almost all of the invasive cervical cancers are preceded by cervical intraepithelial neoplasia ( cin ) . persistent infections with high risk human papilloma virus ( hr - hpv ) types lead to cin and invasive cancer . despite well - defined criteria , the histopathologic diagnosis is subject to high rates of discrepancy among pathologists . ki-67 is a well - known cell proliferation marker , useful for confirmation of the diagnosis in ambiguous cases and cin grading . p16 is a specific biomarker used for identification of dysplastic cervical epithelium with tendency to invasive cervical cancer . the diagnosis of atypical immature metaplasia ( aim ) has poor intra- and inter - observer reproducibility on routine hematoxylin and eosin ( h&e ) stained sections because of its resemblance to cin 3 . ki-67 immunostaining of aim revealed variable results , with a wide range of reactivity and marked overlap between hpv - negative and hpv - positive cases . cytokeratin ( ck ) 17 is a marker for endocervical reserve stem cells which gives rise to metaplasia and expression of ck17 that decreases and disappeares as the metaplastic epithelium matures . antibody to ck17 is used to differentiate between immature squamous metaplasia ( ism ) and high grade cin ( cin3 ) . aim may be re - classified into metaplasia and cin3 based on p16 and ck17 immuoreactivity and mmunohistochemistry . recent studies have shown that ki67 and p16 could be used as progression markers in cervical lesions . the aim of this study was to evaluate and compare staining pattern for ki67 , p16 and ck17 , as adjunct tests , in differentiating cin from benign lesions to increase the diagnostic accuracy in equivocal cases . case selection and immunohistochemical staining 
  a total of 77 formalin - fixed paraffin embedded cervical specimens were selected from the pathology archives of hospitals affiliated with shiraz university of medical sciences shiraz , iran during 2004 to 2009 . initial diagnosis comprised 10 negative dysplasia ( neg ) , 21 ism with or without reactive atypia , and 46 cin , ( 18 cin1 , 11 cin2 , and 17 cin3 ) . all h&e stained sections were first reviewed by 2 independent pathologists blinded to the initial diagnosis . the consensus diagnosis was a gold standard , and defined as diagnostic agreement between the pathologists concerned . for patients with diagnostic disagreement , 12 of 77 cases , immunohistochemical ( ihc ) staining 
  ihc staining for ki-67 , p16 and ck-17 antigens was performed on 5 m sections obtained from formalin - fixed , paraffin embedded blocks , using avidin - biotin peroxidase complex method . the primary antibodies were monoclonal mouse anti ki-67 antigen , clone mib-1 ( dako , code : n1633 , denmark ; diluted 1:2 ) ; mouse monoclonal anti p16 , ( santa cruz , ( jc8 ) sc-56330 , usa ; diluted 1:50 ) and monoclonal mouse anti - ck17 , clone e3 ( dako , code : m7046 , denmark ; diluted 1:30 ) . secondary antibodies included goat anti - mouse and anti - rabbit immunoglobulines ( dako , code : k4061 , denmark ; ready to use ) and dab ( 3,3 diaminobenzidine ; chromogen ( dako ) . immunohistochemical scoring 
  the sections stained by ihc were examined alongside h&e stained specimens , to identify the precise locations of the lesions . ki-67 ( mib-1 ) staining was interpreted positive when a cluster of at least 2 strongly stained epithelial nuclei were present in the upper two thirds of the epithelial thickness anywhere within the lesion . presence of the para - basal cells staining was used as an internal positive control . the p16 was considered positive when it showed nuclear , as well as continuous diffuse cytoplasmic staining of the cells in the basal and para - basal cell layers of the squamous epithelium , variably reaching intermediate and superficial cell layer characterized by diffuse staining pattern . p16 was considered negative when it was completely unstained , or showing focal or sporadic epithelial staining , particularly not of the basal and para - basal cells ( focal staining pattern ) . scoring of ihc results was evaluated on the basis of distribution of immunoreactive cells . however , staining intensity was not graded to avoid subjective interpretation . statistical analysis 
  histologic diagnoses were categorized as ( neg ) , cin1 ( lg - sil ) , cin2 & cin3 ( hg - sil ) , and ism . the concordance between each of the initial and consensus diagnosis with ki67 , p16 and ck17 reactivity was determined by kappa statistical analysis using spss software package , version 15 . kappa values ( k ) of ( 1.0 - 0.75 ) , ( 0.5 - 0.75 ) , ( 0.25 - 0.5 ) and less than 0.25 were considered as thresholds for excellent , good , moderate , and poor concordance respectively . in review of 77 patients with previous diagnoses , the cases were reclassified as 24 negative for dysplasia ( neg ) ; 4 as cin1 ; 5 as cin2 ; 14 as cin3 ; and 30 ism ( figures 1a , 2a , 3a ) . the sensitivity and specificity of the previous diagnoses were 95.6% and 55.5% with 47.8% and 96.8% ppv and np respectively . the overall agreement between previous and consensus diagnosis was 67.5% , ( kappa=0.39 , p<0.001 ) . hematoxylin and eosin ( h&e ) and immunohistochemical staining of ki67 , p16 and ck17 in cin1 . c , diffuse ( one - third ) p16 immunostaining in cin1 and negative staining in normal epithelium . ( 100 )   h&e and immunohistochemical staining of ki67 , p16 and ck17 in immature squamous metaplasia ( ism ) . ( a : 100 , b , c , d : 400 )   h&e and immunohistochemical staining of ki67 , p16 and ck17 in cin3 . a , h&e staining . ( a , b , c , d : 400 ) 
