Triaging equivocal cytology of the cervix : identyfying women at risk
for high-grade cervical lesions
Wensveen, C.W.M.
Citation
Wensveen, C. W. M. (2006, June 13). Triaging equivocal cytology of the cervix : identyfying
women at risk for high-grade cervical lesions. Retrieved from
https://hdl.handle.net/1887/4435
Version:
Corrected Publisher’s Version
License:
Licence agreement concerning inclusion of doctoral thesis in the
Institutional Repository of the University of Leiden
Chapter 6
Abstract
B ack g ro u n d : In this study the MIB-1 immunostaining pattern as an index of cellular proliferation was analysed in smears diagnosed as borderline dysk aryosis in order to establish whether the combination of HPV testing and MIB-1 staining could resolve equivocal cytology.
M ate rial an d M e th o d s: Conventional Pap smears of 10 8 women diagnosed as borderline dysk aryosis were stained with MIB-1 and the proliferation index was assessed. T hese women were evaluated by colposcopy, histological sampling, and HPV, semi-quantitatively evaluated by Hybrid Capture II test.
R e su lts: A ll 64 HPV and MIB-1 negative women had no underlying high-grade CIN or cervical cancer. F orty of the 10 4 women with normal histology or CIN I were positive for HPV, compared to only one positive MIB-1 test (proliferation index of more than 3 5 % ).
C o n clu sio n : A dding a MIB-1-test in HPV positive women with equivocal cytology might reduce the number of colposcopy to predict > CIN II. With this approach only four instead of 4 3 HPV positive women would have been referred to colposcopy. F urther research in a larger volume of patients is necessary to confirm these findings.
I n tro d u ctio n
T here is general agreement that cervical intraepithelial neoplasia (CIN ) is a precursor of cervical carcinoma and that untreated severe dysplasia will progress into invasive lesions1. However, the natural history of CIN is not completely defined. High-risk human papillomavirus (HPV) infection seems to be related etiologically to CIN and cervical carcinoma1-4. T he risk of CIN progression is significantly higher in high-risk HPV positive women then in HPV negative women5 -8. However, it has also been observed that HPV infection is highly prevalent in the general population without clinical or pathologic lesions of the cervix. K enemans showed a HPV prevalence of 2 5 % in women aged 2 0 -2 4 years, and 5 % in women older than 3 5 years9. In a systematic review of 16 studies a pooled specificity of 62 % (95 % CI; 5 6-68 % ) for the standard Hybrid Capture II HPV D N A test ( > 1.0 relative light units cut-off value) was estimated to predict > CIN II in women with equivocal cytology10. Because of this relatively low specificity, the clinical usefulness of the HPV D N A test to predict high-grade lesions of the cervix is still on debate.
is expressed in the nuclei of proliferating cells throughout the cell cycle, except during the G 0 and early G 1 phases, and is recognised by MIB-1 monoclonal antibody11. The borderline cytology has created a management dilemma for clinicians, because it is an equivocal diagnosis. A number of studies have shown that 5-30% of women with this diagnosis harbour undetected cervical cancer precursors or even cervical carcinoma12-14. Although the majority of women with borderline cytology will have trivial lesions, some have significant lesions that warrant either closer surveillance or further investigation.
In this study we analysed the MIB-1 immunostaining pattern as an index of cellular proliferation in smears diagnosed as borderline dyskaryosis in order to establish whether the combination of HPV testing and MIB-1 staining can improve the triage of equivocal cytology.
Methods
Cytology
O ne hundred and eight slides of conventional Pap smears were used from a cohort study of women with borderline dyskaryosis (atypical squamous or glandular cells of undetermined significance), who were enrolled between April 1997 and March 2000 at the gynaecological outpatient clinic of the Medical Centre Haaglanden, The Hague, The Netherlands. All cervical smears were Papanicolaou stained, screened routinely, and independently reviewed to confirm the cytological diagnosis. A colposcopic examination was performed within 12 weeks following the smear, which included a biopsy and HPV test. A biopsy was performed in all women, because we were interested in the colposcopic and histological outcome in women with equivocal cytology. The Medical E thics Committee of the hospital approved this prospective study and informed consent was obtained. In the Netherlands a modified Papanicolaou system (CIS E O -A) is used for classification. This redefined and subdivided the Papanicolaou classes in order to make the terminology correlate with histological terminology15. The Dutch CIS O E -A classification interprets smears using a rating system including information on specimen composition, inflammatory characteristics, and adequacy of the smear. The letters C (composition), I (inflammation), S (squamous), O (other and endometrium), and E (endocervical cylinder epithelium) are used to indicate the composition and morphology of the smears. O f the 108 smears with equivocal cytology, 96 smears showed only atypical squamous cells (AS CU S = S 2/S 3), one only atypical squamous metaplastic cells (atypical repair= O 3), eight only atypical glandular cells (AG U S = E 3/E 4), three a combination of atypical squamous and glandular cells (S 2/3,E 3/4).
