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00000478-200811000-0001500000478_2008_32_1715_negri_immunohistochemistry_11article< 69_0_6_4 >The American Journal of Surgical Pathology© 2008 Lippincott Williams & Wilkins, Inc.Volume 32(11)November 2008pp 1715-1720p16ink4a and HPV L1 Immunohistochemistry is Helpful for Estimating the Behavior of Low-grade Dysplastic Lesions of the Cervix Uteri[Original Articles]Negri, Giovanni MD*; Bellisano, Giulia MD†; Zannoni, Gian Franco MD‡; Rivasi, Francesco MD§; Kasal, Armin BS*; Vittadello, Fabio ScD∥; Antoniazzi, Sonia BS*; Faa, Gavino MD†; Ambu, Rossano MD†; Egarter-Vigl, Eduard MD**Department of Pathology, Central Hospital Bolzano†Department of Cytomorphology, Section Anatomic Pathology, University of Cagliari, Cagliari‡Department of Pathology, Faculty of Medicine Agostino Gemelli, Catholic University, Rome§Department of Pathology, University of Modena and Reggio Emilia∥Explora, Research and Statistical Analysis, Padova, ItalyCorrespondence: Giovanni Negri, MD, Department of Pathology, Central Hospital Bolzano, Via Boehler 5, 39100 Bolzano, Italy (e-mail: ginegri@gmail.com).AbstractAs only a minority of low-grade dysplastic lesions of the cervix uteri will eventually progress to carcinoma, predicting the behavior of these lesions could be of high value in clinical practice. The aim of the study was to evaluate p16ink4a and L1 as immunohistochemical markers of the biologic potentiality of low-grade dysplasia of the uterine cervix. The study included 38 conization specimens with coexisting cervical intraepithelial neoplasia grade 1 (CIN1) and 3 (CIN3) (group A) and 28 punch biopsies from women with CIN1 and proven spontaneous regression in the follow-up (group B). In group A, all CIN3 were p16ink4a positive (p16+) and L1 negative (L1−). The CIN1 of this group were p16+L1− and p16+L1+ in 68.42% and 31.57%, respectively. No other expression pattern was found in this group. In group B, the p16+L1−, p16+L1+, p16−L1+, and p16−L1− patterns were found in 3.57%, 25%, 14.29%, and 57.14%, respectively. Overall, 96.29% p16+L1− CIN1 were found in group A, whereas all the p16−L1+ and p16−L1− CIN1 were found in group B. A significant difference between staining pattern distributions of group A and B was observed (P<0.0001). The results of the study show that p16ink4a and L1 immunohistochemistry can be helpful for estimating the biologic potentiality of low-grade squamous cervical lesions. Particularly in cases in which the grade of the lesion is morphologically difficult to assess, the p16/L1 expression pattern could be useful for planning the clinical management of these women.One major issue in the management of cervical precancers is the evaluation of the progression risk of dysplastic lesions. As only a minority of low grade and not all high-grade dysplastic lesions will eventually progress to carcinoma,13 some women will undergo unnecessary treatment, whereas conization will be delayed in others. The possibility of predicting the behavior of low-grade cervical lesions could therefore be of high value in clinical practice, potentially allowing an individualized management of cervical lesions depending on their progression risk. Unfortunately, morphologic criteria alone are of limited usefulness in distinguishing lesions that will regress from those that will persist or progress.8,13 Recently developed biomarkers may be useful in the evaluation of the biologic potential of these lesions. The value of p16INK4a (p16) as a diagnostic marker of cervical dysplasia and carcinomas of the cervix uteri has already been demonstrated. A diffuse p16 staining is immunohistochemically detected in almost all high-grade precancer and carcinomas of squamous and glandular epithelia of the cervix, whereas reactive conditions are in most cases negative or show only sporadic staining.6,7,10,12 In biopsies of cervical intraepithelial neoplasia grade 1 (CIN1) p16 is diffusely expressed in about 60% of cases and is typically associated with high-risk human papillomavirus infection, although high-risk human papillomavirus can also be demonstrated in p16-negative lesions1,3,7 In fact, expression of p16 has been shown to indicate already advanced interference of the viral oncoproteins with cellular proteins involved in cell-cycle regulation.15,16,18 Thus, p16 may serve as a surrogate marker for