of Gynaecology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China h Department of Epidemiology and Biostatistics, Vrije Universiteit Medical Center, Amsterdam, The Netherlands b

a r t i c l e

i n f o

Article history: Received 10 June 2011 Accepted 14 July 2011 Available online 10 August 2011 Keywords: Cervical cancer screening HPV DNA and mRNA tests LSIL ASCUS Clinical sensitivity and specificity Regression

a b s t r a c t Objective. Different Human Papilloma Virus (HPV) tests are currently used. An integrated comparison of the Amplicor, Cobas4800, PreTect HPV-Proofer and APTIMA HPV tests has not been done. Methods. We compared the high-risk HPV detection power of these HPV tests in 528 consecutive population-based follow-up Liquid-Based Cytology samples (LBC) after ASCUS/LSIL index cytology. Their sensitivity and specificity to detect HPV in LBC, their predictive values of histopathologic CIN2–3 in follow-up punch biopsies and CIN2–3 regression in the subsequent cones was assessed. The HPV subtypes detected by the Linear Array genotyping-test (LA), PreTect HPV-Proofer and Cobas4800 were also compared. The followup histopathology was consensus expert-reviewed and Ki67/p16-supported. The predictive values of the HPV results in LBC by the different tests for presence of CIN2–3 in follow-up biopsies, and regression in subsequent cones, was assessed. Results. Amplicor, Cobas4800 and APTIMA show good agreement for HPV-positivity/negativity. PreTect HPV-Proofer has many discrepancies versus any of the other methods. The sensitivities for Amplicor, Cobas4800 and APTIMA to detect CIN2–3 were very high (96–100%), but rather low for PreTect HPV-Proofer (53%). Specificity in case of CIN1 or less in follow-up biopsies of Amplicor and Cobas4800 is lower than APTIMA and highest for PreTect HPV-Proofer. HPV subtyping by LA agreed in 90% with Cobas4800 but 70% with PreTect HPV-Proofer. Conclusions. The Amplicor, Cobas4800 and APTIMA give comparable results but PreTect HPV-Proofer differs from the other tests, with low sensitivity but higher specificity. None of the methods predicted regression of CIN2–3. © 2011 Elsevier Inc. All rights reserved.

Background Cytology screening is useful to detect cervical intraepithelial neoplasia (CIN) and cancer [1]. Interpretive variability and low sensitivity are problematic for atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) [2–6]. Human Papilloma Virus (HPV) testing is widely performed [7–10], as persistent carcinogenic HPV infection is necessary for cervical

⁎ Corresponding author at: Department of Pathology, Stavanger University Hospital, Box 8100, 4068 Stavanger, Norway. Fax: + 47 51 519920, + 47 51 880895. E-mail address: [email protected] (J.P.A. Baak). 0090-8258/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2011.07.024

neoplasia [11,12]. Much attention is paid to use HPV tests as an adjunct and primary screening tool [4,5,9]. Not all HPV tests detect the same oncogenic HPV types (Supplemental Table 1). Some tests may not fulfill the fully validated laboratory test requirements [13,14]. It is important to determine if newer HPV tests have been adequately validated and are comparable to established HPV methods. Such comparisons have been done to a certain extent [15–22] (Supplemental Table 2). However, an integrated comparison of the DNA-based methods Amplicor and Cobas4800 and the mRNA-based methods PreTect HPV-Proofer (Proofer) and APTIMA for cytologic HPV and follow-up biopsy CIN2–3 detection has not been done. We have analyzed these performances and also regression-or-not of CIN2–3 prediction in the Loop Electrosurgical Excision Procedure

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(LEEP) cone, following the punch biopsies. Moreover, the Linear Array DNA-based genotyping assay (LA), detecting 37 different HPV genotypes was used to assess which genotypes were present in the samples and to compare these with the partial genotyping of Cobas4800 and Proofer [16,21,23,24].

279

analyzed with the LA test and the samples both negative by Amplicor and the other methods, regarded as LA negative. Cobas4800 HPV DNA test

The study has been approved by the Regional Medical Ethics Committee of Health West, Norway (2010/2550).