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Detection of HPV and other (non) S T D's
Specimens for HPV were collected from the cervix. High/ intermediate-risk types of HPV DNA (16/18/31/33/35/39/45/51/52/56/58/59/68) were detected using the Hybrid Capture II™ technology (Digene Corporation, Gaithersburg, MD, USA), which is a signal amplified hybridisation antibody capture microplate assay using chemiluminescence for the quantitative detection of human papillomavirus DNA in cervical specimens. A specimen ratio of > 1 was regarded as positive and a cut-off point of 1.0 pg/ml was used as described in detail previously16.
M IB -1 S taining,
Prior to MIB-1 staining the smears were treated as follows:
1. The smears were placed in a plastic jar containing 10 mmol citrate buffer, pH 6.0. 2. The jar was placed in a microwave oven.
3. The microwave was set at 80% power, 100oC, the time was set for 20 min. 4. The slides were cooled down in the citrate buffer until a temperature of 50oC was
reached.
5. The slides were rinsed in TBS for 2 min17.
During this antigen retrieval, the Papanicolaou stain was removed from the smear. After antigen retrieval the smears were stained for MIB-1 following the standard SAB protocol17, using an antisera dilution of 1:300. After immunostaining, the slides were stained with Gill's haematoxylin. MIB-1-positive nuclei are brown; MIB-1-negative nuclei and (to a lesser extent) cytoplasm are blue.
G rad ing the M IB -1 -Pos itiv e S m ears
When staining positive for MIB-1, the smear can be graded depending on the MIB-1 staining of the epithelial fragments. Note that single positive cells cannot be used for the grading: this proved to be no problem, because in all the cases additional epithelial fragments with MIB-1-positive cells were found. Positive and negative nuclei were counted in 10 of the most abnormal epithelial fragments. The fraction of positive nuclei was calculated for each fragment18.
number positive nuclei
PI = proliferation index = x 100% number all nuclei
The MIB-1 grading corresponds roughly with cytological diagnosis19.
Borderline MIB-1 grading: 1-20% MIB-1-positive nuclei MIB-1 grade I: 21-35% MIB-1-positive nuclei MIB-1 grade II: 36-50% MIB-1-positive nuclei MIB-1 grade III: > 50% MIB-1-positive nuclei For this study we defined a proliferation index of > 20% as a positive MIB-1-test for a CIN lesion. To predict a > CIN II lesion a cut-off point of > 35% was used.
Statistical analysis
Data were collected and analysed using the Statistical package for Social Sciences (SPSS, Inc, Chicago, IL , USA). Chi-square test was used to evaluate the association between pre-malignant lesions and the parameters HPV and the MIB proliferation factor. A p-value of less than 0.05 was considered statically significant. The specificity was assessed for HPV and MIB-1 test to predict > CIN II.
Results
Of the 108 women with equivocal cytology 10.2% (11/108) showed a CIN lesion and 3.7% (4/108) a high-grade lesion of the cervix (> CIN II), three women with CIN II and one with cervical carcinoma. The HPV DNA test was positive in 39.8% (43/108), (Table 1).
Table 1 . HPV* DNA test and histological outcome of 108 women with borderline dyskaryosis
Histological outcome HPV DNA test no CIN # CIN 1 > CIN 2 Total
negative 63 1 1 65
positive 34 6 3 43
Total 97 7 4 108
* HPV= human papillomavirus, # CIN = cervical intraepithelial neoplasia
Nine of the 108 slides (8.3%) showed a MIB-1 proliferation index of more then 20% and 5 (4.6%) more then 35%. The results of the MIB-1 grading are shown in Table 2. The risk of a CIN lesion is significantly higher for HPV positive women than for HPV negative women (odds ratio 8.3; 95% CI 1.7-40.8). For the MIB-1 proliferation index > 20% the risk of a CIN lesion is significantly higher than for women with < 20% MIB-1 grading (OR 10.5; 95% CI 2.3-48.2).