the oncogenic activities of HPV in replication-competent cells of cervical epithelia. In a previous study,11 we evaluated the overexpression of p16 as a possible marker of progression risk of cervical precancerous lesions. In this study, we demonstrated that p16-positive CIN1 have a significantly higher risk of progression compared with p16-negative CIN1. However, a fraction of p16-positive CIN1 may still regress, whereas some p16-negative CIN1 may still progress to CIN3. HPV-L1 (L1) is a capsidic protein that is expressed in the early, productive phase of cervical carcinogenesis and is progressively lost in the later proliferative phase, when p16 gets overexpressed.4 Previous studies have shown that L1 is expressed in up to 43.7% low-grade squamous intraepithelial lesions and 33.3% high-grade squamous intraepithelial lesions.5,9 Furthermore, L1 has already been shown to be prognostic relevant in mild to moderate dysplastic lesions where the L1 expression indicates a higher tendency to regression compared with L1-negative cases.5 As far as we know, however, no progression risk assessment was performed on biopsy specimens and no comparison with p16 expression has been carried out up to date. In this study, we investigated the expression of p16 and L1 on CIN1 biopsy specimens and compared the results with the natural history of the lesions. Furthermore, the expression pattern of p16 and L1 in coexistent CIN1 and CIN3 lesions was assessed.MATERIALS AND METHODSOverall, for this study, 66 histologic samples from 66 women were stained with p16 and L1. The first group of cases (group A) included 38 conization specimens in which CIN1 and CIN3 coexisted in the same section. To assess the coexistence of both lesions, the specimens were reviewed by 2 pathologists (G.N. and G.B.). All CIN1 had an abnormal enlargement of the basal third of the epithelium, associated with typical viral changes in the upper layers. All CIN3 showed a complete replacement of the epithelium by atypical cells and obvious mitotic activity.The second group of cases (group B) included 28 CIN1 punch biopsy specimens from women with cytologically proven regression of the lesion and a negative cytologic follow-up of at least 4 years (mean: 8.1 y). Surgical or noninvasive treatment was excluded for each woman by interviewing the clinicians. Biopsy specimens were provided by 2 different departments, and had originally been diagnosed as CIN1 by several different pathologists. No centralized biopsy review was performed on these cases.All CIN1 punch biopsies were preceded by an abnormal squamous cells of unknown significance or low-grade squamous intraepithelial lesion cytology, all cases of group A by a high-grade squamous intraepithelial lesion cytology or CIN2 or worse punch biopsy. The mean age was 33.13 years and 34.35 years for the groups A and B, respectively.For immunohistochemical analysis, a p16 monoclonal antibody (clone E6H4, CINtec p16INK4a Histology Kit, mtm-laboratories, Heidelberg, Germany) and a L1 monoclonal antibody (HPV L1, cytoactiv diagnostics GmBH, Pirmasens, Germany) were used.Tissue sections were cut at 2 to 4 μm and deparaffinized. Positive and negative controls were used. For p16 staining, after antigen unmasking for 10±1 minutes at 95 to 99°C in Tris buffer, pH 9.0, slides were cooled down in the solution for 20±1 minutes to room temperature. Endogenous peroxidase blocking was performed in 3% hydrogen peroxide for 5±1 minutes. The slides were then first stained with the primary antibody (p16ink4a ready to use for 30±1 min at room temperature) and then with the visualization reagent provided with the kit for 30±1 minutes at room temperature. After development with substrate-chromogene solution (AEC, Dako, Copenhagen) for 10±1 minutes p16-stained slides were finally counterstained with hematoxylin (Dako).For L1 staining after antigen unmasking for 30±1 minutes at 97.5°C in citrate buffer, pH 6.0, slides were cooled down in the solution for 20±1 minutes to room temperature. Endogenous peroxidase blocking was performed in 3% hydrogen peroxide for 5±1 minutes. Protein blocking was performed with Ultra V Block (LabVision Corporation, Fremont). The slides were then first stained with the primary antibody (1:5 for 60 min at room temperature) and then with the visualization reagent (LP Detection System—Polymer, Fremont) 20±1 and +30±1 minutes at room temperature, respectively. After development with substrate-chromogene solution (AEC, Dako, Copenhagen) for 8±1 minutes, L1-stained slides were finally counterstained with hematoxylin (Dako).For the evaluation of the p16-stained slides, both nuclear and cytoplasmic staining was considered. According to the results of a previous study,11 only CIN1 specimens with over 25% of p16-stained cells in the lower third of the dysplastic epithelia (diffuse staining) were judged positive. In CIN3, the whole thickness of the dysplastic epithelium was evaluated.By the evaluation of L1 immunohistochemistry, only nuclear staining was considered. Staining was scored 0, 1+, 2+, and 3+ when no staining or at least 1 high-power field (HPF) with 1 to 3, 4 to 10, and >10 stained nuclei were found, respectively.The Fisher-Freeman-Halton exact test, which is a generalization of Fisher exact test for greater than 2×2 tables, was used to test for differences in frequencies between the groups A and B. Significantly different proportions in distribution (by post hoc χ2 tests in separate 2×2 tables) were analyzed. A value of P<0.05 was considered as statistically significant.The statistical software used was StatsDirect, version 2,6,5 (StatsDirect Ltd, Cheshire, UK).RESULTSThe results of p16 staining in CIN1 lesions are summarized in Table 1. The expression of p16 was cytoplasmic or nuclear and cytoplasmic, and typically located in the lower third of CIN1 (Fig. 1A), whereas in CIN3 the expression was found throughout the epithelium. Overall, p16 was expressed in 46 of 66 CIN1 cases (69.70%). In group A, p16 was diffusely expressed in the lower third of all 38 (100%) CIN1. All 38 (100%) associated CIN3 were diffusely positive throughout the epithelium. In group B, 8 of 28 specimens (28.57%) showed a positive p16 staining.TABLE 1. p16 Expression in 66 CIN1 and 38 CIN3 LesionsGroup A: cases with coexistent CIN1 and CIN3. Group B: CIN1 with spontaneous regression. CIN indicates cervical intraepithelial neoplasia.FIGURE 1. Immunohistochemistry of p16 and L1 in CIN1. A and B, CIN1 with diffuse p16 positivity in the lower third of the dysplastic epithelium (A), L1 (B) is not expressed (high-risk pattern). C and D, CIN1 with strong HPV L1 expression (D), p16 (C) is not expressed (low-risk pattern). E and F, CIN1 with expression of both p16 (E) and L1 (F) (indeterminate pattern).The results of L1 staining in CIN1 lesions are summarized in Table 2. When expressed, L1 showed a nuclear staining in superficial cells of dysplastic epithelia, often with features of koilocytosis (Fig. 1D). Overall, L1 was expressed in 23 of 66 (34.85%) CIN1 cases. In group A, L1 was expressed in 12 of 38 (31.56%) specimens. Staining score was 1+ in 7 of 12 (58.33%) cases, 2+ in 3 (25%), and 3+ in 2 (12.67%). All associated CIN3 were L1 negative. Twenty-six of thirty-eight (68.42%) CIN1 expressed p16 but not L1 (p16+L1−), whereas 12 of 38 (31.58%) CIN1 expressed both p16 and L1 (p16+L1+). In this group, no p16−L1+ or p16−L1− CIN1 were found. All 38 (100%) CIN3 were p16+L1−.TABLE 2. L1 Expression in 66 CIN1 and 38 CIN3 LesionsGroup A: cases with coexistent CIN1 and CIN3. Group B: CIN1 with spontaneous regression. CIN indicates cervical intraepithelial neoplasia.Group B showed a positive L1 staining in 11 of 28 (39.29%) specimens. Staining score was 1+ in 1 (9.09%) cases, 2+ in 4 (36.36%), and 3+ in 6 (54.55%). One of 28 (3.57%) CIN1 expressed p16 but not L1 (16+, L1−), whereas 7 of 28 (25%) CIN1 expressed both p16 and L1 (p16+L1+), 4 of 28 (14.29%) expressed L1 but not p16 (p16−L1+), and in 16 of 28 (57.14%) CIN1 both p16 and L1 were negative (p16−L1−). Careful morphologic evaluation of the immunostained and subsequent slides of p16−L1− cases showed no convincing features of dysplasia.Overall, as shown in Table 3, 26 of 27 (96.30%) p16+L1− CIN1 were found in group A, whereas all the 20 (100%) p16−L1+ and p16−L1− were found in group B. CIN1 with positivity of both p16 and L1 were found in 12 of 38 (31.58%) and 7 of 28 (25%) CIN1 of groups A and B, respectively.TABLE 3. Combined Expression of p16 and L1 in CIN1Group A: cases with coexistent CIN1 and CIN3. Group B: CIN1 with spontaneous regression. CIN indicates cervical intraepithelial neoplasia.Statistical analysis showed that the overall distribution of cases in the 2 groups was significantly