The Cobas4800 system (Roche Molecular Diagnostics, Pleasanton, CA, USA) features fully automated sample preparation combined with real-time PCR technology for amplification and simultaneous detection of 14 hrHPV genotypes (Supplemental Table 1), identification of HPV 16 and 18 and β-globin used as an internal control for sample validity. A minimum of 400 μl ThinPrep liquid-based cytological material was used. The protocol provided by Roche [17] was followed.

Patient material

PreTect HPV-Proofer mRNA test

Originally, 550 cases with a referral = index cytology diagnosis of ASCUS (n = 371), LSIL (n = 157), high-grade intraepithelial lesion (HSIL, n = 10), atypical cells suspect for high-grade lesion (ASCH, n = 5), atypical glandular cells of uncertain significance (AGUS, n = 2) and cases with no referral cytology (n = 5) were diagnosed in the Norwegian population-based Cervical Cancer Screening Program (Supplemental Fig. 1). Only the 528 cases with ASCUS and LSIL were used to compare the different HPV methods. The cytology samples were ThinPrep® liquid-based cervical specimens, (Preservecyt TM, Marlborough, MA, USA) and the follow-up samples were received at the Stavanger University Hospital, Norway, between January and June 2010. For three samples there was insufficient material for testing with the Proofer test (see below). In order to eliminate cytopathologic interpretational inter-observer variation all specimens were evaluated two times, first by a cyto-technician and finally by a cytopathologist. The patients had been referred for follow-up cytologic examination with HPV testing, 6–12 months after the index cytology. Following the Norwegian guidelines, in case of abnormal followup cytology and/or high-risk HPV positivity by Amplicor, a punch biopsy was performed and with CIN2–3 in the punch biopsy, a LEEPcone was done. As this is a prospective study, at the last follow-up (December 31, 2010) only 35 of the 47 patients with a CIN2–3 in their punch biopsy had undergone a LEEP-cone procedure. One more patient had no cone because of pregnancy since the punch biopsy. The punch biopsies and cones were independently classified by two gynecological pathologists (EG, JB, using Ki67 and p16 immunohistochemistry). In case of discrepancies, consensus was obtained during a double-headed microscope session. Regression was defined as a CIN2–3 in a punch biopsy followed by a CIN1-or-less in the cone.

The PreTect HPV-Proofer test (NorChip AS, Klokkarstua, Oslo, Norway) is a real-time multiplex NASBA assay for isothermal amplification of full length mRNA transcripts from 5 hrHPV types (Supplemental Table 1) in addition to the U1A as an internal control for sample validity. The assay was done according to instructions provided by the manufacturer (for details, see [29]). A modified protocol for the MPLC Nucleic Acid Isolation Kit, Large Volume (Roche Diagnostics GmbH, Mannheim, Germany), recommended by Norchip, was used for extraction of total Nucleic Acid (totNA). From each sample, 5 ml ThinPrep solution was pelleted and resuspended in 1000 μl Qiagen lysis buffer (QIAGEN, Valencia, CA, USA). The suspension was used for isolation according to the Roche standard protocol with 50 μl elution buffer volume. The eluate was stored at − 70 °C for further analysis.

Material and methods Ethics

Hybrid Capture II We have not included the Hybrid Capture II (HC2) test (Digene Corporation, Gaithersburg, MD/Qiagen) as it detects the same 13 hrHPV genotypes as the Amplicor and gives very similar results [16,25,26]. Adding HC2 would have been unnecessary costly and noninformative. Amplicor HPV and Linear Array HPV Genotyping Test The AMPLICOR® HPV-test target DNA from 13 hrHPV genotypes and the LA® HPV Genotyping detects 37 genotypes, 12 high risk, one probably high risk, 7 possible high risk, 8 low risk and 9 unclassified, (Supplemental Table 1). For details of the Amplicor and LA tests (Roche Molecular systems, Roche Diagnostics GmbH, Mannheim, Germany), see [27]. Total Nucleic Acid (totNA), isolated according to the protocol described for Proofer and diluted 1:10, was used for the LA test. As cases negative by Amplicor are nearly always also negative by LA, [28] HPV positive cases with any of the four other methods were

