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In this study we investigated the diagnostic application of immunocytochemical staining of p16INK4a (CINtec"!p16INK4a Kit, DAKOCytomation, Denmark) on conventional Pap smear. A total of 137 cervical specimens were enrolled in this study, of which 77 and 60 cervical smears were taken from premenopausal and postmenopausal women, respectively. Two cervical smears were taken simultaneously in 68 women, one for conventional cytology and the other for immunostaining. Additional 69 cervical smears were taken from the archive, decolorized and then used for immunostaining. In premenopausal women 1 out of 14 (7.1%) with negative cytology, 7 out of 24 (29.2%) with low grade squamous intraepithelial lesion (LSIL), all 35 (100%) with high grade squamous intraepithelial lesion (HSIL) and all 4 (100%) with squamous cell carcinoma (confirmed by histopathology) had positive staining to p16INK4a. In postmenopausal women p16INK4a positivity was observed in 4 out of 7 (57.1%) cases of LSIL, 12 out of 14 (85.7%) cases of HSIL and all 4 out of 5 (80%) different cases of carcinoma (1 cervical adenosquamous carcinoma and 3 cervical squamous cell carcinoma in situ confirmed by histopathology), but none of 34 smears with normal cytology. Twenty smears with normal cytology chosen for the negative control in this study were from the group of postmenopausal women and were as expected negative for p16INK4a immunostaining. In the group of postmenopausal women, 16 out of 60 (26.7%) cases the cytological diagnosis was established on the basis of p16INK4a immunostaining as being HSIL. From our preliminary study on a limited number of samples, we can however conclude that p16INK4a immunostaining is a very useful tool for cytological diagnosis enabling to distinguish HSIL from normal, reactive or inflammatory changes. Key words: postmenopausal, cervical, dysplasia, immunostaining, p16INK4a Introduction It is well established that persistant infection with oncogenic Human papillomavirus (HPV) causes preneoplastic and neoplastic changes of the cervical epithelium, leading towards carcinoma, especially in middle aged women1. In postmenopausal age, women have received little attention concerning this problem. Improvements in life standards and better health care have led to increasing population of elderly women who come to their regular gynaecological exam and their Papanicolaou (Pap) smears2-3. In spite of the fact that there is almost 20% of women infected with HPV4, only a few of them develop preneoplastic lesion, and even fewer develop cancer. Smith and al.5 found that 16% of persistent HPV infections exist in the elderly group of women, suggesting that one can predict development of a preneoplastic lesion that can progress toward cancer. However, problems in diagnosis such as aging-related changes, reparative changes and interpretation difficulties may lead towards an incorrect diagnosis. Pap smears of postmenopausal women are often misdiagnosed because of the difficulty in distinguishing atrophic epithelial cell groups only by the morphological criteria. Those criteria are more viable when premenopausal women are concerned, referred to better oestrogen effect, hence better maturation. In this study we investigated whether immunocytochemical staining of p16INK4a, performed on conventional Pap smear could be of any diagnostic help in solving this dilemma. Thus, we performed p16INK4a immunostaining on specimens taken from premenopausal and postmenopausal women in order to make an accurate diagnosis in those cases, which were impossible for us to distinguish on the basis of cytomorphological criteria, i.e. to distinguish a high grade cervical lesion from atrophy or reactive changes. Examinees, Materials and the Methods We analysed 137 cervical smears, which consisted in 69 archival Pap slides and 68 cervical specimens collected from women who attended our clinic in 2006 for their regular check-up. They were divided into two age groups. Seventy-seven premenopausal women were in group I (median age 34.5) and 60 postmenopausal women (median age 63) in group II, of which 20 women with normal cervical smears were used as a control group. Two simultaneous cervical smears were taken by a cytobrush and a wooden spatula, one for conventional cytology and the other for immunostaining. Archival Pap slides were decolorized in 0,5% solution of 36% HCl, rinsed with distilled water and then immunocytochemically stained with CINtec"! p16INK4a Kit (DAKOCytomation, Denmark) according to the manufacturer instructions. There were no differences in staining reaction or