different (P<0.0001 by 2-sided Fisher-Freeman-Halton exact test). Post hoc tests showed significantly different proportions in distribution between groups A and B for p16+L1− (P<0.0001), p16−L1+ (P=0.0284), and p16−L1− (P<0.0001), not for p16+L1+ (P=0.5596).DISCUSSIONAs expressed in different phases of cervical carcinogenesis, L1 and p16 are potentially promising markers of progression risk of CIN1. L1 is expressed in the early, productive phase of cervical carcinogenesis and is progressively lost in the later phases, when p16 gets overexpressed.4 The results of our study show that the combination of both L1 and p16 may be useful in the estimation of the biologic risk of low-grade squamous lesions of the cervix uteri. Compared with p16 alone, the combination with L1 may be particularly useful in assessing those lesions that are still in the productive phase of carcinogenesis. Overall, the results may be summarized in 4 different categories:p16 positive, L1 negative (p16+L1−): These are proliferative, no more productive (L1−) lesions with an already inhibited retinoblastoma protein (pRB) pathway and subsequent p16 positivity. This pattern was evident in all (100%) the CIN3 and most (68.42%) CIN1 in the group A. Only one (3.57%) regressive case of group B showed this pattern, with a highly significant difference between both groups. Thus, this pattern might be defined as “high-risk” pattern, which is typically found also in high-grade lesions of the cervix.p16 positive, L1 positive (p16+L1+): These lesions are still productive (L1+), although the pRB pathway is already inhibited (p16+). Although the L1 score was more often low in group A than in group B, this pattern was evident without significative differences in both groups (31.58% of group A vs. 25% of group B). Thus, this pattern might be defined as “indeterminate” pattern.p16 negative, L1 positive (p16−L1+): These are productive lesions with a still working pRB pathway (p16−). These lesions may be associated with low-risk HPV infection, although may also be caused by high-risk HPV.3,7 In our study, this pattern was present only in the regressive group B (14.29%) and was never associated with a CIN3. Thus, this pattern might be defined as “low-risk” pattern.p16 negative, L1 negative (p16−L1−): These specimens showed neither features of productive lesions (L1−) nor of inhibition of the pRB pathway (p16−). In fact, these cases were found only in group B (57.14%). Careful morphologic evaluation of the immunostained and subsequent slides showed no obvious features of dysplasia or HPV (enlargement and atypia of the lower third of the epithelium, koilocytosis). In some specimens the dysplasia may have been lost in the subsequent sections for immunohistochemistry, in other cases marked inflammatory changes were present and may have mimicked a dysplastic lesion. This is a well-known issue that is already well demonstrated by the low diagnostic concordance of CIN.8,17 As in our study no cases with both L1 and p16 negativity were found in group A, this pattern might be classified as “low risk” or, unless the original section shows obvious dysplastic features, as “no evidence of CIN.”Particularly the high-risk, low-risk, and no evidence of CIN patterns seem potentially useful for the routine, being significantly associated with a definite biologic behavior. Indeed, 96.30% of CIN1 with high-risk pattern were found in group A, whereas all low risk or no evidence of CIN patterns were found in group B. Compared with p16 alone, adding L1 permits a better evaluation particularly of p16-negative samples which in some cases may actually represent mimics of dysplasia. However, it is important to bear in mind that even if some patterns may be associated with a higher or lower risk of progression or coexistence with a high-grade lesion, a definitive conclusion concerning the biologic behavior of CIN1 based only on these biomarkers is still not possible. It may be conceivable, for example, that an initially p16−L1+ lesion becomes p16+L1+ or even p16+L1− short after the biopsy, and also “progressive” lesions may eventually regress. Thus, expression patterns of p16 and L1 alone should not influence directly the management of low-grade lesions. In cases in which the grade of the lesion is morphologically difficult to assess, however, the p16 and L1 pattern may be helpful for