APTIMA HPV mRNA assay The APTIMA is based on target capture transcription-mediated amplification of E6/E7 mRNA from 14 hrHPV types (Supplemental Table 1) in a single tube. Samples were diluted 1:2.9 in specimen transport media prior to analysis on the DTS®400 system (Gen-Probe, San Diego, CA 92121) and 400 μl of diluted specimen was tested in the APTIMA HPV assay [30,31]. Statistical analysis We compared: 1. hrHPV detection in follow-up cytology samples by the different methods; 2. the specificity of the different methods to predict CIN 1 or less and sensitivity to predict CIN2–3 in the follow-up biopsies; 3. the significance of the different methods to predict spontaneous regression-or-not of CIN2–3 in the follow-up biopsies and cone excisions. SPSS version 17 (SPSS Inc., Chicago, IL) and MedCalc (MedCalc Software, Mariakerke, Belgium) were used. Cross tables were made for the different methods in the follow-up cytology specimens and compared with the Kappa test. As the different tests do not all detect all the hrHPV genotypes, samples were regarded as false-negative if LA was positive for the HPV genotypes detectable by the other test and the other test still was negative. Cross tables were made for the different methods to detect hrHPV as compared to the LA results, positive or negative with any genotypes detectable by that method and compared by Kappa measure of agreement. Results Eighty percent of the women were N30 years, the median age was 37, mean 40, Range: (25–69) years. Description of the cytology and histology Of the 528 index cytology samples, 371 were ASCUS and 157 LSIL (Supplemental Fig. 1). In 312 of these 528 (59%) cases, the follow-up

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Table 1 Distribution of follow-up biopsy diagnosis according to index (referral) and follow-up cytology. Follow-up punch biopsy

Index cytology

ASCUS LSIL Total

Follow-up cytology

Normal ASCUS LSIL HSIL ASCH Invalid Total

Table 2 Detection of HPV positive/negative as detected in follow-up cytology by the different methods according to the index cytology ASCUS or LSIL. Method

Normal

CIN 1

CIN2–3

Metaplasia

Inadequate

Total

24 (34%) 19 (30%) 43 (32%) 16 (55%) 13 (39%) 6 (14%) 4 (29%) 3 (25%) 1 (50%) 43 (32%)

18 (26%) 19 (30%) 37 (28%) 5 (17%) 12 (36%) 15 (34%) 1 (7%) 4 (33%)

25 (36%) 22 (34%) 47 (35%) 5 (17%) 7 (21%) 22 (50%) 9 (64%) 4 (33%)

2 (3%) 4 (6%) 6 (4%) 3 (10%)

1 (1%)

70 (52%) 64 (48%)

0 37 (28%)

0 47 (35%)

0 1 (2%)

1 (1%)

Amplicor

HPV results

Index cytology diagnosis ASCUS

LSIL

Total

Negative

258 (70%) 109 (29%) 4 (1%) 371 (70%) 268 (72%) 103 (28%) 371 (70%) 319 (86%) 42 (11%) 9 (3%) 370 (70%) 280 (75%) 91 (25%) 371 (70%) 256 (82%) 115 (53%) 371 (70%)

61 (39%) 95 (61%) 1 (1%) 157 (30%) 64 (41%) 93 (59%) 157 (30%) 118 (75%) 34 (22%) 3 (3%) 155 (30%) 79 (50%) 78 (50%) 157 (30%) 56 (18%) 101 (47%) 157 (30%)

319 (60%) 204 (39%) 5 (1%)

Positive Invalid

134 Total

0 1 (3%)

29

0

44

0

14

Cobas4800

Negative

33 Positive Total

0 1 (8%) 1 (50%) 6 (4%)

PreTect HPV-Proofer 0

12

0 1 (1%)