intensity of reaction between decolorized and freshly taken smears. According to DAKO instructions at least 1% of cells should be stained for the reaction to be considered positive. Cells are regarded as stained if brownish-like granules were found in nuclei and/or in a cytoplasm. Pap smears were classified by the Bethesda system6 into a normal epithelium, low (LSIL) and high grade intraepithelial cervical lesion (HSIL) and squamous cell carcinoma (SCC). Among postmenopausal women, there was one case of recurrent adenosquamous carcinoma and metastatic transitional cell carcinoma. In 16 postmenopausal women we could not diagnose based on the morphological criteria; in those women diagnosis was established after the p16INK4a immunostaining. Fifty-five women underwent colposcopy and biopsy, and a consequent cold knife conisation or hysterectomy was performed; in each case histological verification was made. Unfortunately, the patient with metastatic transitional cell carcinoma died. The distribution of positivity rate versus diagnosis was analyzed with the standard Chi square (2) test. The statistical significant differences was set at p<0.0001. Results The distribution of cytological diagnoses and p16INK4a immunostaining in premenopausal women is as follows: 18.2% (14/77), 31.2% (24/77), 45.5% (35/77) and 5.2% (4/77) women had a negative cytology, LSIL, HSIL, SCC, respectively (Table 1). P16INK4a immunostaining was observed in 7.1% (1/14), 29.2% (7/24) and 100% (35/35 and 4/4) of premenopausal women with negative cytology, LSIL, HSIL and SCC, respectively. The observe frequencies were significantly different (2= 52.24, p<0.0001). The positive p16INK4a immunostaining of one of the 4 SCC later histologically confirmed, and of normal metaplastic cells is shown in Figure 1 and 2. In this age group we did manage to establish the diagnosis in every patient s smear owing to viable morphological signs and well established criteria for every grade of intraepithelial lesion. The distribution of cytological diagnoses and p16INK4a immunostaining in postmenopausal women is as follows: 52.3% (34/100), 10.8% (7/100), 21.5% (14/100) and 7.7% (5/100) women had a negative cytology, LSIL, HSIL, CC, respectively (Table 2). P16INK4a immunostaining was observed in 57.1% (4/7), 85.7% (12/14) and 80% (4/5) of premenopausal women with LSIL, HSIL and CC, respectively, while none with those with normal cytology. The observe frequencies were significantly different (2= 40.97, p < 0.0001). In 16 postmenopausal women we had difficulties for classifying a certain cell groups as neoplastic because of their resemblance to mucosal atrophy or because of the presence of strong degenerative, inflammatory or reactive changes. In 8 women, after p16INK4a immunostaining we were able to distinguish HSIL from the primary diagnosis, reactive changes (Figure 3). Among these women there was one case of adenosquamous carcinoma in which a positive reaction of p16INK4a made us consider the possibility of recurrent carcinoma. Discussion In spite of good screening methods based on Pap smear and well organized screening programmes in many countries, women all around the world continue to develop cervical disease. The incidence rate of cervical cancer in developing countries is increasing due to the aging population, while in Western Europe first peak is between 30 and 40 years of age and the second peak occurs around 60 years of age7. Genital infection with oncogenic or high-risk (hr) HPV is established as one of the main events in pathogenesis of cervical and other genital tumours1. After the first contact with the virus, viral replication is limited to superficial layers of the cervical epithelium that represents the acute, transitory phase. If viral E6 and E7 oncogene products are over-expressed in a basal or parabasal layers, they will interfere with the regulation of the host cell cycle, inducing genetic instability. The probability of progression of cervical precancerogenic lesions is considered greater with the longer persistence of papillomavirus infection. Almost 100% of all cervical cancers can be attributed to certain hrHPV types8. The highest proportion of cervical squamous cancers is associated with HPV type 16, followed by type 18 which is more common in adenocarcinomas, than HPV types 31, 33, 45, 52, 58 and others1,7. There are lots of regulating proteins involved in the cell cycle control. One of them, p16INK4a, has been shown to be over-expressed after the cell has been infected by hrHPV types. Expression of the viral E7 oncogene leads to functional inactivation of tumour suppressor retinoblastoma protein (pRB), which in turn, results in strong over-expression of the cyclin-dependent kinase inhibitor p16INK4a. This indicates that an active expression of the viral E7 oncogene is present in dysplastic cells4. There are a lot of data about HPV prevalence in younger women, but not much about elderly population. Smith et al.5 showed that, opposite to a common belief, the prevalence rate of HPV in postmenopausal women was almost as high as in younger women and the association between hrHPV and consecutive abnormal Pap smear was 36%. Bosch and Harper7 on the other hand summarised the results from cohort studies and found that hrHPV infection persisted in 50% cases. Bruner and Davey9 had similar results in their group of women aged 60 years and over in contrast with 20% of hrHPV positive infection in younger groups (40 and 50 year old women). That leads towards conclusion of Massad et al.10 that older women more frequently have a higher abnormality grade, while younger women had more lesions connected with transient infection. In our study, following Bethesda recommendations we classified lesions as normal including inflammatory and reactive changes, as LSIL presented with or without koilocytotic changes, as HSIL and as carcinoma. Among premenopausal women with normal cervical smears p16-positive metaplastic cells were noted in one patient. It is known that some metaplastic and endocervical normal cells could be positively stained due to unfinished differentiation of the cells. Staining intensities of the normal, metaplastic cell are weaker than in dysplastic10,12, and staining was observed only in the cytoplasm, while the nucleus showed no staining reaction. After p16INK4a immunostaining, most (70.8%) of dysplastic cells in LSIL were stained negative in a group of premenopausal women. There were only 7 (29.2%) LSIL smears with a stained reaction in the nucleus and cytoplasm of the cells (Figure 1). All of these premenopausal women with HSIL were hrHPV positive (data not shown). Our results showed p16INK4a overall positivity of 61% that is similar to the results of Bose et al.13, although higher percentage up to 78% were reported previously14,15. We agree with the statement of Bose that p16INK4a does not have a role in the diagnosis of LSIL but it might be useful for confirming the diagnosis of HSIL since our study showed positive staining in all HSIL. The role of p16INK4a as a predictive and prognostic factor is to be confirmed by additional large scale studies. Correct cytological diagnosis of cervical abnormalities in older women is a much greater problem because of the morphological similarities between atrophic epithelial cells and cells from high-grade squamous lesion. Therefore one should keep in mind several cytomorphologic findings such as: clean smear background, maturation index, nuclear/cytoplasmatic ratio, presence of hyperchromasia and then incorporate that in physiologic and pathologic changes of women s genital tract. Saad et al.16 divided impact factor analysis to individual (degree of cell maturation, patient age, and smear background) and the other ones that dramatically influenced accurate diagnosis. The following factors were suggested: nuclear membrane features, hyperchromasia with nuclear/cytoplasmatic ratio favouring nucleus and abnormal single cell arrangement. These cytomorphologic findings lead us to an  uncertain cytological diagnosis in postmenopausal smears with profound inflammatory or degenerative changes. Performing p16INK4a immunostaining showed positive staining in the nucleus and cytoplasm suggesting that some of these  uncertain lesions were likely to to be HSIL, which was further confirmed by histology. In cytological negative smears of postmenopausal women p16INK4a staining was negative as well. From our preliminary study on a limited number of samples, it appears that although p16INK4a is not helpful in the diagnosis of LSIL in younger women, in postmenopausal women it may be useful for confirming cervical lesion even if cytological diagnosis was mild dysplasia. To avoid  uncertain diagnosis when the rate of possible false-negative diagnoses as well as false-positive cytology is high, we think that the use of p16INK4a staining would be helpful for identification of adequate diagnosis. For elderly women with squamous atypia an oestrogen treatment is often suggested, maturation effect can make an abnormal cell more viable and abnormal reactive changes disappear. That involves repeated patient s discomfort of physical exam as well as a certain dose of stress. To minimize that we suggest taking two simultaneous smears during Pap sampling; it will