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Table 2 . MIB-1 grading, HPV* testing and histological outcome of 108 women with cervical smears diagnosed as borderline dyskaryosis
Histological Outcome
No CIN# CIN I > CIN II n=97 n=7 n=4
MIB-1 grading (%) HPV neg HPV pos HPV neg HPV pos HPV neg HPV pos Total
No staining 43 20 1 4 0 0 68 1-20% 19 10 0 2 0 0 31 21-35% 1 3 0 0 0 0 4 36-50% 0 1 0 0 0 2 3 > 50% 0 0 0 0 1 1 2 Total 63 34 1 6 1 3 108
* HPV= human papillomavirus, #CIN = cervical intraepithelial neoplasia
We could not estimate accurately the sensitivity and positive predictive value of the HPV and MIB-1 test to predict high-grade CIN, because of the small number of these high-grade cervical lesions (4/108). No high-grade CIN or cancer was found in all 64 HPV and MIB-1 negative women. Of the 104 women with normal histology or CIN I forty were HPV positive and 64 were HPV negative (specificity 62%), (Table 1). However, only one of these 104 women was MIB-1 positive compared to 103 negative MIB-1 tests (specificity 99%), (Table 2).
Finally, in Table 3, the correlation between HPV outcome and MIB-1 pattern > 35% is shown (p=0.08). The one case of HPV negative and MIB-1 positive showed a CIN II. Both tests were positive for the other two women with CIN II and the one with cervical carcinoma. For only one case both tests were positive with a normal histology (Table 2). After one year the cervical smear was normal and HPV could not be detected in this woman.
Table 3 . Correlation between HPV* and MIB-1 in 108 women with borderline dyskaryosis
MIB-1 > 35% neg pos Total
MIB neg 64 39 103
MIB pos 1 4 5
Total 65 43 108
Discussion
Many women with equivocal cytology are positive for high-risk HPV, which is 39.8% of the 108 smears in this study. The HPV test was more often positive in women with histological > CIN II than in women with normal histology or CIN I.
The sensitivity of the Hybrid Capture II HPV DNA test could not be estimated accurately in this study of women with equivocal cytology, because only a few women (4/108) showed a histological outcome of > CIN II. However, 104 women showed normal histology or CIN I. Therefore, we could calculate the specificity. The specificity for the HPV test to detect high-grade lesions of the cervix was 62% (64/104). The pooled specificity of HC II at the standard > 1.0 cut-off point reported in a systematic review of 16 studies was 62% and comparable with our findings (95% CI; 56-68%)10. Dunton at al.20 determined the clinical usefulness of the MIB-1-test in women with abnormal cytology to identify women harbouring high-grade lesions of the cervix. They investigated 101 women with cytology diagnosed as borderline (45) or mild dyskaryosis (56). High-grade CIN or cancer was diagnosed in 24.7% (25/101) of the women with abnormal smears. The MIB-1-test showed a sensitivity of 96%, a specificity of 67%, a positive predictive value of 49%, and a negative predictive value of 98% to detect the underlying > CIN II. In their study the MIB-1-test seemed to be a significant predictor of high-grade CIN (odds ratio 21.5; 95% CI 5-0-92.0)20.
Bekkers et al.21 investigated 108 women with cytology diagnosed as borderline (13), mild (40) or moderate/severe dyskaryosis (55), who were referred for colposcopy and a large loop excision of the transformation z one to identify women with CIN III. The percentage of the underlying high-grade CIN (CIN II and III) and CIN III was respectively 80.6% (87/108) and 50.9% (55/108). They found that the mean of the MIB-1-test increased with the degree of CIN. A higher specificity rate for the MIB-MIB-1-test (62%) than for the HPV test (48%) to predict CIN III was measured21.
Both MIB-1 and HPV test showed a high negative predictive value to detect > CIN II. In this context, it is of interest to mention that there was one patient with a negative HPV test and positive MIB-1 proliferation test in this study showing a CIN II. Therefore repeating a HPV negative smear diagnosed as borderline dyskaryosis after six months can be considered. In this study, forty of the 43 women with a positive HPV test have been referred to a colposcopy clinic and had normal histology or CIN I, compared to only one of the five women with a positive MIB-1-test (PI > 35%). When all 43 HPV positive smears were consequently stained for MIB-1 two cases with histological outcome of CIN II and the one case of cervical cancer were identified. This implies that the other 40 HPV positive cases could safely be monitored with cytology and subsequent HPV testing and did not really need immediately colposcopy and biopsy. Therefore, an additive MIB-1-test might reduce the number of colposcopies without missing underlying high-grade lesions of the cervix.
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References
(1) Cox J T. Epidemiology of cervical intraepithelial neoplasia: the role of human papillomavirus. Baillieres Clin Obstet Gynaecol 1995 Mar;9(1):1-37.
(2) Guido R. Guidelines for screening and treatment of cervical disease in the adolescent. J Pediatr Adolesc Gynecol 2004 Oct;17(5):303-11.
(3) Walboomers J M, J acobs MV, Manos MM, Bosch FX , Kummer J A, Shah KV, Snijders PJ , Peto J , Meijer CJ , Munoz N. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999 Sep;189(1):12-9.