2

Negative Positive Invalid

134 Total APTIMA

cytology after 6–12 months was normal (of which 29% were HPV positive by Amplicor). For the remaining 216 cases, 111 were ASCUS, 65 LSIL, 17 HSIL, 16 ASCH and 7 inadequate for cytology. At the time of last follow-up in December 2010, 134 cases had a punch biopsy, seventy follow-up biopsies had referral cytology ASCUS (24 normal, 18 CIN1, 25 CIN2–3, 2 metaplasia and 1 inadequate) and 64 were LSIL (19 normal, 19 CIN1, 22 CIN2–3 and 4 metaplasia), (Table 1). Of the 47 CIN2–3 cases 34% were CIN2 and 66% CIN3. In the 37 cases with a LEEP-cone after punch biopsy, the diagnoses were normal in 6 cases (16%), 6 CIN1 (16%), 23 CIN2–3 (62%) and 2 metaplasia (5%), (Supplemental Fig. 1). Of the 47 cases with a punch biopsy diagnoses of CIN2–3, 35 had a LEEP-cone and 12 (32%) showed spontaneous regression (i.e., CIN1 or less in follow-up cone excision). In the remaining 12 cases with punch biopsy diagnoses of CIN2–3, a LEEP-cone was not performed (see Material and methods).

Description of the HPV in the follow-up cytology The number of HPV negative and positive cases with the different methods in the follow-up cytology specimens is listed in Table 2. Cobas and APTIMA had no invalid samples, Amplicor had 5/528 (1%) and Proofer had 12/528 (2%) invalid samples due to an invalid internal control (see Material and methods section). For the Amplicor and Cobas4800, the number of HPV positive cytology specimens is quite similar (204/528=39% and 196/528=37%), for the APTIMA the number was only slightly lower (169/528=32%). In contrast, the Proofer had the lowest number of HPV positives (76/513=15%). When all methods were taken together, 237 (45%) cases were positive with any of the methods although 21/237 (9%) of these were negative with the LA. These 21 cases were HPV positive by only one of the other methods, indicating that the LA negative result most probably was correct. As detected by LA, 66/216 (31%) had a single genotype while 150/216 (69%) had multiple genotypes. The most frequent genotypes in the positive samples were: 21% HPV 16, 13% HPV 66, 13% HPV 51, 13% HPV 52, 11% HPV 45, 10% HPV 31, 10% HPV33 and 8% HPV 18. The distribution of genotypes for the 12 regression cases as analyzed in the cytology specimens taken prior to the biopsies are as follows: 1 (HPV 16, 56, 66), 1 HPV 35, 1 HPV 56, 1 (HPV 6, 35, 42), 1 (HPV 42, 52, 81, 82, cp6108), 1 (HPV 16, 18, 51), 1 (HPV 18, 56, 58, 82, 83), 1 (HPV 16, 39), 1

Negative Positive Total

Linear Array

Negative Positive Total

a

528 332 (63%) 196 (37%) 528 437 (83%) 76 (15%) 12 (2%) 525a 359 (68%) 169 (32%) 528 312 (59%) 216 (41%) 528

Three samples did not have sufficient material for PreTect HPV-Proofer analysis.

(HPV 51, 66, 81, 83), 1 (HPV 16, 33), 1 (HPV 33, 51, 53, 59, 68, 73) and 1 (HPV 51, 53, 59, cp6108). Comparison of HPV detection (as positive/negative) by the different methods The agreement between Amplicor and Cobas4800 is almost perfect (κ = 0.85), Amplicor and Proofer have fair agreement (0.33) and Amplicor and APTIMA substantial agreement (κ = 0.74). Cobas4800 and Proofer have fair agreement (kappa = 0.34) while Cobas4800 and APTIMA have an almost perfect agreement (κ = 0.81). Proofer and APTIMA have moderate agreement, (kappa = 0.41) (Table 3). Comparison of the different methods with the LA (as positive or negative with any genotype detectable by the different methods) shows almost perfect agreement with Amplicor, Cobas4800 and APTIMA (κ =0.93, 0.94 and 0.82 respectively), but moderate agreement with

Table 3 Correlation of the different HPV methods with each other for detection of HPV positivity or not. Amplicor Amplicor Cobas4800 PreTect HPV-Proofer APTIMA