allow to perform normal Pap and p16INK4a immunostaining. In this way, we will be able to distinguish more accurately normal, non-specific reactive or inflammatory changes from neoplastic changes in postmenopausal women Furthermore, p16INK4a immunostaining would be useful in a routine laboratory practice for analysis and follow-up of abnormal cervical smears in general. Further population-based studies will be necessary to analyse predictive value of p16INK4a protein in cervical smears in both groups of women. Acknowlegments We are most grateful to the cytotechnologists and other staff of our laboratory for their help in preparation of the samples and other technical support in this study. References 1. " INTERNATIONAL AGENCY FOR RESEARCH ON CANCER (IARC). IARC Handbook of Cancer Prevention. Cervical Cancer Screening, Vol.10, (IARC Press, Lyon, France, 2005). 2. " FLYNN K, RIMM DL, 24 (2001) 132. 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Krivak Bolan a Laboratory for Cytology and Clinical Genetic, Department of Gynaecology and Obstetrics, University Hospital Merkur, Zaj eva 19, Zagreb, Croatia E-mail: HYPERLINK "mailto:ines.krivak@kb-merkur.hr"ines.krivak@kb-merkur.hr Table 1 P16INK4a FINDINGs according to diagnosis of premenopausal women p16INK4aN (%) of cytological diagnosisNormalLSILHSILCarcinomaTotalNegative13 (92.9)17 (70.8)0030 (39)Positive1 (7.1)7 (29.2)35 (100)4 (100)44 (61)Total14 (18.2)24 (31.2)35 (45.5)4 (5.2)77 (100) LSIL  low grade squamous intraepithelial lesion, HSIL  high grade squamous intraepithelial lesion, *cervical squamous cell carcinoma in situ confirmed by histopathology, 2 = 52.24, p < 0.0001 TABLE 2 P16INK4a FINDINGs according to diagnosis of postmenopausal women p16INK4aN (%) of cytological diagnosisNormalLSILHSILCarcinomaTotalNegative34 (100)*3(42.9)2(14.3)1** (20.0) 40 (66.7)Positive04(57.1)12 (85.7)4*** (80.0) 20 (33.3)Total 34 (52.3)7(10.8)14 (21.5)5 (7.7)60 (100)LSIL  low grade squamous intraepithelial lesion, HSIL  high grade squamous intraepithelial lesion, *include 20 women as control group, **transitional cell carcinoma confirmed by histopathology, ***1 case of cervical adenocarcinoma and 3 cases of cervical squamous cell carcinoma in situ confirmed by histopathology, 2-square = 40.97, p < 0.0001 Fig 1. p16INK4a staining of malignant squamous cells found in a smear of a premenopausal womanFig 2. p16INK4a staining of normal metaplastic cells found in a smear of a premenopausal womanFig 3. p16INK4a staining of high grade squamous intraepithelial lesion (HSIL) found in a smear of a postmenopausal woman PROCJENA P16INK4a U LEZIJAMA GRLIA MATERNICE KOD PREMENOPAUZALNIH I POSTMENOPAUZALNIH }ENA SA}ETAK este su pogreane citoloake dijagnoze cervikalnih razmaza postmenopauzalnih ~ena zbog sli nosti u morfoloakom izgledu stanica dubljih slojeva epitela i neoplasti no promijenjenih stanica. U naaoj studiji istra~ili smo primjenu imunocitokemijskog bojanja sa p16INK4a (CINtec"!p16INK4a Kit, DAKOCytomation, Denmark) na cervikalnim razmazima u svrhu rjeaavanja tog problema. Imunocitokemijski smo obojali uzorke 77 ~ena u generativnoj dobi i 60 uzoraka ~ena u postmenopauzi. Kontrolnu skupinu su sa injavali 20 urednih citoloakih razmaza bez izra~enih upalnih ili degenerativnih promjena iz skupine ~ena u postmenopauzi. Kod 68 pacijentica prilikom uzimanja uzoraka u inili smo 2 razmaza: jedan za citoloaku analizu a drugi za imunocitokemijsko bojanje. `ezdesetdevet predhodno uzetih uzoraka smo odbojali i pripremili za imunoreakciju. Kod ~ena generativne dobi 14 pacijentica je imalo uredan Papa-test i opa~en je p16INK4a pozitivitet kod jednog uzorka (7,1%); 24 uzoraka je ocijenjeno kao SIL (skvamozna intraepitelna lezija) niskog stupnja i meu njima je bilo 7 (29,2%) p16INK4a pozitivno obojenih uzoraka . Svih 39 uzoraka SIL visokog stupnja, od kojih 4 karcinoma plo astih stanica in situ naknadno histoloaki utvrenih pozitivno se obojilo. Pozitivitet p16INK4a kod razmaza starijih ~ena je rastao u slijedeem nizu: 0%, 57.1%, 85.7% i 80% kod 34 urednih razmaza, 4 razmaza niskog i 12 visokog SIL te 4 karcinoma naknadno histoloaki utvrenih (1 adenokarcinom, 3 karcinoma plo astih stanica in situ). U skupini od 16 pacijentica, to na citoloaka dijagnoza SIL-a visokog stupnja postavljena je tek nakon imunocitokemijske reakcije. Na temelju naaeg preliminarnog istra~ivanja na malom broju uzoraka, mo~emo zaklju iti da uklju ivanje p16INK4a imunocitokemijskog bojanja u redovitu laboratorijsku praksu mo~e pomoi u razlikovanju reaktivnih ili degenerativnih promjena od neoplasti nih lezija cervikalnog epitela.   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