(4) Ferenczy A, Franco E. Persistent human papillomavirus infection and cervical neoplasia. Lancet Oncol 2002 J an;3(1):11-6.
(5) Ho GY , Bierman R, Beardsley L, Chang CJ , Burk RD. Natural history of cervicovaginal papillomavirus infection in young women. N Engl J Med 1998 Feb 12;338(7):423-8.
(6) Ho GY , Burk RD, Klein S, Kadish AS, Chang CJ , Palan P, Basu J , Tachezy R, Lewis R, Romney S. Persistent genital human papillomavirus infection as a risk factor for persistent cervical dysplasia. J Natl Cancer Inst 1995 Sep 20;87(18):1365-71.
(7) Moscicki AB, Shiboski S, Broering J , Powell K, Clayton L, J ay N, Darragh TM, Brescia R, Kanowitz S, Miller SB, Stone J , Hanson E, Palefsky J . The natural history of human papillomavirus infection as measured by repeated DNA testing in adolescent and young women. J Pediatr 1998 Feb;132(2):277-84. (8) Remmink AJ , Walboomers J M, Helmerhorst TJ , Voorhorst FJ , Rozendaal L, Risse EK, Meijer CJ , Kenemans P. The presence of persistent high-risk HPV genotypes in dysplastic cervical lesions is associated with progressive disease: natural history up to 36 months. Int J Cancer 1995 May 4;61(3):306-11.
(9) Kenemans P. HPV genotype as a prognostic factor for progression to cervical carcinoma in young women. Eur J Obstet Gynecol Reprod Biol 1994 May 31;55(1):24-5.
(10) Arbyn M, Paraskevaidis E, Martin-Hirsch P, Prendiville W, Dillner J . Clinical utility of HPV-DNA detection: triage of minor cervical lesions, follow-up of women treated for high-grade CIN: an update of pooled evidence. Gynecol Oncol 2005 Dec;99(3 Suppl 1):S7-11.
(11) Key G, Becker MH, Baron B, Duchrow M, Schluter C, Flad HD, Gerdes J . New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated against bacterially expressed parts of the Ki-67 cDNA containing three 62 base pair repetitive elements encoding for the Ki-67 epitope. Lab Invest 1993 J un;68(6):629-36.
(12) Manos MM, Kinney WK, Hurley LB, Sherman ME, Shieh-Ngai J , Kurman RJ , Ransley J E, Fetterman BJ , Hartinger J S, McIntosh KM, Pawlick GF, Hiatt RA. Identifying women with cervical neoplasia: using human papillomavirus DNA testing for equivocal Papanicolaou results. J AMA 1999 May 5;281(17):1605-10.
(13) Solomon D, Schiffman M, Tarone R. Comparison of three management strategies for patients with atypical squamous cells of undetermined significance: baseline results from a randomized trial. J Natl Cancer Inst 2001 Feb 21;93(4):293-9.
(14) Cox J T, Lorincz AT, Schiffman MH, Sherman ME, Cullen A, Kurman RJ . Human papillomavirus testing by hybrid capture appears to be useful in triaging women with a cytologic diagnosis of atypical squamous cells of undetermined significance. Am J Obstet Gynecol 1995 Mar;172(3):946-54. (15) Hanselaar AG. Criteria for organized cervical screening programs. Special emphasis on The
Netherlands program. Acta Cytol 2002 J ul;46(4):619-29.
(16) Schiffman M, Herrero R, Hildesheim A, Sherman ME, Bratti M, Wacholder S, Alfaro M, Hutchinson M, Morales J , Greenberg MD, Lorincz AT. HPV DNA testing in cervical cancer screening: results from women in a high-risk province of Costa Rica. J AMA 2000 J an 5;283(1):87-93.
(17) Kok LP BM. Microwave cookbook for microscopists: art and science of visualization. 3rd revised ed. ed. Leiden: Coulomb Press leyden; 1992. p. 266.
(18) Boon ME, Vinkestein A, van Binsbergen-Ingelse A, van HC. Significance of MiB-1 staining in smears with atypical glandular cells. Diagn Cytopathol 2004 Aug;31(2):77-82.
(19) Boon ME, Kleinschmidt-Guy ED, Wijsman-Grootendorst A, Hoogeveen MM. Upgrading unsatisfactory cervical smears with the MiB-1 method. Diagn Cytopathol 1996 Nov;15(4):270-6. (20) Dunton CJ , van Hoeven KH, Kovatich AJ , Oliver RE, Scacheri RQ , Cater J R, Carlson J A, J r. Ki-67
antigen staining as an adjunct to identifying cervical intraepithelial neoplasia. Gynecol Oncol 1997 Mar;64(3):451-5.
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