Kappa N Kappa N Kappa N Kappa N

523 0.85 523 0.33 508 0.74 523

Cobas4800

PreTect HPV-Proofer

APTIMA

0.85 523

0.33 508 0.34 513

0.74 523 0.81 528 0.41 513

528 0.34 513 0.81 528

513 0.41 513

528

I.T. Ovestad et al. / Gynecologic Oncology 123 (2011) 278–283 Table 4 Results of the different methods as compared to Linear Array, testing HPV positive or negative for any of the genotypes detectable by the other method. Linear Array results for any genotype detectable by the method

Amplicor

Negative Positive

Cobas4800

Total Kappa Negative

PreTect HPV-Proofer

APTIMA

310 (96%) 2 (1%) 312

14 (4%) 192 (99%) 206

296 (86%) 9 (13%) 305

48 (14%) 62 (87%) 110

310 (88%) 2 (1%) 312

42 (12%) 164 (99%) 206

Total 308 193 501

324 194 518

344 71 415

Detection of CIN2–3 in the histological punch biopsy and spontaneous regression of CIN2–3 in the LEEP-cone The sensitivities (with 95% confidence interval) for Amplicor, Cobas4800 and APTIMA to detect CIN2–3 were very high: 100% (92.5– 100), 96% (85.5–99.5) and 98% (88.7–99.9), respectively, but much lower for Proofer 53% (38.1–67.9). The specificity for detection of CIN1 or less was low for Amplicor and Cobas4800: 18% (9.9–27.6) and 26% (17.0– 37.3), somewhat higher for APTIMA: 38% (26.9–49.0) and Proofer had the highest specificity: 76% (65.4–85.1) (Table 5, Supplemental Fig. 2). Further analysis of Proofer negative CIN2–3 biopsies (n =22) shows that 7/22 (32%) were CIN2 and 15/22 (68%) CIN3, (Table 6). Supplemental Table 3 shows LA results versus the other methods in the 43 normal biopsies. The predictive value of CIN regression for Cobas4800 and Proofer were not significant, furthermore all CIN2–3 were positive for both Amplicor and APTIMA. Therefore, none of the four methods predicted regression. Discussion

0.60

Positive Total Kappa

9 (3%) 186 (96%) 195

0.94

Positive Total Kappa Negative

Positive

299 (97%) 7 (4%) 306 0.93

Positive Total Kappa Negative

Negative

281

352 166 518

0.82

Proofer, κ=0.60. Compared to LA, the RNA methods had many more negatives than the DNA methods: Proofer, n =48 and APTIMA, n =42, contrasting n=9 for Amplicor and n =14 for Cobas4800 (Table 4). Comparison of partial genotyping for Cobas4800 (HPV16 and 18) and Proofer (HPV 16, 18, 31, 33 and 45) versus Linear Array genotyping We compared the specific genotypes detected by the LA with the partial genotyping results from Cobas4800 (HPV16 and 18) and Proofer (HPV16, 18, 31, 33 and 45). Overall agreement was 212/237 (90%) for Cobas4800 and 161/229 (70%) for Proofer.

The detection of hrHPV and the sensitivity to predict CIN2–3 by the Amplicor, Cobas4800 and APTIMA show almost perfect agreement, while Proofer differs significantly from the other tests, with considerably fewer HPV positives and lower sensitivity. The partial genotyping results of the PreTect HPV Proofer showed many discrepancies with the LA Genotyping test, while the Cobas4800 had good agreement with the LA. A number of the samples which were HPV positive by the DNA tests were negative by the RNA tests (Table 4). An up-regulation of the oncogenic proteins E6 and E7 is a marker for increased risk of cervical cancer and hypothetically the RNA tests should better predict spontaneous regression of CIN2–3 in the cones. Our results do not support this hypothesis. We have shown before that the differences in the natural history of a high grade CIN (i.e. regression or persistence) relates to the balance between viral and host/immunological factors and their importance may overshadow the degree of HPV-DNA transcription [27,32], explaining why the mRNA is not a sensitive predictor for regression-or-not. Although the Proofer and APTIMA both are mRNA based tests, their agreement is only moderate (kappa = 0.41), with 22% discrepancies.