 ki67 immunostaining 
  the patterns of positive ki67 staining were regarded as scattered ( 5.2% ) or / and diffuse ( 97.3% ) ; ( figures 1b , 2b , 3b ) . other 7 cases of ism were only positive for ki67 with scattered patterns ( figure 2b ) . the sensitivity and specificity of ki67 staining are 95.6% and 85.1% respectively with 73.3 positive predictive value ( ppv ) and 97.8% negative predictive value ( npv ) . the overall agreement regarding ki67 with consensus diagnosis was 88.3% ( kappa=0.74 , p<0.001 ) . p16 immunostaining 
  all cases of hg - sil were positive with strongly diffuse staining . the staining was both nuclear and cytoplasmic , and mostly involved full - thickness of the epithelium ( figure 3c ) . also p16 was positive in 2 of 4 cin1 patients , of which one was diffuse basal and the other diffuse one - third thickness ( figure 1c ) . of ism cases , 73.3% were negative for both p16 and ki67 . additionally , p16 staining was entirely negative for neg and ism cases ( figure 2c ) . the sensitivity and specificity of p16 staining were 91.3% and 98.1% , with 95.4% ppv and 96.3% npv , respectively . the overall agreement between p16 and consensus diagnosis was 96.1% ( kappa=0.90 , p<0.001 ) , which were higher than those of ki67 and consensus diagnosis . all of the p16 positive cases were also positive for ki67 , but none of the p16 positive cases were negative for ki67 ( table 1 ) . the agreement between ki67 and p16 was higher than ki67 and consensus diagnosis , but lower than p16 and consensus diagnosis . correlation between p16 and ki67 immunostaining 
  ck17 immunostaining 
  ck17 was positive in 3 of 24 neg , 3 of 4 cin1 , 1 of 5 cin2 , 9 of 14 cin3 , and 30 of 30 ism cases ( figures 1d , 2d , 3d ) . the sensitivity and specificity of ck17 negativity for cin detection were 39.1% and 40.7% with 21.9% ppv and 61.1% npv , respectively . the overall agreement between ck17 with consensus diagnosis was 46.7% ( kappa=-0.015 , p=0.89 ) . there was poor negative correlation between ck17 negative staining and consensus diagnosis in cin detection . the evaluation of cin is subjective in relation to intra- and inter - observer variability regarding interpretation of histomorphologic features . variability in diagnosis of cin by assessment of h&e staining in the current and some other studies are presented in table 2 . interobserver variability for the assessment of h&e stained sections * nondysplastic tissue includes normal , inflammation , mature and immature metaplasia , atypical cells . * * 71% in normal and 3% in the other nondysplastic tissues diagnosis of cin1 on the basis of h&e staining alone is subject to a high level of intra - observer variability . many studies show that ihc staining for ki67 and p16 is a very useful adjunctive aid in the diagnosis of equivocal cervical biopsies . in the previous studies , ki67 expression has been found to be associated with the grade of dysplasia , indicating that ihc for ki67 is a useful adjunctive test in the evaluation of low - grade lesions of the cervix . the advantage of mib-1 staining over hpv testing is its higher specificity , since the staining is negative in subclinical hpv infections . other advantages of this marker are simplicity , availability , reproducibility , and low - cost laboratory techniques . although presence of mib-1 positive nuclei in the upper two thirds of epithelial thickness is outstanding criteria for mib-1 positivity , there are few false positive interpretations of the staining , such as tangential sectioning with the presence of positive nuclei in the superficial layers of the epithelium , mib-1 positive lymphocytes throughout the epithelial thickness in the cervicitis , mib-1 positive cells in the upper layers of epithelium in the ism and areas of repair . any ki67 positivity in an atrophic epithelium , especially when diffuse , is consistent with sil , since atrophic epithelium has virtually no staining . another study showed sensitivity of 71.4% , 94.7% , and 7.7% for ki67 in lsil , hsil and non - dysplastic lesions , respectively . in the present study , the respective sensitivity and specificity of ki67 were 95.6% and 85.1% . ihc staining for p16 yields greater accuracy of cin grading with less variability and helps to avoid unnecessary diagnostic and surgical procedures related to pregnancy - associated morbidity and