deciding the appropriate management of the women, for example, suggesting a immediate repetition of biopsy, rather than a cytologic follow-up, to rule out a high-grade lesion.Some limits that are common to most progression studies are also potentially present in this work. Regressive cases are often difficult to recruit because of the necessity of a long follow-up and the need of excluding a surgical or conservative treatment, which implies a complete knowledge of the clinical history of the woman. Although this was done in this study, the tendency to “regression” is obviously influenced also by the diagnostic accuracy of CIN1 diagnoses. Unfortunately, diagnostic agreement in cervical biopsies is generally poor8,17 and may be influenced by different factors, for example, the experience of the pathologist or the knowledge of a previous abnormal smear or molecular HPV test. Indeed, in a previous work concerning progression risk of CIN1,11 the rigorous exclusion from the study of samples that did not show all typical criteria of dysplasia led to the exclusion of many cases that had been previously classified as CIN1. Even if this approach may be more rigorous, we did not perform such a selection in the present study, to preserve the heterogeneity of the CIN1, which reflects the usual condition in a routine laboratory, particularly when several pathologists who may use more or less strict diagnostic criteria are involved. A revision of the slides was conversely performed in the group with coexisting CIN3, because only the most severe lesion had been described in the original diagnosis, therefore making the revision of all cases necessary to assess the presence of both lesions. Because coexisting with a CIN3, these CIN1 might represent a subset of low-grade lesions with a potentially more aggressive behavior and already show some of the genetic changes usually associated with a higher risk of neoplastic progression.9 However, as previous studies claimed that coexisting CIN1 and CIN3 may sometimes be independent,2,14 the CIN1 of group A may not always represent true progressive lesions. Although this may be a potential limit of this study, even CIN1 followed only years later by a CIN3 are not a warranty of being the actual precursors of the high-grade lesion, therefore, making this limit unavoidable independently of the study design. Indeed, in group A, both CIN1 and CIN3 showed in most cases the same p16 and L1 expression pattern, with a significant difference from group B. As far as we know, no data concerning the expression of these biomarkers in coexistent low-grade and high-grade squamous lesions of the cervix have been available up to now.In conclusion, the results of our study show that combining p16 and L1 may allow a distinction between different risk patterns for low-grade lesions. These can be helpful for estimating the biologic potentiality of low-grade squamous cervical lesions. Particularly in cases in which the grade of the lesion is morphologically difficult to assess, the p16/L1 expression pattern could be helpful for the clinical management of these women. Even if this small panel still does not replace the morphologic evaluation of cervical biopsies, it may already contribute to a better understanding of the biologic potentiality of cervical lesions.REFERENCES1. 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[CrossRef] [Medline Link] [Context Link] p16; HPV; L1; cervical dysplasia; biologic behaviorovid.com:/bib/ovftdb/00000478-200811000-0001500003376_2005_121_99_agorastos_intraepithelial_|00000478-200811000-00015#xpointer(id(R2-15))|11065213||ovftdb|SL0000337620051219911065213P46[CrossRef]10.1016%2Fj.ejogrb.2004.11.024ovid.com:/bib/ovftdb/00000478-200811000-0001500003376_2005_121_99_agorastos_intraepithelial_|00000478-200811000-00015#xpointer(id(R2-15))|11065405||ovftdb|SL0000337620051219911065405P46[Medline Link]15949888ovid.com:/bib/ovftdb/00000478-200811000-0001500006693_2006_19_384_benevolo_immunohistochemical_|00000478-200811000-00015#xpointer(id(R3-15))|11065213||ovftdb|SL0000669320061938411065213P47[CrossRef]10.1038%2Fmodpathol.3800551ovid.com:/bib/ovftdb/00000478-200811000-0001500006693_2006_19_384_benevolo_immunohistochemical_|00000478-200811000-00015#xpointer(id(R3-15))|11065405||ovftdb|SL0000669320061938411065405P47[Medline 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