Table 5 Comparison of HPV positivity or negativity by the different methods in follow-up cytology, 6–12 months after an index cytology diagnosis of ASCUS or LSIL. The presence of histological CIN1 or less and CIN2–3 in epithelium, assessed by gynecological pathologists' review in the follow-up punch biopsies, was used to calculate the specificity and sensitivity of the methods. Follow-up punch biopsy Normal Amplicor

Cobas4800

PreTect HPV-Proofer

APTIMA

a

95% confidence interval.

CIN 1

CIN2–3

Total

Negative

10

4

0

14

Positive

33

33

47

113

Total Negative

43 12

37 9

47 2

127 23

Positive

31

28

45

104

Total Negative

43 34

37 27

47 22

127 83

Positive

8

7

23

38

Total Negative

42 16

34 14

45 1

121 31

Positive

27

23

46

96

Total

43

37

47

127

Sensitivity (95% CI)a

Specificity (95% CI)a 18% (9.9–27.6)

100% (92.5–100) 26% (17.0–37.3) 96% (85.5–99.5) 76% (65.4–85.1) 53% (38.1–67.9) 38% (26.9–49.0) 98% (88.7–99.9)

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Table 6 22 CIN2–3 cases testing negative with PreTect HPV-Proofer (PP) as compared to APTIMA results and according to follow-up cytology, follow-up biopsies and regression or persistence in follow-up cones. Index cytology

Follow-up cytology

Follow-up biopsy

ASCUS ASCUS ASCUS ASCUS ASCUS ASCUS ASCUS ASCUS ASCUS ASCUS ASCUS ASCUS LSIL LSIL LSIL LSIL LSIL LSIL LSIL LSIL LSIL LSIL

LSIL LSIL LSIL ASCUS LSIL ASCUS Normal LSIL ASCUS ASCH Normal HSIL LSIL LSIL LSIL LSIL LSIL LSIL HSIL LSIL LSIL LSIL

CIN2 CIN2 CIN2 CIN2 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN2 CIN2 CIN2 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3

Genotypes detectable by PP

Genotypes not detectable by PP

APTIMA result

Regression/persistence

51

Positive Positive Positive Negative Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive

No follow-up Persistence Regression No follow-up Persistence Persistence Persistence Regression No follow-up Persistence Regression Persistence No follow-up Persistence Regression Regression Persistence No follow-up Regression No follow-up Regression Regression

18, 35, 53, 67, 81, 82, 84 51, 66, 81, 83 Neg 35, 51 56, 82 31, 39 42, 52, 81, 82, cp6108 31, 51, 61 52, 56, 66 16, 39 52, 62 35, 39, 52, 54 33, 59, 62, 68, 81 51, 53, 59, cp6108 6, 35, 42 35, 53, 73, 82 35 35 51 56 16, 56, 66