psychological distress . several studies show that p16 is a sensitive and specific marker with a better predictive value than hpv - dna testing for high grade sil . the results of our study is concordant with the results of previous studies ( table 3 ) . comparison of p16 staining in the previous reports and the present study * nondysplastic tissue includes normal , reactive , inflammation , mature and immature metaplasia , and atypical cells cin1 lesions show a more variable reactivity , with percentages of diffuse positive staining ranging from 20 - 50% . p16 staining is considered as a useful and reliable diagnostic adjunct for distinguishing biopsies with and without cin2 or is more severe but not so useful for discriminating between cin1 and non - cin . ki67 staining was inferior to p16 and its inclusion with p16 shows no marked improvement in clinical performance over p16 per se . in our study , sensitivity and specificity for p16 were 91.3% and 98.1% respectively . one study reveals that unlike non - progressive cases with negative cin1 , all cin1 biopsies from patients who progressed to cin 2 - 3 were positive for p16 ,   in this context , cin1 lesions with positive p16 showed a markedly higher tendency to progress to cin2 - 3 , indicating that p16 may have a significant role in the evaluation of cin1 lesions , excluding about half of the cases from an invasive clinical follow up . supplementary use of p16 staining significantly improves the accuracy of grading cin lesions by a single pathologist , equivalent to an expert consensus diagnosis . some authors re - classified aim lesions in consensus diagnosis based on ki67 and p16 ihc and hpv tests . almost two - thirds of aim cases could be re - classified as benign based on negative p16 staining . another one - third could be re - classified as hsil regarding positive ki67 and p16 staining . another study showed a strong uniform cytoplasmic ck17 positivity of the proliferating cells together with p16 negativity in ism lesions . the lesions featuring both metaplastic changes and atypia with staining of both p16 and ck17 are classified as high - grade dysplasia . the current study revealed that ck17 can be positive in immature squamous metaplasia , and in some cin lesions . for differentiation between metaplastic lesions with or without dysplasia , it would be helpful to consider another marker such as p16 . based on h&e stained sections , consensus diagnosis was reached in one of the cases with ism . however , ihc study showed ki67 and p16 positivity , which was compatible with cin2 . in another case , consensus diagnosis was cin1 but ihc staining for ki67 , and p16 were negative , a finding consistent with non - dysplastic lesion . the limitation of this study was the number of cin cases collected from the files hospitals concerned . it should be considered that cin cases in this region are not as common as those in western countries . we recommend using ki67 and p16 markers as complementary tests for differentiation between dysplastic and non - dysplastic lesions . ki67 can be positive in some immature squamous metaplastic lesions , thus p16 is useful to rule out dysplasia . testing for p16 is proposed to rule out dysplasia which is positive in almost all hsil cases . however , it may be positive or negative in lsil . a complementary study including more cases and follow up examinations is warranted for better evaluation and definitive prognostic significance of these biomarkers .
background : cervical intraepithelial neoplasia ( cin ) is a premalignant lesion capable of progressing to cervical cancer . despite the existing well - defined criteria , 
 the histomorphologic diagnosis is subject to high rates of discordance among pathologists . 
 the aim of this study was to evaluate ki-67 ( mib-1 ) , ck17 and p16 ink4a ( p16 ) markers by immunohistochemical methods in differentiating cin from benign cervical lesions . 
 methods : the present study reviewed and re - classified 77 cervical biopsies , originally diagnosed as 31 non - cin , and 46 cin , as 54 non - cin , and 23 cin based on at least two similar diagnoses . immunostaining by ki67 , p16 and ck17 markers was performed on all cases and the results were compared with pervious and consensus diagnosis . 
 results : the overall agreement between pervious and consensus diagnosis was 67.5% ( kappa=0.39 , p<0.001 ) . 
 the sensitivity and specificity of ki67 immunostaining were 95.6% and 85.1% respectively , while for p16 the corresponding values were 91.3% and 98.1% . 
 the overall agreement , for both p16 and ki67 , with consensus diagnosis were significant ( p<0.001 ) . 
 the sensitivity and specificity of ck17 negative staining in cin detection were 39.1% and 40.7% respectively . 
 conclusion : ki67 and p16 markers are recommended as complementary tests for differentiating between dysplastic and non - dysplastic lesions . 
 ck17 does not discriminate between immature metaplasia with and without dysplasia .