Technically the methods differ, as Proofer detects 5 and APTIMA 14 hrHPV subtypes. The analytical sensitivity for Proofer is also lower, which is compensated by using a much larger sample volume for isolation of totNA [33,34]. Our lower clinical sensitivity for Proofer, confirm other studies [18,21,35]. A large report tested 953 women referred for colposcopy [16]. The sensitivities for detecting CIN2+ for Amplicor and APTIMA (98% and 97%, respectively) were similar to our results, but for Proofer their sensitivity result was better (71% versus 53%). This is probably due to differences in distribution of hrHPV genotypes in HSILs (which are frequent in colposcopy material) and ASCUS/LSIL (as we have studied) [36]. HSILs often contain HPV16, 18 and other genotypes detected by Proofer. In ASCUS/LSIL, many lesions are positive for hrHPV genotypes not discovered by Proofer. The specificity to predict normal histology in punch biopsies for Amplicor, APTIMA and Proofer respectively is similar to our results. We found that only 27% of the CIN2–3 lesions (Table 6) not detected by Proofer had hrHPV types detectable by this method, and the difference therefore is quite understandable. Another study of colposcopy referred patients (n = 1551), also concluded that Proofer misses many CIN2–3 cases because of the limited number of targeted genotypes in the test [22]. In a study of 531 patients with follow-up biopsies [17], the Cobas4800 and LA agreed in 95% (κ = 0.92) in detecting less than CIN1 versus CIN2–3. We found a comparable, almost perfect overall agreement (Table 4). In 1578 women with ASCUS cytology, the clinical sensitivity for Cobas4800 to predict CIN2 or worse was 90%, versus 87% for the HC2 [37], also in agreement with our study. We found 29% HPV16 and 18 positive follow-up samples, comparable to another study of mildly abnormal cytology [38]. This is much lower than the 60–70% in CIN2–3 lesions detected by HSIL cytology [36]. An average of 43% (range 23–74%) with ASCUS and 76% LSIL (range 55–89%) cytology were hrHPV positive in a meta-analysis [39]. The percentages of hrHPV positives, especially for LSIL, drops with increasing age. In our study with mean age of 40, 39% were hrHPV positive, 29% of the ASCUS and 61% of the LSILs. The prevalence for CIN2–3 was 9% in our study, comparable to other studies [40]. For low grade and borderline abnormal lesions cytological changes might partly be caused by low-risk HPV and therefore not detected by hrHPV tests. Therefore HPV testing is more sensitive than cytology alone, and has become an important adjunct to cytology screening. Testing negative for hrHPV has a high Negative Predictive Value for CIN2–3 in the

cone

cone

cone

cone

cone cone

next 3–5 years [41,42]. We used a virological reflex test to compare the strength of the different HPV tests to detect women at risk for developing CIN2–3 within the context of a screened population following the Norwegian guidelines for HPV testing. The interval of 6–12 months between referral and follow-up cytology may give any transient and clinically irrelevant infection a chance to clear and improve the specificity. The bias this may have caused for regressing lesions is hypothetical and not relevant as it has regarded all the five test methods used. For women with ASCUS and LSIL cytology, the 2-year cumulative risk for CIN3 or worse for HPV16 positives were 33% and 39%, respectively and for HPV18 positives 9% and 15% [38]. Identification of HPV16- or HPV18-positive cases, the two most common hrHPV types in cervical cancer, could be an additional risk stratification, increase the clinical value of HPV testing and decide the management of patients[37]. The low specificity of the HPV tests for detection of CIN2–3 [16,19,22] is a concern. A major challenge will be implementing programs that do not over-treat non-HPV16 or -18 positive women without obvious longterm persistence of HPV. Repeated HPV testing would reveal potential persistent lesions with other HPV genotypes. 15–40% of CIN2–3 lesions regress, independent of lesion size [27,43,44]. For intervals less than 9 weeks between the punch biopsy and LEEP, regression is low (5%) while intervals exceeding 9 weeks have a higher regression rate (38%). A mean interval of 10 weeks, as in the current study, is quite common in many clinics and generally sufficient to allow spontaneous regression. However, the range was considerable and it would be interesting to evaluate the different HPV tests in a colposcopy referred group of patients with standardized long intervals between the biopsy and the LEEP. ThinPrep plus an HPV test is more costly than the conventional PAP smear. Based on assessment of quality-adjusted life years and improvement of health benefits, age-based screening with HPV testing as a triage test for equivocal, uncertain cytology results in younger women, and as a primary screening test in older women, are more cost-effective than current screening practices [45–49]. However, the costs and benefits vary from one country to the other. We conclude that only reliable and reproducible HPV tests with high sensitivity and specificity, validated for use in the intended population, should be implemented for use in a screening population. The sensitivity of the Proofer test at the moment does not fulfill these requirements. Supplementary materials related to this article can be found online at doi:10.1016/j.ygyno.2011.07.024.

I.T. Ovestad et al. / Gynecologic Oncology 123 (2011) 278–283 Conflict of interest statement We hereby declare that none of the authors or the study sponsors has conflicts of interest regarding the study design, collection of data, analyzing and interpretation of data, writing the report or in the decision to submit the report for publication. None of the authors has any financial interest in publishing these data, nor any financial or advisory relationship with a company producing the equipment or chemicals described in this article.

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